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Relaxin Family Peptides and Their Receptors

January 2013
Physiological Reviews 93(1):405-80

January 2013
93(1):405-80

DOI:10.1152/physrev.00001.2012

Source
PubMed

Authors:

Ross Bathgate

University of Melbourne

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There are seven relaxin family peptides that are all structurally related to insulin. Relaxin has many roles in female and male reproduction, as a neuropeptide in the central nervous system, as a vasodilator and cardiac stimulant in the cardiovascular system, and as an antifibrotic agent. Insulin-like peptide-3 (INSL3) has clearly defined specialist roles in male and female reproduction, relaxin-3 is primarily a neuropeptide involved in stress and metabolic control, and INSL5 is widely distributed particularly in the gastrointestinal tract. Although they are structurally related to insulin, the relaxin family peptides produce their physiological effects by activating a group of four G protein-coupled receptors (GPCRs), relaxin family peptide receptors 1-4 (RXFP1-4). Relaxin and INSL3 are the cognate ligands for RXFP1 and RXFP2, respectively, that are leucine-rich repeat containing GPCRs. RXFP1 activates a wide spectrum of signaling pathways to generate second messengers that include cAMP and nitric oxide, whereas RXFP2 activates a subset of these pathways. Relaxin-3 and INSL5 are the cognate ligands for RXFP3 and RXFP4 that are closely related to small peptide receptors that when activated inhibit cAMP production and activate MAP kinases. Although there are still many unanswered questions regarding the mode of action of relaxin family peptides, it is clear that they have important physiological roles that could be exploited for therapeutic benefit.

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RELAXIN FAMILY PEPTIDES

AND THEIR RECEPTORS

R. A. D. Bathgate, M. L. Halls, E. T. van der Westhuizen, G. E. Callander, M. Kocan,

and R. J. Summers

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences & Department of Pharmacology, Monash

University, Victoria, Australia; Florey Neurosciences Institutes and Department of Biochemistry and Molecular

Biology, The University of Melbourne, Victoria, Australia; and Université de Montréal, Institut de Recherche en

Immunologie et Cancérologie, Pharmacologie Moléculaire, Montréal, Canada

LBathgate RAD, Halls ML, van der Westhuizen ET, Callander GE, Kocan M,

Summers RJ. Relaxin Family Peptides and Their Receptors. Physiol Rev 93: 405–

480, 2013; doi:10.1152/physrev.00001.2012.—There are seven relaxin family

peptides that are all structurally related to insulin. Relaxin has many roles in female

and male reproduction, as a neuropeptide in the central nervous system, as a

vasodilator and cardiac stimulant in the cardiovascular system, and as an antiﬁbrotic agent.

Insulin-like peptide-3 (INSL3) has clearly deﬁned specialist roles in male and female reproduc-

tion, relaxin-3 is primarily a neuropeptide involved in stress and metabolic control, and INSL5

is widely distributed particularly in the gastrointestinal tract. Although they are structurally

related to insulin, the relaxin family peptides produce their physiological effects by activating a

group of four G protein-coupled receptors (GPCRs), relaxin family peptide receptors 1–4

(RXFP1–4). Relaxin and INSL3 are the cognate ligands for RXFP1 and RXFP2, respectively,

that are leucine-rich repeat containing GPCRs. RXFP1 activates a wide spectrum of signaling

pathways to generate second messengers that include cAMP and nitric oxide, whereas RXFP2

activates a subset of these pathways. Relaxin-3 and INSL5 are the cognate ligands for RXFP3

and RXFP4 that are closely related to small peptide receptors that when activated inhibit cAMP

production and activate MAP kinases. Although there are still many unanswered questions

regarding the mode of action of relaxin family peptides, it is clear that they have important

physiological roles that could be exploited for therapeutic beneﬁt.

I. INTRODUCTION: THE DISCOVERY OF... 405

II. MOLECULAR BIOLOGY, SYNTHESIS,... 413

III. MOLECULAR BIOLOGY, STRUCTURAL... 417

IV. EXPRESSION AND SECRETION OF... 425

V. LOCALIZATION OF RELAXIN FAMILY... 429

VI. SIGNAL TRANSDUCTION PATHWAYS... 431

VII. PHYSIOLOGICAL ROLES OF... 443

VIII. POTENTIAL THERAPEUTIC... 456

IX. MAJOR UNANSWERED QUESTIONS... 459

I. INTRODUCTION: THE DISCOVERY OF

RELAXIN FAMILY PEPTIDES AND THEIR

RECEPTORS

A. The Discovery of the Relaxin Family

Peptides

Relaxin was discovered and named by Dr. Frederick Hisaw

following observations of the reproductive endocrinology

of the gopher and later the guinea pig. He noticed that there

was softening and expansion of the pubic ligament just

prior to delivery in the pregnant female that facilitated par-

turition. In 1926 he showed that the injection of serum from

pregnant guinea pigs or rabbits caused relaxation of the

pubic ligament of virgin guinea pigs when given shortly

after estrus (220). The following year the same relaxing

factor was shown to be present in pig corpus luteum and

rabbit placenta (221). The hormone was formally named

relaxin after it was extracted from pig corpus luteum in

1930 (165). For the next 15 years or so, relatively little

work was done with relaxin, but post World War II there

was a reawakening of interest in its physiological role that

established properties that would be useful in understand-

ing its roles in pregnancy and parturition. These included,

in estrogen-primed animals, expansion of the public liga-

ment of mice (193, 195), relaxation of the uterine myome-

trium in guinea pigs (295), and cervical softening in cattle.

The subsequent puriﬁcation of relaxin from animal sources

(initially pigs but then many other species) led to the deter-

mination of the peptide structure, biological actions and

generation of reliable bioassays. The information obtained

from the determination of the peptide structure enabled the

use of recombinant DNA techniques to clone the rat (244)

and pig (192) relaxin cDNAs that demonstrated a close

structural relationship with insulin (FIGURE 1A). Screening

Physiol Rev 93: 405–480, 2013

doi:10.1152/physrev.00001.2012

of a human genomic library resulting in the cloning of hu-

man gene-1 relaxin (RLN1) (245) followed by the isolation

of a second gene, human gene-2 relaxin (RLN2), from a

human luteal cDNA library (246). It has subsequently been

shown that humans and higher primates have RLN1 and

RLN2 genes, whereas other mammals have only RLN1.

Importantly, the peptide encoded by the human RLN2 gene

and the RLN1 gene from other mammals encodes the re-

laxin peptide circulating during pregnancy. The function of

the RLN1 gene in humans and higher primates is unknown.

The ability to clone human relaxin genes and species ho-

mologs led to the production of relaxin by recombinant

techniques and the development of the relaxin knockout

A B

B-chains:

A-chains:

INSL3

INSL6

INS

IGF2

IGF1

INSL4 INSL5

RLN2

RLN1

RLN3

R3 R2

CysB10

CysB22

CysA24

CysA10

CysA15

CysA11

CysA11 CysA15

A-chains B-chains

FIGURE 1. Relationship between relaxin family peptides, insulin, and insulin-like peptides. A: CLUSTALW

(v1.83) alignment of human relaxin/insulin family peptides. Relaxin and insulin family peptide sequences

are from the UniProt/SwissProt database: human relaxin-1 (P04808), human relaxin-2 (P04090),

human relaxin-3 (Q8WXF3), human INSL3 (P51460), human INSL4 (Q14641), human INSL5 (Q9Y5Q6),

human INSL6 (Q9Y581), human IGFI (P01343), human IGFII (P01344), and human insulin (P01308).

Conserved cysteine residues are black with gray background. Solid black lines indicate disulﬁde bonds.

Identical amino acid residues are indicated by *. The insulin-like growth factors are not cleaved from the

pro-hormone state, thus indicating that the sequence of these peptides is continuous. B: an unrooted

phylogenetic tree derived using the neighbor joining method in CLUSTALW. Human peptide sequences

were edited to delete the C and signal peptide sequences. The resulting domains corresponding to the B

and A chains were further edited to minimize gaps, and then concatenated (562). The phylogenetic tree

was formatted using the program Dendroscope (249). C: A-chains and B-chains of the cognate relaxin

family peptides for RXFP1–4 superimposed on one another. This demonstrates the extraordinary degree

of structural similarity between the peptides that differ mainly in the mid and COOH-terminal regions of

the B-chain. R2 (magenta), human relaxin-2; R3 (blue), human relaxin-3; I3 (red), INSL3; I5 (green),

INSL5.

BATHGATE ET AL.

406 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

mouse. Furthermore, cloning strategies or database search-

ing using the conserved relaxin peptide structure led to the

discovery of a variety of related peptides. These include

insulin-like peptide 3 (INSL3) (2), INSL4 (94) by differen-

tial cloning, and INSL5 (100) and INSL6 (236) by ex-

pressed sequence tags (EST) database screening. The most

recent addition, relaxin-3, was discovered by screening the

human genomic database (46). In humans, the seven relaxin

family peptides that have been identiﬁed are encoded by

genes that possess a similar structure (FIGURE 1, AAND C)

and synthesize similar prepropeptides (241). The mature

peptides are all predicted or proven to be processed from

the prepropeptides by convertases to produce two chain

peptides that all have an A-chain linked to a B-chain by two

disulﬁde bonds with an additional intrachain disulﬁde in

the A chain (see section II).

Although many of the relaxin family peptides were discov-

ered as a result of their roles in reproduction, it is now clear

that they perform a wide variety of physiological functions

in humans. Some such as relaxin have widely differing roles

between species, whereas others such as relaxin-3 and

INSL3 have well conserved roles in all mammalian species.

B. The Relaxin Peptides

As mentioned above, a relaxin gene is present in all mam-

mals and is responsible for the production of the relaxin

peptide that is circulating in the blood during pregnancy.

However, the levels of relaxin in the blood during preg-

nancy are very different between species and correlate with

the varied roles for relaxin in pregnancy and parturition in

different species (see sect. IVA). In humans, blood levels of

relaxin are highest in the ﬁrst trimester, and consequently,

relaxin is not associated with parturition in humans. Nev-

ertheless, it is likely that in humans, relaxin has a role in the

early stages of pregnancy and also in the cardiovascular

changes that accompany pregnancy. Furthermore, it is be-

coming increasingly clear that relaxin is produced in many

tissues in mammals as a paracrine or autocrine factor to

exert many different physiological roles (see sect. IVA).

Relaxin-3 is the most recently identiﬁed relaxin family pep-

tide and was discovered following a search of the human

genomic database (46). It is classiﬁed as a relaxin peptide

based on the RxxxRxxI/V relaxin-binding motif in the B-

chain, despite an otherwise relatively low sequence homol-

ogy to other relaxin peptides (FIGURE 1A). Unlike the other

relaxins, which show considerable species heterogeneity

apart from the relaxin binding motif and key structural

residues, the relaxin-3 sequences are well-conserved across

species (562, 574). It is now widely accepted that relaxin-3

is the ancestral peptide of the relaxin family peptides (562)

and that in mammals is primarily a neuropeptide expressed

almost exclusively in brain (46). Functions likely to be as-

sociated with relaxin-3 include stress, memory, and appe-

tite regulation (see sect. VIIC) (25, 329, 358, 516).

C. INSL3, 4, 5, and 6

INSL3 (formerly Leydig insulin-like peptide) was discov-

ered in the Leydig cells of the testis (2) and is highly ex-

pressed in these cells in all species that possess the INSL3

gene (47). Although there is evidence for INSL3 expression

in other tissues (see sect. IVB), this occurs at much lower

levels than in the Leydig cells. As such, INSL3 has a critical

role in testis descent, and INSL3 knockout mice are cryp-

torchid and as a consequence infertile (379, 596). INSL3

plays an important role in the development of the guber-

naculum, which is important in the ﬁrst stage of testis de-

scent, and it also appears to have a role in the maintenance

of fertility in females (490) (see sect. VIIB).

Compared with the other relaxin family peptides, very little

information is available regarding INSL4–6. INSL4 is highly

expressed in the placenta (94) and may play a role in bone

development (303), but its target receptor has yet to be iden-

tiﬁed. INSL5 on the other hand has quite a wide distribution

pattern (see sect. IVD) with particularly high expression in the

gastrointestinal tract (100). Although INSL5 knockout mice

have been developed, the associated phenotype is currently not

well characterised; nevertheless, there may be functional ef-

fects on fat and glucose metabolism [patent WO2005124361

(A3)]. This is of interest given that the related peptides insulin

and the insulin-like growth factors, IGF-I and IGF-II, have

well-deﬁned roles in glucose metabolism. INSL5 activates

RXFP4 with high potency but unlike relaxin-3 does not acti-

vate RXFP1 or RXFP3 and in fact is a weak antagonist at

RXFP3 (315). It is now widely accepted that INSL5 is the

cognate ligand for RXFP4 (212, 315). INSL6 was discovered

by screening EST databases (236, 275, 317) and like INSL3 is

found predominantly in the testis. Speciﬁc localization of pep-

tide expression in spermatocytes and spermatids suggests a

role for INSL6 in male fertility, and male INSL6 knockout

mice show a marked reduction in sperm numbers and motility

(85). The receptor for INSL6 is currently unknown.

D. Receptors for the Relaxins and

Insulin-like Peptides

The search for the cognate receptors for the relaxin family

peptides proved demanding and was complicated by the close

similarity between relaxin family peptides and insulin which

together with studies that showed an increase in tyrosine phos-

phorylation of a 220-kDa protein in response to relaxin (407)

suggested that they were tyrosine kinases. However, the key

insights came from an unlikely direction when it was noticed

that INSL3 knockout mice (379, 596) shared the same pheno-

type of abnormal testis descent as mice with disruptions in the

G protein-coupled receptor encoded by the GREAT gene

(later shown to be the mouse ortholog of human LGR8) (406).

This suggested that the targets for relaxin family peptides

might be GPCRs and subsequently led to the deorphanization

of LGR7 and LGR8 (239). Construction of constitutively ac-

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

407Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

tive mutants of LGR7 and LGR8 showed that these receptors

likely signaled by activating the adenylyl cyclase pathway and

further stimulation of these receptors by relaxin resulted in

increases in cAMP (239). A subsequent study demonstrated

that, as anticipated from the studies above, LGR8 is the recep-

tor for INSL3 (65, 301). Two other orphan receptors

GPCR135 and GPCR142 were shown to be the targets for

relaxin-3 and INSL5, respectively (313, 314). These receptors

have now been classiﬁed as relaxin family peptide (RXFP)

receptors: RXFP1 (LGR7*), RXFP2 (LGR8*), RXFP3

(GPCR135*), and RXFP4 (GPCR142*) (*these are the previ-

ously used names for the RXFP receptors prior to reclassiﬁca-

tion by NC-IUPHAR) (FIGURE 2D)(42). Cognate receptors for

INSL4 and INSL6 have yet to be identiﬁed, and these peptides

do not interact with any of the RXFP receptors.

RXFP1 is the cognate receptor for human relaxin-2 in humans

that in addition to the classical 7-transmembrane spanning

regions has a large extracellular domain containing 10 leucine-

rich repeats (LRR) and a unique NH

-terminal low-density

lipoprotein receptor type A (LDLa) module (239) (FIGURE 3).

As beﬁtting the roles of relaxin in reproduction, RXFP1

mRNA and protein are found in a wide range of reproductive

tissues including ovary, uterus, placenta, mammary gland,

prostate, and testis (FIGURE 4). The receptor is also found in

the heart, kidney, lung, liver, and blood cells as well as in a

number of areas of brain such as cortex, organum vasculosum

of the lamina terminalis (OVLT), and subfornical organ (SFO)

(see TABLE 1 and sect. VA). Thus relaxin not only has auto-

crine and paracrine roles but also acts as a neuropeptide. In-

teraction of relaxin with RXFP1 involves at least three stages:

high-afﬁnity binding between the 〉-chain and the LRR region;

lower afﬁnity binding to the transmembrane exoloops, and

ﬁnally an interaction involving the LDLa module that is essen-

tial for signaling (199, 501) (FIGURE 3). Although RXFP1 cou-

ples to numerous signal transduction pathways (see sect. VIA),

many early studies indicated that treatment with relaxin

caused increases in cAMP levels in tissues (69, 95, 96, 446).

This link with cAMP signaling was strengthened in the studies

that deorphanized LGR7 (RXFP1) where it was shown that a

constitutively active receptor mutant generated cAMP (239)

(FIGURE 3). This has been conﬁrmed in numerous studies that

show that RXFP1 couples to at least three G proteins to induce

a complex pattern of cAMP production (198, 202, 204). In

addition, RXFP1 is known to activate Erk1/2, tyrosine ki-

nase(s), gene transcription, and nitric oxide (NO) signaling

and can also interact with the glucocorticoid receptor (GC).

The full implications of the pleiotropic effects of relaxin are

still to be elucidated.

RXFP2 shares very close structural similarity with RXFP1

(239) and is the cognate receptor for INSL3 (301). It has a

much more restricted distribution, physiological function,

and signaling proﬁle than RXFP1 (FIGURE 4;TABLE 1).

RXFP2 is expressed in the gubernaculum as anticipated

from its critical role in testicular descent but is also ex-

pressed in the testis and ovary relating to roles in gonadal

function (276) and in bone relating to a role in bone metab-

olism (161) (see TABLE 1 and sect. VIIB). RXFP2 signaling

also involves adenylyl cyclase activation, but this is re-

FIGURE 2. Evolutionary relationships between human RXFP receptors and other family A GPCRs. Receptors showing similarity to RXFP1 or

RXFP3 were identiﬁed by using complete transmembrane domain amino acid sequences (from the beginning of helix 1 through to the COOH

terminus) to conduct BLAST searches against the human REFSEQ protein database. Sequences showing the greatest similarity with each of

RXFP1 or RXFP3 were then aligned using CLUSTALW, and a phylogenetic tree derived by the neighbor-joining method, also in CLUSTALW.

A: the phylogenetic tree shows that RXFP1 and RXFP2 are closely related to each other, with 59% sequence identity and 80% similarity based

on conservative amino acid changes. They form a subgroup that also includes the three glycoprotein hormone receptors for thyroid stimulating

hormone (TSHR), luteinizing hormone/chorionic gonadotropin (LHCGR), and follicle stimulating hormone (FSHR). These receptors show

somewhat higher homology to RXFP1 and RXFP2 (27–29% identity, 49–51% similarity) than a second group comprising the leucine-rich

repeat-containing receptors LGR4, LGR5, and LGR6 (22–25% identity, 46–47% similarity). RXFP1 and RXFP2 are also related to family A

bioamine receptors such as

␤

-adrenoceptors (ARs) and adenosine receptors, as well as additional peptide receptors. B: the RXFP3 and RXFP4

amino acid sequences that display 43% sequence identity and 60% similarity are most closely related to a subgroup comprising the angiotensin

II type 1 receptor (AGTR1), the apelin receptor (APLNR) and the chemokine receptor (CCR1), and a second subgroup containing neuropeptide

receptors including members of the somatostatin and opioid receptor families and the neuropeptide B/W receptor (NPBWR1). They are also related

to various chemokine receptors (CXCR7, CXCR4, CCR1, and CCR9) and to the formyl peptide receptors (FPR2 and FPR3), C: the RXFP1/RXFP2 and

RXFP3/RXFP4 receptor subgroups are not highly related to each other, and in fact, the only signiﬁcant homology between RXFP1 and RXFP3 is

within the highly conserved TM7 motif NSxLNP(I/V)Y that is essential for G protein coupling as well as neighboring key residues in helix 8. RXFP1

and RXFP4 show slightly higher homology, although again the regions of similarity encompass only short motifs in TM3, TM6, and TM7. Instead,

both receptor subgroups are related to other family A GPCRs such as the

␤

-AR and the adenosine A2B receptor. In essence, these bioamine

receptors occupy a bridge between the distinct RXFP1/RXFP2 and RXFP3/RXFP4 subgroups. For example, the

␤

-AR has homologies with

each of the RXFP1 and RXFP3 receptors, with 23% identity/39% similarity to RXFP1 and 21% identity/38% similarity to RXFP3. D: RXFP1

and RXFP2 are closely related to each other and have a characteristic extracellular leucine-rich repeat region and a unique low-density lipoprotein

receptor class A module at the NH2 terminus. In contrast, RXFP3 and RXFP4 are both similar to small peptide (e.g., bradykinin, angiotensin

II) liganded receptors. The primary ligands for each RXFP are shown in FIGURE 1. Whereas the RXFP1/RXFP2 and RXFP3/RXFP4 receptors

belong to separate subtrees, the endogenous ligands do not show a corresponding split in lineage. Based on BLASTP and also PSI-BLAST

searches using the human relaxin-2 B chain sequence, human relaxin-3 is somewhat more similar than INSL3 (57% identity, 67% similarity

versus 52% identity, 62% similarity, respectively), while INSL5 is the least related. Searches using the A chain sequence indicate that human

relaxin-3 and INSL3 are equivalent in their homology to human relaxin-2, and again INSL5 is more distantly related. Thus human relaxin-3 is

closely related to human relaxin-2 despite the corresponding primary receptors residing in different subgroups. This is not surprising given the

distinct nature of the ligand binding domains in each receptor subgroup and indicates that the capacity to bind related peptides has arisen by

a process of convergent evolution.

BATHGATE ET AL.

408 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

SSTR1

SSTR5

SSTR2

RXFP4

RXFP3

RXFP1

RXFP2

TSHR

FSHR

LHCGR

LGR5

LGR6

LGR4

APLNR

CCR1

AGTR1

OPRL1

OPRM1 NPBWR1 ADRB2

ADORA2B

NPFF2 RXFP1 RXFP2

TSHR

LHCGR

FSHR

LGR5

LGR6

LGR4

GHSR

OXTR

SSTR1

SSTR4

AGTR1

AGTR2

ADORA2B

DORA2A

ADORA1

ADRB1

ADRB2

SSTR1

SSTR5

SSTR2

SSTR4

OPRM1

OPRL1

OPRK1

OPRD1

NPBWR1

CCR1

CXCR4

CCR9

FPR3FPR2

CXCR7

AGTR2

AGTR1

APLNR RXFP3 RXFP4

Extracellular

Intracellular

RXFP3/4 RXFP1/2

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

409Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

stricted to a subset of the G proteins utilized by RXFP1 (see

sect. VIB). Although in the cell systems often used to study

RXFP2 activation of the receptor causes an overwhelming

increase in cAMP levels, in endogenously expressing sys-

tems both increases and decreases in cAMP are seen. For

instance, in gubernacular cells (301) or osteoblasts (161),

activation of RXFP2 causes increased cAMP, but in male

germ cells and oocytes, the opposite effect is observed (276).

This may reﬂect the particular expression pattern of signal-

ing proteins in the various cells (202). Interestingly, some

species relaxins have been shown to activate RXFP2 in vitro

(45, 239, 301, 453) but the physiological signiﬁcance (if

any) of this interaction is not clear. There is no evidence that

suggests that relaxin can activate RXFP2 in vivo. Mouse

relaxin does not activate RXFP2 (45), indicating that stud-

ies in the RXFP1 knockout mouse will not resolve possible

physiological actions of relaxin at RXFP2.

RXFP3 and RXFP4 are both similar to type 1 small peptide

receptors and are distinctly different in structure from

RXFP1 and RXFP2 (FIGURE 2D). RXFP3 was originally

named SALPR or somatostatin and angiotensin-like pep-

Leucine-rich repeat region

Primary high affinity binding site

LDLa module

LDLa-less receptor binds but

does not signal

ECL region

Secondary lower affinity

binding site

IL3-Gαs coupling

Asp637 constitutive activity

Helix 8 Interaction

with AKAP79

C-tail Ser704 β-arrestin-2 binding

Final 10 residues + Arg752 - Gαi3 activation

RXFP1

FIGURE 3. Structural features of the relaxin family peptide receptor RXFP1. The RXFP1 receptor has a large

extracellular domain that contains an NH

-terminal low-density lipoprotein receptor type A (LDLa) module

connected to a leucine-rich repeat (LRR) region tethered to the transmembrane domains of the receptor by a

unique hinge region (237). The primary high-afﬁnity binding site for relaxin is located in the LRR region, but the

peptide also interacts with a lower afﬁnity binding site located on the extracellular loops (199, 501). An intact

LDLa module at the NH

terminus is essential for signaling (231, 280, 456) which involves the third

intracellular loop. Protein-protein interactions important for signaling occur with helix 8 and the COOH-terminal

tail of RXFP1 (196, 198, 201, 204).

BATHGATE ET AL.

410 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

tide receptor (352). Similarly, RXFP4 was originally

thought to be a bradykinin receptor (63) before both

RXFP3 and RXFP4 were paired with their cognate ligands

relaxin-3 (314) and INSL5 (315). Importantly, relaxin-3

will bind to and activate RXFP4 as well as RXFP1; in ad-

dition, INSL5 is a low-afﬁnity antagonist of RXFP3. The

receptor expression proﬁles (FIGURE 4) match potential

roles as a neuropeptide receptor in the case of RXFP3 and a

gut hormone receptor in the case of RXFP4 (see sect. VII, C

and D). Relatively little is known of the signaling proﬁles

exhibited by RXFP3 (see sect. VIC) and RXFP4 (see sect.

VID). RXFP3 couples to G

i/o

and inhibits adenylyl cyclase

(314, 546), and receptor stimulation also activates ERK1/2

phosphorylation (546). Although earlier studies indicated

that only relaxin-3 and its 〉-chain could activate RXFP3,

more recent work suggests that relaxin can also activate

speciﬁc pathways by binding to a subtly different site on

RXFP3 (545). Understanding this ligand-directed signaling

bias will be important for the development of RXFP3 as a

therapeutic target.

Relaxin-RXFP1 Relaxin-3-RXFP3

INSL3-RXFP2 INSL5-RXFP4

Brain

Heart

Vasculature

Breast

Lung

Skin

Kidney

Liver Gut

Bone

Uterus

Ovary

Testis

Prostate

FIGURE 4. Expression patterns of relaxin family peptides and their cognate RXFP receptors in the major

organ systems of the body. Although there appears to be in some cases overlap between the distribution of

ligand/receptor pairs and in some cases there has been shown to be interaction between particular peptides

and several RXFP receptors in vitro, this does not appear to occur in vivo. Thus human relaxin-2 interacts with

RXFP1 and RXFP2 and human relaxin-3 interacts with RXFP1, RXFP3, and RXFP4 in vitro, but there is no

evidence for such interactions occurring in a physiological setting.

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

411Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

Table 1. Tissue localization of RXFP1-4 receptors

RXFP1 RXFP2 RXFP3 RXFP4

Tissue/Species Rat Mouse Human Rat Mouse Human Rat Mouse Human Human

Ovary mRNA

Protein

mRNA

2,54

mRNA

Oviduct mRNA

mRNA

Protein

Uterus mRNA

mRNA

3,14,30

mRNA

2,4,8

mRNA

Protein

2,20,21

Protein

4,8,25

Uterine smooth

muscle mRNA

38,46

mRNA

Protein

20,21,38,46

Protein

Endometrium mRNA

Protein

mRNA

10,18,19,49

Protein

18,19

mRNA

Cervix, vagina Protein

2,20,22

mRNA

3,30

Protein

Placenta mRNA

mRNA

2,19

mRNA

Nipple Protein

mRNA

3,30

Protein

Breast Protein

Protein

4,25

mRNA

Testis mRNA

1,6,51

mRNA

3,15,30,47

mRNA

1,17,51

mRNA

36,39

mRNA

Protein

Prostate mRNA

mRNA

Gubernaculum mRNA

6,12

mRNA

Brain mRNA

1,5,6,9,51

mRNA

3,30

mRNA

Protein

mRNA

36,39

mRNA

Protein

20,21

Protein

Brain regions mRNA

mRNA

28,29

mRNA

33,29,31

mRNA

41,42,52

mRNA

43⫺45,53

mRNA

36,39

Protein

28,29

Protein

33,29

Protein

41,42,52

Protein

44,53

Pituitary mRNA

mRNA

Kidney mRNA

mRNA

Heart mRNA

1,5,9,51

mRNA

3,30

mRNA

Protein

21,24

Lung mRNA

7,30

Liver

Intestine mRNA

mRNA

Colon mRNA

mRNA

Pancreas mRNA

36,39

Adrenal mRNA

mRNA

36,39

Thyroid mRNA

2,16

mRNA

Thymus mRNA

mRNA

Salivary glands mRNA

36,39

mRNA

Muscle mRNA

Blood cells mRNA

Continued

BATHGATE ET AL.

412 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

II. MOLECULAR BIOLOGY, SYNTHESIS,

STRUCTURE, AND IDENTIFICATION OF

FEATURES IMPORTANT FOR ACTIVITY

OF RELAXIN FAMILY PEPTIDES

A. Molecular Biology of Relaxin Family

Peptides

Although the relaxin peptide was initially described in the

1920s, the ﬁrst relaxin gene sequence was not cloned until

the early 1980s. At this time, cloning of porcine (192) and

rat (244) relaxin was achieved by screening of corpus lu-

teum cDNA libraries. These studies conﬁrmed that relaxin,

like insulin, is synthesized as a preprohormone with four

distinct regions: a signal peptide, a 〉-chain, a C-chain, and

a COOH-terminal A-chain. Relaxin cDNA and gene se-

quences were subsequently cloned from a number of mam-

malian species (43). Additionally, multiple new mammalian

relaxin gene sequences are now available in the genome

databases. All of these sequences show a similar gene struc-

ture and possess two exons interrupted by an intron, which

is situated in the middle of the C-peptide sequence.

The ﬁrst human relaxin gene was cloned by screening a

genomic library using the porcine relaxin cDNA sequence

and was designated the human RLN1 gene with the peptide

product termed H1 relaxin (human relaxin-1) (245).

How-

ever, a year later, the same researchers isolated an addi-

tional relaxin gene by screening a corpus luteum cDNA

library (246). This gene was designated RLN2 and the gene

product termed H2 relaxin (human relaxin-2) (see footnote 1).

The RLN2 product sequence was identical to that of the

relaxin peptide that was subsequently isolated from the hu-

man corpus luteum (126, 566), and two relaxin genes are

only present in higher primates (562). Thus the human re-

laxin-2 peptide is the equivalent of the relaxin peptide ﬁrst

discovered by Hisaw in lower species. The function of the

human relaxin-1 peptide is currently unknown, although a

synthetic human relaxin-1 peptide based on the RLN1 se-

quence activates the relaxin receptor (46, 514). The human

RLN1 and RLN2 genes are located sequentially on chro-

mosome 9 at 9p24 adjacent to the INSL4 and INSL6 genes.

In contrast to the cloning of the relaxin genes, the other

members of the relaxin peptide family were discovered as

novel cDNA clones in differential cloning projects or by

screening of EST or genomic databases. All of the new

members retain the relaxin-like preprohormone peptide

structure and a similar gene structure to RLN with two

exons interrupted by an intron situated in the middle of the

C-peptide sequence. INSL3 was discovered by researchers

searching for genes highly expressed in the pig (2) and

In this review the terminology species relaxin-1, relaxin-2, relax-

in-3, etc. is used for the relaxin peptides.

Table 1.—Continued

RXFP1 RXFP2 RXFP3 RXFP4

Tissue/Species Rat Mouse Human Rat Mouse Human Rat Mouse Human Human

THP-1

monocytes mRNA

mRNA

Protein

Bone marrow mRNA

Skin mRNA

mRNA

Northern blot (237);

RT-PCR, immunohistochemistry (239);

lacZ reporter expression (293);

immuno-histochemistry (255);

5,6,7

RT-PCR (290)

(298) (444);

RT-PCR, immunohistochemistry (325);

9,10,11

RT-PCR (443) (354) (406);

Northern blot (301);

13,14,15,16

RT-PCR (383) (477) (444)

(228);

Northern blot, in situ hybridization (276);

receptor autoradiography (67);

RT-PCR, immunohistochemistry (322);

20,21

receptor autora-

diography (403) (515);

immunohistochemistry (300);

23,24

receptor autoradiography (573) (401);

immunohistochemistry (288);

receptor

binding (411);

27,28,29

in situ hybridization, receptor autoradiography (333) (416) (189);

RT-PCR, Northern blot analysis (455);

31–33

in situ

hybridization, receptor autoradiography (469) (460);

RT-PCR (552);

receptor autoradiography (519);

mRNA by RT-PCR (314);

mRNA by

RT-PCR (313);

qRT-PCR, Western blotting, immunohistochemistry (550);

mRNA by RT-PCR (352);

receptor autoradiography (312);

in situ

hybridization, receptor autoradiography (508);

in situ hybridization, receptor autoradiography (329);

in situ hybridization (62);

in situ hybrid-

ization, receptor autoradiography (507);

in situ hybridization (306);

qRT-PCR, Western blotting, immunohistochemistry (551);

RT-PCR (259);

RT-PCR (286);

qRT-PCR (372);

qRT-PCR (89);

RT-PCR, immunohistochemistry (167);

in situ hybridization, receptor autoradiography (486);

in situ hybridization, receptor autoradiography (487);

qRT-PCR (342).

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

413Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

mouse testis (424), and it was initially called Leydig insulin-

like peptide (Ley-I-L) due to its high expression in the tes-

ticular Leydig cells. Subsequent cloning of the INSL3 gene

from numerous mammalian species has conﬁrmed its high

expression in the Leydig cells of mammals (259). The hu-

man INSL3 gene is located on chromosome 19 at 19p13.2.

INSL4 was independently identiﬁed as a highly expressed

gene in the human placenta by two groups and was initially

called placenta insulin-like peptide (EPIL) (94) or placentin

(289). The INSL4 gene, like the RLN1 gene, is only present

in higher primates, although there is evidence to suggest

that INSL4 emerged prior to RLN1 (56).

The advent of the EST databases provided an opportunity

to search for insulin-like genes using the conserved A- and

B-chain core sequences. Human INSL5 was identiﬁed from

a colon EST library (100), whereas an independent group

discovered mouse INSL5 from a mouse colon EST clone

(236). The mouse clone was originally called relaxin/insu-

lin-like factor 2 (RIF2) and was discovered together with

mouse INSL6 that was called RIF1. Two other independent

groups also discovered mouse (275) and human (317)

INSL6 in testis cDNA libraries. Both INSL5 and INSL6

have been subsequently cloned from numerous mammalian

species and shown to be highly expressed in the colon and

testis, respectively (42). The human INSL5 gene is located

on chromosome 1 at 1p31.3.

The human and mouse RLN3 genes were discovered by

searching the human genome sequence using the relaxin B-

chain (46). The human RLN3 gene is located on chromosome

19 at 19p13.2, ⬃3 mB from the INSL3 gene. The RLN3 gene

has subsequently been identiﬁed in all of the sequenced mam-

malian genomes and all other vertebrates including ﬁsh, birds,

and amphibians (49, 241, 562). Indeed, bioinformatic analy-

ses showed that RLN3 emerged prior to the divergence of ﬁsh,

as RLN3 orthologs are present in Takifugu rubripes and

Danio rerio (562). Phylogenetic analyses also suggest that te-

leosts possess two orthologs that group closely with mamma-

lian relaxin-3, but not with the main circulating form of re-

laxin (410). The high homology between orthologs in the ma-

ture peptide region demonstrates that RLN3 is under strong

purifying selection, unlike the gene encoding relaxin (563).

This is thought to reﬂect the differences between relaxin and

relaxin-3 receptor interactions and function. Together, the

highly conserved sequence homology and expression of relax-

in-3 in the brain indicate that it is an important neuropeptide

(see sect. IVC).

The RLN3 gene encoding human relaxin-3 is likely to be

the ancestral gene from which a series of gene duplications

gave rise to other members of the relaxin family (562). It is

not possible to assign any order to the divergence between

insulin and the relaxin family peptides; however, insulin,

IGF-I, and IGF-II clearly form a separate subgroup. An

analysis based on peptides from 15 species showed that

human relaxin-3 and INSL5 are closely related to each

other and cluster within a subtree that is distinct from the

group comprising human relaxin-1, human relaxin-2,

INSL3, INSL4, and INSL6 (562). The analysis shown in

FIGURE 1Bbased on human peptide sequences also demon-

strates the close relationship between human relaxin-3 and

INSL5; however, these peptides are clustered within a sub-

tree that also contains human relaxin-1, human relaxin-2,

and INSL6. From this analysis, INSL3 is not more closely

related to human relaxin-2 than the human relaxin-3/

INSL5 peptides. The previous analysis is based on a much

larger data set and provides a comprehensive view of re-

laxin family peptide evolution across diverse species (562).

The discrepancy with the tree based only on the human

peptides reﬂects the short input sequences available for

comparison, and the fact that the level of homology be-

tween key members of the relaxin peptide family is almost

the same. BLASTP searches (National Center for Biotech-

nology Information) provide measures of amino acid iden-

tity as well as similarity based on conservative changes be-

tween residues that are structurally conserved. The phylo-

genetic tree shown here (FIGURE 1) reﬂects the fact that human

relaxin-2 is slightly more homologous to human relaxin-3 than

to INSL3. Compared with the core 23-amino acid human

relaxin-2 B chain sequence, human relaxin-3 has 12 identical

residues and an additional 2 conservative changes, while

INSL3 has 10 identical residues and 2 further conservative

changes. Compared with the core 20-amino acid A chain se-

quence of human relaxin-2, INSL3 has 8 identical residues and

no additional conservative changes, and human relaxin-3 has

7 identical residues. The combined B/A chain CLUSTALW

analysis places human relaxin-2 closer to human relaxin-3

than INSL3; however, this is due to an overall difference in

homology of one amino acid. This analysis is not improved by

including the C peptide regions, as these show much greater

divergence in both length and sequence than the A and B

chains. Based on the CLUSTALW and BLASTP analyses, it is

reasonable to conclude that human relaxin-1, human relax-

in-2, human relaxin-3, INSL5, INSL6, and INSL3 all form a

subtree with at least 42% identity and 47% similarity between

members, and that not all branch positions within this group-

ing are likely to be signiﬁcant.

B. Structure of Relaxins and Insulin-like

Peptides

As mentioned previously (see sect. IIA), the conserved prepro-

hormone structure of the relaxin family peptides (consisting of

a signal peptide, 〉-chain, C-chain, and COOH-terminal A-

chain) necessitates a degree of processing to produce the ma-

ture, active peptide. It is thought that the signal peptide is

removed, followed by the enzymatic removal of the C-peptide,

to form a mature heterodimeric peptide with two disulﬁde

bonds between the A- and B-chains and an additional intra-A-

chain bond. However, the cleavage of the C-peptide has only

been directly demonstrated for some of the peptides.

BATHGATE ET AL.

414 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

The native relaxin peptide has been isolated and characterized

from numerous species, which allowed the conﬁrmation of a

two-chain, three-disulﬁde bonded structure similar to insulin

(FIGURE 1A)(42). A similar native structure, generated by en-

zymatic removal of the connecting C-peptide, has been con-

ﬁrmed for bovine INSL3 (76) and porcine relaxin-3 (314).

Although there is also some evidence for cleavage of the C-

peptide during INSL6 processing (323), a mature, native pep-

tide has yet to be isolated for both INSL4 and INSL5. Struc-

tural studies on recombinant human relaxin-2 (144) and syn-

thetic INSL3 (435), human relaxin-3 (434), and INSL5 (211)

have enabled the determination of the three-dimensional

structures of the peptides (FIGURE 1C).

The crystal structure of the human relaxin-2 peptide con-

ﬁrmed that the overall fold is similar to insulin (144). Further-

more, human relaxin-2 forms an asymmetrical dimer in a

manner resembling insulin, although the human relaxin-2

dimer occurs in a different orientation. Indeed, all relaxin fam-

ily peptides share a common overall fold but have differences

around their termini (FIGURE 1C)(433). Nuclear magnetic

resonance (NMR) studies of human relaxin-3 show that it has

a broadly similar structure to human relaxin-2 and insulin.

However, when compared with the X-ray crystal structure of

human relaxin-2, human relaxin-3 has a more hydrophobic

core with a condensed B-chain

␣

-helix. This “tightness” al-

lows the B-chain tryptophan at position 27 to interact with the

hydrophobic core of the peptide (434). In contrast, the B-chain

␣

-helix in human relaxin-2 is one helix turn longer, forcing the

tryptophan to face away from the core of the molecule where

it is solvent exposed (FIGURE 1C)(434). This orientation of the

COOH terminus of the B-chain of human relaxin-2 is similar

to that of the COOH terminus of INSL5 determined by NMR

(212). In contrast, the NMR structure of INSL3 reveals a

COOH-terminal orientation that is more similar to human

relaxin-3, with the COOH terminus contacting the hydropho-

bic core (434). Despite this, the COOH termini of human

relaxin-3 and INSL3 still show disparity in the precise orien-

tation of the COOH terminus of the B-chain, that has direct

ramiﬁcations for the key role of the COOH-terminal trypto-

phan in the activity of these peptides (see sect. IIC). Interest-

ingly, a synthetic INSL4 peptide based on a putative heterodi-

meric mature peptide structure is largely unstructured in solu-

tion (79, 310), bringing into question whether INSL4 is

actually produced as a prohormone in vivo.

C. Structure-Activity Relationships of

Relaxin Family Peptides

1. Structural features of relaxin necessary for

biological activity

Early studies on relaxin highlighted that both A- and B-

chains are necessary for activity since treatment of relaxin

with reducing agents destroyed its biological activity (174).

The initial effort to identify relaxin peptides in a range of

different species was the ﬁrst step in determining the struc-

ture-activity relationships of relaxin family peptides, their

receptors, and downstream signaling effectors. Alignment

of the amino acid sequences of relaxin peptides from rat,

tiger shark, dogﬁsh, rabbit, horse, skate, minke whale, dog,

human, dolphin, and cow allowed the identiﬁcation of key

conserved residues, i.e., those most likely to be important

for the structure-activity relationships of relaxin (470).

Aside from the essential cysteine residues, these alignments

highlighted a conserved RxxRxxI/V motif located in the

B-chain

␣

-helix (FIGURE 5). The X-ray crystal structure of

relaxin indicates that the arginine residues are located on

the ﬁrst and second loop of the

␣

-helix and face away from

the core of the molecule (FIGURE 1C)(144). The outward-

facing arginines form the receptor binding site for relaxin,

in conjunction with an isoleucine or valine residue that is

located on the same face of the B-chain

␣

-helix (452, 513).

Substitution of the arginine, isoleucine, or valine residues in

this motif markedly reduces or obliterates activity (FIGURE

5) (452, 513), highlighting that these residues are critical for

the activity of the relaxin peptide.

Analysis of the relaxin family peptides showed that the

A-chain of the relaxins had greater sequence divergence

than the B-chains (470) (FIGURE 1A). Other than the con-

served cysteine residues, only G14 seems to be highly con-

served (FIGURE 5) and is essential for maintaining chain

ﬂexibility and structure (73). Therefore, it was assumed that

the relaxin peptide A-chains would play a less important

role in receptor binding and activation than the B-chain.

Truncation of the A-chain of human relaxin-2 results in a

peptide that progressively loses the ability to bind to and

activate both RXFP1 and RXFP2 (232). It was thought that

this might be due to the loss of structure in the B-chain

resulting from the truncation. However, a recent study

demonstrated that targeted point-mutations within the hu-

man relaxin-2 A-chain can selectively alter receptor binding

and activation proﬁle: alanine substitution at the A16 and

A17 positions, (T16A and K17A, respectively) did not

markedly alter the cAMP response to RXFP1 activation,

but enhanced cAMP accumulation following peptide bind-

ing to RXFP2 compared with wild-type control (FIGURE 5);

furthermore, alanine substitution of residues A22 and A23

(R22A and F23A, respectively) markedly reduced the human

relaxin-2 activity at RXFP2 without reducing binding or ac-

tivity at RXFP1 (FIGURE 5) (410). Additionally, double ala-

nine substitutions at positions A19 and A20 (S19A and L20A,

respectively) also reduced peptide binding and activity at

RXFP1, again suggesting that residues of the human relaxin-2

A-chain are important for receptor binding and activation

(FIGURE 5) (73). This study also highlights differences in the

mechanisms by which relaxin can bind to and activate RXFP1

compared with RXFP2 (FIGURE 1C). Differences in the mech-

anism of binding and activation between RXFP1 and RXFP2

are further supported by the species-speciﬁc nature of these

structure-activity relationships; thus neither mouse nor rat re-

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

415Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

laxin can bind to or activate RXFP2 (454), and human relax-

in-3 has only a poor afﬁnity for RXFP2 (45).

2. Relaxin-3: structure and function

While relaxin-3 requires both A- and B-chains to bind to

and activate RXFP1, it only requires the B-chain to bind

and activate RXFP3 and RXFP4 (314) as the B-chain of

relaxin-3 is a low-afﬁnity agonist at both receptors (313,

314). Replacement of the human relaxin-3 A-chain with the

INSL5 A-chain (R3/I5) does not inﬂuence RXFP3 binding

or activation, but the chimeric peptide displays markedly

reduced ability to bind to and activate RXFP1 (see sect. IIC)

(312). Furthermore, truncation of the human relaxin-3 A-

chain produces a peptide that no longer binds to or activates

RXFP1 but retains full activity at RXFP3 (232).

Mutagenesis of key human relaxin-3 B-chain residues suggests

that R8, R16, I15, and F20 are important for human relaxin-3

binding to both RXFP3 and RXFP4 (FIGURE 5) (299), whereas

R12 is only important for human relaxin-3 binding to RXFP3

(FIGURE 5) but not to RXFP4 (299). Additionally, the muta-

tion of R26A and W27A causes a loss of inhibition of forsko-

lin-stimulated CRE activation by the peptide (299), suggesting

that these residues are essential for RXFP3-mediated inhibi-

tion of cAMP (FIGURE 5). These mutagenesis studies facili-

tated the development of a high-afﬁnity RXFP3 antagonist

(⌬R3/I5) that has a truncated human relaxin-3 B-chain (re-

Human relaxin-2 A-chain

T16A or K17A enhanced cAMP accumulation

at RXFP2 but not RXFP1

S19A/L20A reduced binding and activity at

RXFP1

R22A or F23A markedly reduced activity at

RXFP2 without reducing binding or activity

at RXFP1

Human relaxin-2 B-chain

R13, R17 and I20 involved in binding to

RXFP1

Truncation of B-chain N-terminus results in

progressive loss of binding to RXFP1 and

RXFP2. Truncation of C-terminus past G24

and W27 results in loss of binding for RXFP1

and RXFP2 respectively

Human relaxin-3

A-chain

Truncation of A-chain

N-terminus-progressive

loss of binding to RXFP1

but RXFP3 binding

not affected until

truncation > R8.

Human relaxin-3

B-chain

R8, I15, R16 and F20

involved in binding to

RXFP3 and RXFP4

R12 involved in binding

to RXFP3

R26 and W27 involved in

activation but not binding

to RXFP3 and RXFP4

INSL3 A-chain

C10S/C15S or C10del/C15del devoid of cAMP

signalling activity but retained affinity for RXFP2

N-terminus truncation (AAATNPA; AAATNPAR or

AAATNPARY) devoid of cAMPsignallingbut retained

binding to RXFP2

INSL3 B-chain

W27critical for RXFP2 binding and activation

H12, R16, V19 and R20 all contribute to binding

together with W27

N-terminus truncation (PTPEMREK) - loss of cAMP

signallingbut retained binding affinity at RXFP2



FIGURE 5. Structure-activity relationships for the relaxin family peptides human relaxin-2, human relaxin-3,

and insulin-like peptide 3 (INSL3). The three peptides share common structural features including the two

disulﬁde bonds between the A and B chains and the intrachain disulﬁde bond in the A chain. In spite of these

similarities, all of the peptides bind to their cognate receptors in characteristic ways. Binding of human

relaxin-2 to RXFP1 involves the RxxxRxxI/V motif in the B chain (144, 470), and mutations in the A chain can

alter activity at both RXFP1 and RXFP2 (232, 410); binding of INSL3 to RXFP2 involves a similar motif but

displaced one turn along the

␣

-helix of the B chain (74–76), whereas NH

-terminal deletions alter functional

responses while retaining binding (74). The human relaxin-3 B chain contains motifs associated with both

binding and activity (299, 434), and the A chain can be shortened without affecting activity (232). Similar

motifs that occur across peptides probably account for the cross-reactivity seen for some peptides at other

RXFP receptors.

BATHGATE ET AL.

416 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

moving R26 and W27), a G23A mutation, and the A-chain of

INSL5 (299) to produce a peptide that does not bind to

RXFP1. More recently, a single-chain antagonist has been de-

veloped that utilizes a modiﬁed B-chain of this peptide. This

has the 〉-chain of ⌬R3/I5 with the cysteine residues mutated

to serine (C10S-C22S) (213).

3. INSL3: structural features associated with

speciﬁc interaction with RXFP2

INSL3 interacts with RXFP1 only at extremely high concen-

trations (199, 239), suggesting that it is unlikely to bind to or

activate RXFP1 in a physiological setting. Mutagenesis studies

on synthetic INSL3 peptides have demonstrated that INSL3

utilizes numerous amino acids in the B-chain to bind to and

activate RXFP2. W27 of the INSL3 B-chain is critical for

RXFP2 binding and activation (74, 77), although other single

amino acid replacements with either alanine or valine

throughout the INSL3 B-chain suggested that there were also

other important residues for receptor binding in addition to

W27 (FIGURE 5). However, while these individual substitu-

tions only slightly reduced the binding of INSL3 to RXFP2, in

combination they produce a large effect (75, 435). Thus the

combined mutation of H12A, R16A, V19A, R20A, and

W27A results in a mutant that cannot bind to RXFP2, high-

lighting the necessary contribution of all of these residues (FIG-

URE 5) (435). There have also been a number of INSL3 muta-

tions that have been identiﬁed in both normal and patient

populations (see sect. VIIB1).

Studies on INSL3 B-chain analogs have suggested that while

the B-chain alone can still bind to RXFP2, these single chain

peptides cannot activate the receptor and therefore act as

functional antagonists (121, 464). A similar effect is pro-

duced within the peptide dimer by truncation of the INSL3

A-chain: while deletion of up to six residues has no effect on

the full agonist activity of the peptide, deletion of up to 10

residues from the NH

terminus of the INSL3 A-chain pro-

duces a peptide that still binds to RXFP2, but no longer

increases cAMP accumulation (74). These derivatives are

speciﬁc competitive inhibitors of INSL3 at RXFP2 (74).

Similarly, as with the native INSL3 A-chain, deletion of

eight NH

-terminal residues of the B-chain produces an

INSL3 peptide that retains binding afﬁnity for RXFP2, but

lacks the cAMP signaling function (74). Replacing any of

these eight B-chain residues individually with alanine does

not affect receptor signaling, nor does replacing all eight

residues of the A-chain with alanine (74). Alteration of the

cysteine residues of the intra-chain A-chain disulﬁde bond

(C10S/C15S; or C10del/C15del) also produced peptides

that retain RXFP2 binding but do not activate cAMP sig-

naling (FIGURE 5) (587). All of these studies highlight that

while both the A- and B-chains are necessary for INSL3

activation of RXFP2, the mechanism of action is different

from the relaxin-RXFP1 interaction (232).

III. MOLECULAR BIOLOGY, STRUCTURAL

FEATURES, AND FUNCTIONAL

DOMAINS OF RXFP RECEPTORS

The relaxin family peptide receptors are four highly con-

served family A GPCRs that fall into two groups based on

their architecture and signaling properties. The RXFP1 and

RXFP2 genes have introns, and there are a large number of

splice variants many of which result in proteins whose phys-

iological function is yet to be established. In contrast, the

genes encoding RXFP3 and RXFP4 are intronless. Both

RXFP1 and RXFP2 have a large extracellular NH

termi-

nus containing a leucine-rich repeat (LRR) domain that is

responsible for high-afﬁnity ligand binding (this is comple-

mented by a low-afﬁnity binding interaction within ex-

oloop 2) and, uniquely, an LDLa module that is essential for

signaling and inﬂuences receptor translocation. While

RXFP1 and RXFP2 show similarities in ligand binding and

signaling, only RXFP1 has a COOH terminus containing

motifs that induce the formation of highly sensitive protein

signaling complexes termed signalosomes. The third intra-

cellular loop is essential for G protein coupling in all RXFP

receptors. RXFP1 and RXFP2 couple to G

␣

and to G

␣

which modulates this effect, but only RXFP1 is able to

couple to G

␣

involving R752 in the COOH-terminal tail

to produce a delayed surge in cAMP accumulation. On the

other hand, RXFP3 and RXFP4 couple solely to G

␣

and

␣

proteins with the pattern of coupling dependent on the

cell type in which the receptor is expressed.

A. Molecular Biology of Relaxin Family

Peptide Receptors

1. Leucine-rich repeat containing receptors RXFP1

and RXFP2

RXFP1 and RXFP2 are family A G protein-coupled receptors

(GPCRs), similar to the rhodopsin receptor, that speciﬁcally

belong to the leucine-rich repeat-containing GPCR (LGR)

subfamily (reviewed in Ref. 18). Studies of LGRs from differ-

ent species suggest that the three subtypes of LGRs (A, B, and

C) originated during the early evolution of metazoans (241).

Generally, LGRs contain a leucine-rich repeat (LRR) domain,

a hinge region, seven transmembrane-spanning domains, and

a COOH-terminal tail. Type A LGRs include the FSH recep-

tor, the LH receptor, and the TSH receptor, each of which

contains nine LRRs before the hinge region. The type B LGRs

consist of three members: LGR4, LGR5 and LGR6. Both

LGR4 and LGR5 have 17 LRRs, whereas LGR6 has 13 LRRs.

All the receptors from this subgroup have a different hinge

region to the type A LGRs. The type C LGRs, RXFP1 (LGR7)

and RXFP2 (LGR8), are differentiated by the presence of a

LDLa module at the extreme NH

terminus, leading into 10

LRRs and a unique hinge region prior to the transmembrane-

spanning domain (FIGURE 3).

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

417Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

There is a high degree of conservation within the sequence

of RXFP1 and RXFP2 from ﬁsh to mammals (240), with

human and rodent orthologs exhibiting ⬎90% sequence

similarity (238, 239, 455). Human RXFP1 is located on

chromosome 4q32.1, and RXFP2 is located on chromo-

some 13q13.1; the receptors share 60% amino acid se-

quence identity and 80% homology.

The genes for RXFP1 and RXFP2 are very large, covering

⬎60 kB and both contain 18 exons. Alternative splicing

occurs commonly within LGRs, and there are a number of

splice variants of all type A LGRs (188, 319, 366, 377,

530), as well as LGR4 and LGR6 (375). To date, 29 splice

variants of RXFP1 and RXFP2 have been described result-

ing in multiple potential protein products including frag-

ments consisting only of the transmembrane or the extra-

cellular region (282, 375, 456). A selection of these variants

has been characterized in transfected cell systems, and al-

though some of the transmembrane-containing products

translocate to the plasma membrane, others are retained

inside the cell. Most of the transmembrane containing vari-

ants that are expressed at the cell surface are unable to bind

ligand (375). However, a RXFP2 variant that lacks only the

exon that encodes the LDLa module is able to bind ligand

but does not activate cAMP production, suggesting that this

variant may be a native binding protein that could modulate

INSL3 action in vivo (456). Products encoding only the

extracellular portions of the receptors, including variants

that express the LDLa module alone, can be secreted from

the cell (456). These variants could therefore act as func-

tional RXFP1 antagonists by binding and preventing re-

laxin activation of RXFP1. It is postulated that the native

variants that are expressed in rodent tissues could act as

endogenous antagonists (457). Some of the other splice

variants have also been demonstrated to modulate the func-

tion of the wild-type receptors. Three RXFP1 splice variants

occur in human fetal membranes, and in vitro studies in

transfected cells suggest that these variants can form het-

erodimers with RXFP1 and consequently modulate the

function of the receptor (282).

Construction of phylogenetic trees to examine the evolu-

tionary relationship between human RXFP receptors and

other family A GPCRs conﬁrms that RXFP1 and RXFP2

are closely related to one another, with 59% sequence iden-

tity and 80% similarity based on conservative amino acid

changes, and also to the three glycoprotein hormone recep-

tors for thyroid stimulating hormone (TSHR), luteinizing

hormone/chorionic gonadotropin (LHCGR), and follicle

stimulating hormone (FSHR). RXFP1 and RXFP2 are

somewhat less related to the LRR-containing receptors

LGR4, LGR5, and LGR6 (22–25% identity, 46–47% sim-

ilarity) (FIGURE 2A). LGR4 and LGR5 have recently been

shown to regulate Wnt/

␤

-catenin signaling in response to

R-spondins (90). RXFP1 and RXFP2 are also (somewhat

surprisingly) related to family A bioamine receptors such as

␤

-adrenoceptors (ARs) and adenosine receptors, as well as

additional peptide receptors (FIGURE 2A).

2. Small peptide receptor-like receptors RXFP3 and

RXFP4

RXFP3 and RXFP4 were deorphanized 1 year after RXFP1

and RXFP2 (42, 313–315) and are most closely related to

small peptide receptors, with a relatively short NH

-termi-

nal domain common among family A GPCRs (FIGURE 2B).

As a consequence of this short NH

-terminal domain,

RXFP3 and RXFP4 are signiﬁcantly smaller (⬃400 resi-

dues) than RXFP1 and RXFP2 (⬃700 residues). Human

RXFP3 is located on chromosome 5p13.2 and RXFP4 on

chromosome 1q22. Unlike RXFP1 and RXFP2, the genes

do not contain introns and thus have no splice variants.

RXFP3 and RXFP4 are closely related to one another (43%

sequence identity and 60% similarity) and to a subgroup

comprising the angiotensin II type 1 receptor (AGTR1), the

apelin receptor (APLNR) and the chemokine receptor

CCR1, and to a second subgroup containing neuropeptide

receptors including members of the somatostatin and opi-

oid receptor families and the neuropeptide B/W receptor

(NPBWR1) (FIGURE 2B). They are also more distantly re-

lated to various chemokine receptors (CXCR7, CXCR4,

CCR1, and CCR9) and to the formyl peptide receptors

FPR2 and FPR3 (FIGURE 2B). However, in spite of the close

similarity between the cognate ligands for these receptors,

the RXFP1/RXFP2 and RXFP3/RXFP4 receptor subgroups

are not highly related to each other (FIGURE 2C), and in fact,

the only signiﬁcant homology between RXFP1 and RXFP3

is within the highly conserved TM7 motif NSxLNP(I/V)Y

that is essential for G protein coupling as well as neighbor-

ing key residues in helix 8. RXFP1 and RXFP4 show

slightly higher homology, although again the regions of

similarity encompass only short motifs in TM3, TM6, and

TM7. Instead, both receptor subgroups are related to other

family A GPCRs such as the

␤

-AR and the adenosine A2B

receptor. In essence, these bioamine receptors occupy a

bridge between the distinct RXFP1/RXFP2 and RXFP3/

RXFP4 subgroups (FIGURE 2C). For example, the

␤

-AR has

homologies with each of the RXFP1 and RXFP3 receptors,

with 23% identity/39% similarity to RXFP1 and 21% iden-

tity/38% similarity to RXFP3.

B. Structural Features and Mode of Ligand

Activation of the LRR-Containing

Receptors RXFP1 and RXFP2

1. General features of RXFP1 and RXFP2

The high degree of similarity between RXFP1 and RXFP2

has facilitated the identiﬁcation of the functional domains

of the two receptors. Initial work used relaxin-3 because of

its selectivity for RXFP1 over RXFP2, and utilizing chime-

BATHGATE ET AL.

418 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

ric receptors revealed that the peptide needs to interact with

both the ectodomain and exoloop 2 of the transmembrane

domain of RXFP1 in order for the full binding and cAMP

signaling proﬁle to be achieved (501). The bimodal interac-

tion of relaxin with RXFP1 was later shown to be a feature

of both receptors that both contain two ligand-binding

sites: a high-afﬁnity site within the ectodomain and a lower

afﬁnity site within the transmembrane region (FIGURE 3)

(199). Evidence for two distinct binding sites was also pres-

ent in functional assays, with the high-afﬁnity ectodomain

site inducing cAMP accumulation with higher efﬁciency

than the lower afﬁnity transmembrane site (TABLE 2) (199).

Studies with chimeric and truncated receptors showed that

both sites are necessary for optimal binding and signal

transduction (199). In addition to the LRR-region of the ect-

odomain, the NH

-terminal LDLa module is essential for sig-

naling, although to date there is no evidence for a role of this

domain in ligand binding (TABLE 2) (231, 280, 456). Thus

RXFP1 and RXFP2 have two ligand binding sites, and recep-

tor binding followed by activation is currently thought to re-

quire the LRR, extracellular loops, and the LDLa module.

In a manner similar to many other class A GPCRs, recent

BRET studies have also shown that both receptors form

homo- and heterodimers (282, 510, 511). For RXFP1 and

RXFP2, dimerization occurred in the absence, and was in-

dependent, of ligand occupation of the receptor (510, 511).

Furthermore, dimerization between the haloreceptor and a

number of splice variants (encoding only the LDLa module,

and up to 8 LRR) was evident, and dimers were present at

all stages of receptor translocation from the endoplasmic

reticulum to the plasma membrane (282), suggesting an

important role of dimerization over the lifetime of the re-

ceptor. Dimerization was also evident between the halore-

ceptor and a transmembrane-only domain receptor, al-

though the BRET

ratio was decreased compared with that

obtained for haloreceptor dimers (510, 511). This suggests

that while the transmembrane domain is sufﬁcient for

dimerization, the ectodomains play an important role in

stabilizing the oligomer.

A functional consequence of dimerization is that both re-

ceptors display negative cooperativity (510, 511). In con-

trast to allosterism, where binding of a molecule to a single

site inﬂuences the properties of a second site, the two bind-

ing sites do not have a ﬁxed afﬁnity in proteins exhibiting

negative cooperativity. Instead, the afﬁnity of each remain-

ing unoccupied receptor binding site decreases as occu-

pancy increases. There are two intriguing, functionally rel-

evant consequences of negative cooperativity at RXFP1 and

RXFP2: 1) this phenomenon increases the functional range

of the ligand over a bigger concentration range, and 2) the

ligand residence time at the receptor shortens as the free

ligand concentration increases, potentially allowing selec-

tive activation of different signaling pathways (476). The

negative cooperativity concentration-response curve for

RXFP2 was bell-shaped, suggesting that binding occurs in a

trans manner within the dimer: at low concentrations,

INSL3 binds to the high-afﬁnity site on receptor 1 and the

low-afﬁnity site on receptor 2, with partial ligand dissocia-

tion and increased free ligand concentrations; another

INSL3 molecule binds in the same manner to the remaining

two sites, accelerating the dissociation of the ﬁrst INSL3

molecule; and ﬁnally, at very high concentration of free

ligand, this effect is lost due to two additional INSL3 mol-

ecules binding to the free high- and low-afﬁnity sites, thus

preventing further cross-linking (510). Contrastingly, the

negative cooperativity concentration-response curve for re-

laxin binding to RXFP1 was linear, although the absence of

a protein structure precludes conclusions regarding the

functional consequences of this observation (511)].

2. A unique feature of RXFP1 and RXFP2: the LDLa

module

The LDLa module was originally deﬁned by the structure of

the LDL receptor (110, 571) but has since been identiﬁed in

a number of other proteins including the very-low-density

lipoprotein receptor (177), the LDL receptor-related pro-

tein (218), renal glycoprotein gp330 (223, 326), the C9

component of complement (123, 494), the Tva receptor for

Rous sarcoma virus (39), and retina- and brain-speciﬁc neu-

ropilin, and tolloid-like protein 1 transmembrane protein

(500). RXFP1 and RXFP2 are the only known human GPCRs

to contain this module (42). The NMR solution structure of

the RXFP1 LDLa module was recently solved (FIGURE 3) and

shows the typical fold generated by the essential six cysteine

residues, and the anticipated incorporation of a calcium ion by

a largely conserved motif of acidic residues (231).

Mutagenesis studies of the LDLa region have highlighted the

essential role of this module in receptor signaling. Mutation of

conserved residues, those that compromised correct folding,

or indeed entire deletion of the LDLa module of either RXFP1

or RXFP2 resulted in receptors that when occupied are unable

to increase cAMP, despite these mutant receptors maintaining

intact ligand binding proﬁles (231, 280, 456). Mutation of

two conserved cysteines (C47A and C53A), or mutation of a

residue within the calcium-binding motif (D58E), resulted in

mutant RXFP1 receptors that were unable to increase

cAMP accumulation in response to either human relaxin-1

or human relaxin-2 (280). Other mutations that affect the

ability of the LDLa module to correctly fold (C5S and

C18S, which disrupts the ﬁrst disulphide bond) also results

in a receptor that is unable to increase cAMP accumulation

(TABLE 2) (231). Additionally, when the RXFP1 LDLa mod-

ule is swapped for the LDLa module contained within the

second ligand-binding domain of the LDL receptor, the chi-

meric receptor was unable to increase cAMP accumulation

in response to human relaxin-2 but bound ligand normally

(231). These data together with further mutagenesis of spe-

ciﬁc residues in the RXFP1 LDLa module indicate that this

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

419Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

domain mediates signaling by a speciﬁc side-chain-driven

interaction (231).

The LDLa module may also play a role in receptor matura-

tion and delivery to the cell surface. A high proportion of

full-length RXFP1 receptors were found to exist in an im-

mature form containing high mannose-type N-linked oligo-

saccharides, consistent with retention of the receptor within

the endoplasmic reticulum (280). This has previously been

reported for other glycoprotein hormone receptors includ-

AKAP79 β-arrestin

RXFP1 + + + + + + + ++

LDLa- + + - - - - - NT NT

LRR + - - - - - - NT NT

TM - + - - - - - NT NT

t703 + + + - + + - NT NT

t747 + + + - + + - NT NT

R752A +++ -++-

NT NT

S755A ++++++-

NT NT

S704A + + + + + + + +-

D637A + + + + + NT NT NT NT

C47A

C53A

++ - --NTNT

NT NT

D58E ++ - --NTNT

NT NT

C5S

C18S

++ - --NTNT

NT NT

D231N E233Q

E277Q D279N -NTNTNTNTNTNT

NT NT

--+NTNTNTNT

NT NT

Gαi3 GαoB Gαs

NFκB

cAMP

CRE

Binding

ECL

Binding

LRR

IC3 Fragments

615-629, 619-

629K Palmitate

TABLE 2. Functional domains of the human RXFP1 receptor determined using receptor fragments or

site-directed mutagenesis. The effect of deletion of regions or site-directed mutagenesis was determined on

labeled relaxin binding to the leucine-rich repeat region (LRR), extracellular loops (ECL); on cAMP generation

determined by activation of CRE or NF

␬

B reporter genes; on signaling mediated by G

␣

,orG

␣

; and on

binding to AKAP79 or

␤

-arrestin. The modiﬁed RXFP1 receptors utilized included RXFP1 lacking the low-density

lipoprotein receptor type A module (LDLa⫺), consisting of the NH

-terminal domain of RXFP1 (LRR), the

transmembrane spanning region (TM), truncated RXFP1 receptors (t703 and t747), receptor variations

generated by site-directed mutagenesis, and fragments of intracellular loops of RXFP1. ⫹, Response or

interaction present; ⫺, no response or interaction; ⫾, reduced response or interaction. NT, not tested. Data

are from References 199, 204, 231, 280, 456, 475, and 501.

BATHGATE ET AL.

420 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

ing the LH receptor (18, 419, 520) and the FSH receptor

(115, 428) and is suggested to be a form of control over cell

surface expression. Interestingly, RXFP1 lacking an LDLa

module was expressed as the mature form, as was a chime-

ric RXFP1 receptor containing the LDLa module of RXFP2

(280). Furthermore, mutation of a conserved glycosylation

site within the LDLa module results in a receptor with a

reduced ability to generate cAMP, which was attributable

to a decrease in cell surface expression (280, 572). In

RXFP2, mutations of amino acid residues that form the

disulﬁde bond or coordinate Ca

2⫹

binding in the LDLa

module (C71Y and D70Y) also reduced cell surface expres-

sion (64). Taken together, this suggests that the LDLa mod-

ule may impair, or negatively regulate, receptor expression

at the plasma membrane.

3. The LRR region and its importance for receptor

translocation, ligand binding, and signal

transduction

Glycosylation is a posttranslational modiﬁcation common

to many GPCRs and is important for receptor delivery to

the cell surface, ligand binding, and signal transduction. In

addition to the glycosylation sites within the LDLa module,

RXFP1 contains a number of glycosylation sites within the

LRRs (asparagines at N105, N250, N303, and N346) that

are all important for both cell-surface localization of the

receptor and full cAMP signaling efﬁcacy, but have no role

in ligand binding (572). As yet, the glycosylation status of

RXFP2 has not been studied in detail.

Both RXFP1 and RXFP2 contain two peptide-binding sites:

a high-afﬁnity site in the LRR region and a lower-afﬁnity

site in the extracellular loop region (TABLE 2) (199, 501).

Modeling of the LRR region of RXFP1 based on the crystal

structure of the porcine ribonuclease inhibitor (a protein

with LRRs) allowed in silico peptide docking and targeted

receptor mutagenesis (78). These studies revealed that the

site most likely to bind relaxin occurs at a 45° angle across

ﬁve of the parallel LRRs (78). Relaxin binding to these

LRRs occurs through synchronized chelation of two argi-

nines in the relaxin B-chain (R13 and R17: part of the

relaxin binding motif), neutralization of charge from acidic

groups within the concave face of the LRRs (E277 and

D279 in LRR8, and D231 and E233 in LRR6), and gener-

ation of hydrogen bonding (78). This is stabilized by a hy-

drophobic interaction between the relaxin B-chain isoleu-

cine (I20) and a receptor-based cluster of W180 and I182

within LRR4, and L204 and V206 within LRR5 (78). De-

letion of any of these residues within the receptor abolished

relaxin binding (78). Although these residues are also pres-

ent on RXFP2, evidence suggests that human relaxin-2

binds to RXFP2 in an INSL3-like manner (458).

As outlined in section IIC, INSL3 utilizes different residues

in the B-chain to interact with RXFP2 compared with the

relaxin-RXFP1 interaction (TABLE 3). However, human re-

laxin-2 contains most of these residues and can mimic the

INSL3 interaction. By using the NMR solution structure of

INSL3, and molecular modeling of the RXFP2 LRR based

on the crystal structure of the Nogo receptor (47% amino

acid sequence homology), a recent study identiﬁes the

RXFP2 residues that directly interact with the INSL3 B-

chain (459). With the use of targeted mutagenesis of both

peptide and receptor, in conjunction with in silico docking

of the INSL3 B-chain to the LRR of RXFP2, seven residues

within RXFP2 were identiﬁed. Thus within the B-chain of

INSL3, residue R16 is predicted to interact with RXFP2

D227, INSL3 H12 with RXFP2 W177, INSL3 V19 with

RXFP2 I179, INSL3 R20 with RXFP2 E229 and D181, and

INSL3 W27 with RXFP2 F131 and Q133 (459). The pri-

mary determinants of INSL3 binding are the peptide residues

R20 and W27 within the B-chain. Although ﬁve of the identi-

ﬁed RXFP2 residues are conserved in RXFP1, the two non-

conserved residues (D181 and F131) interact with essential

residues within the INSL3 binding motif: R20 and W27 (459).

Thus this model potentially provides an explanation for the

lack of INSL3 binding at RXFP1.

4. Evidence for low-afﬁnity relaxin family peptide

binding to extracellular loops in RXFP1 and

RXFP2

In addition to the relatively well-deﬁned high-afﬁnity ligand-

binding site within the LRRs, both RXFP1 and RXFP2 have

an additional lower afﬁnity binding site presumed to be within

the transmembrane exoloops of the receptors. The existence of

this site was ﬁrst suggested by a study that utilized chimeric

receptors, which exploited the inability of relaxin-3 to either

bind to or activate RXFP2 (501). The binding afﬁnity and

cAMP signaling efﬁcacy of relaxin-3 was reduced at an

RXFP1/2 chimera (RXFP1 ectodomain, fused to the trans-

membrane and COOH terminus of RXFP2) compared with

RXFP1. However, replacement of the second extracellular

loop (ECL2) of RXFP2 with that of RXFP1 restored relaxin-3

binding and signaling, thereby identifying ECL2 as a second-

ary binding site (TABLE 3).

This additional low-afﬁnity site has since been conﬁrmed

for other relaxin peptides at both RXFP1 and RXFP2.

These studies used the same chimeric receptors described

above and exploited the preference of rat relaxin for RXFP1

over RXFP2, and the preference of INSL3 for RXFP2 over

RXFP1 (199). Thus primary binding is directed by the ectodo-

main, with an inﬂuence of the low-afﬁnity site within the

transmembrane ECLs. This mechanism is also reﬂected in

cAMP signaling efﬁcacy proﬁles; thus the high-afﬁnity site sig-

nals to cAMP accumulation with high efﬁciency, and the low-

afﬁnity site with low efﬁciency. The precise location of the sites

has yet to be determined, although it is presumed to be within

the ECLs and may involve an interaction with the A-chain of

the relaxin family peptides (TABLE 3).

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

421Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

5. Functional roles of the intracellular loops

The third intracellular loop appears to play an important

functional role in coupling RXFP1 to G

␣

and thus is

important in controlling increased cAMP accumulation

in response to receptor activation (see sect. VIA). Peptide

fragments derived from the NH

-terminal region of the

third intracellular loop [IC3; residues 615–629, and

619–629-Lys(Palm)] increased adenylyl cyclase (AC) ac-

tivity independently of relaxin (475) (TABLE 2). The same

peptides functionally “antagonized” the cAMP response

to relaxin activation of RXFP1 endogenously expressed

in rat striatum and rat cardiac muscle (475). Further-

more, a synthetic peptide derived from the COOH termi-

nus of G

␣

(residues 385–394) inhibited both the AC

activity stimulated by relaxin and the increased cAMP

stimulated by the IC3 peptides alone (475), suggesting

that G

␣

interacts with the IC3 of RXFP1 (FIGURE 3).

Many other GPCRs interact with G proteins by their

intracellular loops, and there is much evidence to support

this interaction site (143, 217, 284, 431). It is tempting to

speculate that IC3 not only directs coupling to G

␣

, but

also directs coupling to G

␣

for both RXFP1 and

RXFP2 (see sect. VIA): truncation of either receptor after

helix 8 did not affect these components of the cAMP

signaling response, suggesting a common interaction site,

although this remains to be demonstrated. Similarly, the

constitutive activity of RXFP1 mediated by G

␣

(and

␤␥

) activation of adenylyl cyclase 2 (AC2) (see sect.

IIIB6; Ref. 201) is also likely to depend on G

␣

coupling

to ICL3.

6. The COOH-terminal tail of RXFP1 controls both

cAMP signaling through the delayed pathway and

signalosome formation

Only RXFP1, but not RXFP2, increases cAMP accumula-

tion by coupling to G

␣

(198). Activation of adenylyl cy-

clase 5 (AC5) to increase cAMP occurs downstream of

␣

,byaG

␤␥

-PI3K-PKC

␨

pathway (see sect. VIA). The

coupling of RXFP1 to this pathway involves the ﬁnal 10

amino acids of the receptor COOH terminus and absolutely

Receptor Relaxin INSL3 Relaxin INSL3

RXFP1 9.75 5.68 9.39 ND

RXFP2 8.96 9.71 7.88 8.27

RXFP1/2 9.48 8.57 9.08 8.5

RXFP2/1 9.18 10.05 8.17 8.82

9.66 7.67 - -

9.37 9.69 - -

9.59 ND 8.91 ND

9.85 ND 9.07 ND

9.28 ND 8.78 ND

Binding - pKD, pKi cAMP - pEC50

RXFP1

/2ECL1

RXFP2-BP

RXFP1-BP

RXFP1

/2ECL2

RXFP1

/2ECL3

N.B. Top 6 rows are means of published values

TABLE 3. Functional domains of RXFP1 and RXFP2 receptors determined using receptor chimeras. RXFP1

residues are outlined in black, with RXFP2 in gray. Binding is determined using radiolabeled relaxin and

expressed as ⫺log [concentration]. cAMP accumulation was measured in response to relaxin or INSL3. The

top 6 rows are the means of published data. ND, not detectable. Data are from References 199 and 501.

BATHGATE ET AL.

422 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

requires R752 within these residues (FIGURE 3), as trunca-

tion of the COOH terminus or substitution of R752 selec-

tively removes coupling to the G

␣

pathway (TABLE 2)

(204). As yet, the precise mechanism by which the terminal

10 residues of RXFP1 direct G

␣

coupling is unclear. There

are a number of possibilities including direct G

␣

coupling,

␣

coupling induced by receptor phosphorylation, or re-

cruitment of scaffolding proteins for colocalization of the

receptor with G

␣

. However, it would be unprecedented

for G protein coupling to occur directly with the RXFP1

COOH-terminal tail.

The COOH-terminal tail of RXFP1 also controls a small

degree of constitutive activity (201) and only RXFP1, but

not RXFP2, constitutively couples to AC2 by an associ-

ation between helix 8 and A-kinase anchoring protein 79

(AKAP79). This protein complex also facilitates G

␣

and

␤␥

-mediated stimulation of AC2 to increase cAMP ac-

cumulation in response to subpicomolar concentrations

of relaxin (see sect. VIA). The level of cAMP generated by

this complex is tightly controlled by the activity of pro-

tein kinase A (PKA)-stimulated phosphodiesterase (PDE)

4D3, which is localized to the receptor COOH terminus

by an association between

␤

-arrestin 2 and S704 of

RXFP1. This signalosome, directed and maintained by

the RXFP1 COOH-terminal tail, mediates a cAMP re-

sponse to low concentrations of peptide and may provide

a novel cellular response to low levels of relaxin in some

physiological situations.

The RXFP1 COOH-terminal tail also contains a number of

potential consensus sequences for phosphorylation and

protein-protein interactions and is the region of most vari-

ation between RXFP1 and the highly related RXFP2 (203),

thus representing an area of functional divergence between

the two receptors which may relate to the more varied phys-

iological roles of relaxin in relation to INSL3.

C. The Mode of Ligand Activation of the

Small Peptide Receptor-like Receptors

RXFP3 and RXFP4

As noted previously, the small NH

-terminal domains of

RXFP3 and RXFP4 are markedly different from RXFP1

and RXFP2. The precise mode by which relaxin-3 and

INSL5 bind to and activate their receptors is currently

unknown. However, current evidence suggests that the

B-chain of relaxin-3 contains all the RXFP3 contact

points. It is postulated that the residues in the B-chain

central helix R8, R16, I15, and F20 that are important for

human relaxin-3 binding may interact with the RXFP3

ECLs or NH

-terminal domain, whereas residues R26 and

W27, which are essential for receptor activation, may inter-

act with the RXFP3 transmembrane helices (291, 594). The

precise residues in both RXFP3 and RXFP4 that interact

with these amino acids are currently unclear, although one

study has extensively examined the potential roles of the

extracellular domains and transmembrane helices in recep-

tor function (TABLE 4) (594). This study examined the

binding and activation of RXFP3 and RXFP4 chimeras

by INSL5 and human relaxin-3 to determine regions of

the receptor necessary for ligand interaction. A chimera

of RXFP3 (transmembrane domain, loops, and COOH-

terminal tail) with the NH

-terminal region of RXFP4

(CR1) showed increased afﬁnity for INSL5 compared

with RXFP3 but no increase in GTP

␥

S binding and little

change in

125

I R3/I5 binding. The reverse chimera, con-

sisting of RXFP4 (transmembrane domains, loops and

COOH-terminal tail) and the NH

-terminal region of

RXFP3 (CR13), had similar afﬁnity to the wild-type

RXFP4 receptor for human relaxin-3, but lower afﬁnity

for INSL5 (594), suggesting that the NH

terminus of

RXFP4 is important for INSL5 and possibly human re-

laxin-3 binding (TABLE 4).

The roles of the ECLs of RXFP3 were also investigated

using RXFP3 and RXFP4 chimeras. Chimeras of RXFP3,

with ECL1 or ECL3 replaced with the corresponding

loops of RXFP4, produced receptors that behaved essen-

tially like the RXFP3 wild-type receptor, suggesting that

ECL1 and ECL3 were not important in INSL5 binding to

either RXFP3 or RXFP4 (594). Receptors consisting of

RXFP3 with the ECL2 of RXFP4 (CR3) had higher af-

ﬁnity for INSL5, and a slightly lower afﬁnity for human

relaxin-3, compared with RXFP3, similar to the ﬁndings

with CR1 (594). The potency of human relaxin-3 at CR3

was unchanged, whereas the potency of INSL5 was in-

creased (594). Receptors consisting of RXFP4 with the

ECL2 of RXFP3 (CR14) had similar afﬁnity for human

relaxin-3, but lower afﬁnity for INSL5 (594), suggesting

that ECL2 was important for INSL5 binding to RXFP4.

Functionally, CR14 behaved similarly to CR13, showing

a slight decrease in the potency of human relaxin-3 and a

larger decrease in the potency of INSL5 (594), suggesting

that the ECL2 of RXFP3 and RXFP4 are important for

ligand binding and may also have a role in activation

(TABLE 4).

Replacement of both the NH

terminus and ECL2 of

RXFP3 with the NH

-terminal region and ECL2 of

RXFP4 produced a chimeric receptor (CR5) that bound

human relaxin-3 and INSL5 in a similar manner to wild-

type RXFP4 (594), suggesting that both the ECL2 and

the NH

-terminal region of RXFP4 are important for

INSL5 binding. However, stimulation of chimeric CR5

with INSL5 does not increase GTP

␥

S binding, suggesting

that the NH

terminus and ECL2 of RXFP4 (though

important for binding) are not sufﬁcient for receptor ac-

tivation (TABLE 4). The reverse receptor, consisting of

RXFP4 with the NH

-terminal region and ECL2 replaced

with the equivalent regions of RXFP3 (CR15), had a

reduced afﬁnity for INSL5 but no change in the afﬁnity

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

423Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

for human relaxin-3. Correspondingly, while human re-

laxin-3 stimulation of CR15 increased GTP

␥

S binding,

INSL5 was ineffective, suggesting that the NH

terminus

and ECL2 of RXFP4 are more important for ligand bind-

ing than for receptor activation (TABLE 4). Together,

these studies with chimeric receptors conﬁrm that the

-terminal region and ECL2 are both important for

human relaxin-3 and INSL5 binding, and thus may inﬂu-

ence the activation of RXFP3 and RXFP4 receptors

(594).

The importance of the transmembrane (TM) spanning

regions of RXFP3 and RXFP4 was also investigated using

chimeras. Systematic replacement of the TM domains of

RXFP4 with those of RXFP3 showed that replacement

of TM3 or TM5 of RXFP4 with the corresponding regions

of RXFP3 (CR19 or CR21, respectively) caused a decrease

in afﬁnity and a complete loss of INSL5 activity (594). This

suggests that TM3 and TM5 are involved in INSL5 binding

to RXFP4 and essential for activation. Although the reverse

chimera of RXFP3 with both TM3 and TM5 of RXFP4

(CR10) showed increased afﬁnity for INSL5 (compared to

wild-type RXFP3), INSL5 still did not activate GTP

␥

S bind-

ing (594). This suggests that TM3 and TM5 alone are not

enough to allow INSL5 activation of RXFP3. The chimera

of RXFP3 with TM2, TM3, TM5, and ECL2 of RXFP4

(CR12) exhibited similar afﬁnity for both human relaxin-3

and INSL5 as the wild-type RXFP4 receptor (594), suggest-

ing that all of these regions were important for human re-

laxin-3 and INSL5 binding. Functionally, human relaxin-3

had the same potency for GTP

␥

S binding at CR12 as the

wild-type RXFP4, and while INSL5 displayed increased po-

tency at CR12, it was still slightly lower than at RXFP4

(594). This suggests that TM2, TM3, TM5, and ECL2 are

all involved in ligand binding and activation of RXFP3 and

RXFP4 (TABLE 4).

Receptor Domain INSL5 INSL5

RXFP3 None 9.29 7.01 9.48 ND

CR1 8.84 7.99 9.10 ND

CR2 ECL1 9.17 7.03 9.21 ND

CR3 ECL2 8.94 7.94 9.02 ND

CR4 ECL3 9.19 6.92 9.32 ND

CR5 8.94 8.53 8.89 ND

CR8 8.67 8.58 8.88 8.54

CR12 8.79 8.29 9.08 8.45

RXFP4 None 8.81 8.66 9.01 8.94

CR13 8.95 7.66 9.11 7.74

CR14 ECL2 8.76 7.50 8.86 7.76

CR15 8.87 6.84 8.93 <7

CR16 9.04 7.06 9.09 <6

CR21 TM5 8.76 7.66 8.71 <6

Binding - pIC50 GTPγS binding - pEC50

Relaxin-3 Relaxin-3

N-term-TM5

N-term/ECL2

N-terminus

N-term-TM3

N-term-TM5

N-term/ECL2

N-terminus

TABLE 4. Functional domains of RXFP3 and RXFP4 receptors determined using receptor chimeras. RXFP3

residues are outlined in black, with RXFP4 in gray. Binding was determined using

125

I-labeled R3/I5 and

receptor activation by GTP

␥

s binding. Data are from Reference 594.

BATHGATE ET AL.

424 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

IV. EXPRESSION AND SECRETION OF

RELAXIN FAMILY PEPTIDES IN

HUMANS AND OTHER SPECIES

A. Production and Secretion of Relaxin in

Mammals

Although relaxin was originally discovered on the basis of

its actions on the pregnant female reproductive tract in

lower species, it is clear that it also has additional roles in a

wide variety of tissues in the nonpregnant female and the

male. Thus, in those species where it has been examined,

relaxin is found and acts as a neuropeptide in brain and is

expressed in peripheral tissues such as heart, kidney, lung,

liver, and pancreas (FIGURE 4). In reproductive tissues, re-

laxin expression is found in the corpus luteum, uterus,

mammary gland, testis, and prostate. Whereas the general

distribution of relaxin in nonpregnant mammals points to

autocrine or paracrine functions, in pregnancy relaxin is

secreted in large amounts and has endocrine functions. The

source of this relaxin varies but can be the corpus luteum,

uterus, or placenta or combinations of these depending on

the species. The actions of relaxin during pregnancy are also

quite variable across species but are generally related to its

plasma secretion proﬁle; thus in some species where levels

are high in late pregnancy it plays an important role in

modifying the reproductive tract in readiness for parturi-

tion, whereas in others where the plasma proﬁle is different,

such as humans, its role is less clear.

The following sections outline the known sources of relaxin in

various species. We have not attempted to compare the rela-

tive expression of relaxin in different tissues due to the variety

of techniques that have been used to determine the sites of

expression. It should be noted that on occasion only one tech-

nique was used to demonstrate tissue expression, and in addi-

tion, the antibodies utilized were not always characterized for

cross-reactivity between different relaxin family peptides.

Therefore, some of these data may need to be conﬁrmed using

independent techniques.

1. Detection of relaxin mRNA and protein in human

reproductive tissues

In nonpregnant women, human relaxin-2 mRNA expres-

sion in the ovary has been identiﬁed by Northern blotting,

RT-PCR, and Southern blotting (191, 246, 258). It is likely

that ovarian relaxin is produced in the corpus luteum since

human relaxin-2 is found in luteal extracts (391), luteal cyst

ﬂuid (321), and in bathing ﬂuid from dispersed corpora

lutea (451). Relaxin expression examined using immuno-

histochemistry reveals expression in the corpus luteum

(579), speciﬁcally to luteal cells (351), granulosa cells of

lutenized follicles (577), and cells from the lutenized theca

interna of developing follicles (61). In women, there is good

evidence that this relaxin is released into the circulation

during the menstrual cycle, since a small peak of relaxin

immunoreactivity occurs in the plasma 9–10 days follow-

ing ovulation (497).

Relaxin is also expressed in the secretory endometrium

(578, 579), and the glandular and luminal epithelia and

decidualized stromal cells of the endometrium (71). Relaxin

mRNA expression has been detected by RT-PCR in glan-

dular epithelial and stromal cells from cultures of human

endometrium (409). Both human relaxin-1 and human re-

laxin-2 mRNA were found by RT-PCR and qRT-PCR in

the uterus during the menstrual phase of the cycle (67).

In human pregnancy, the corpus luteum is the major source

of immunoreactive circulating relaxin (336, 391, 392). The

levels peak in the ﬁrst trimester, and ovarian venous blood

levels are three to four times higher at 6 wk gestation than in

the luteal phase of the menstrual cycle (283). The circulat-

ing relaxin from the corpus luteum is human relaxin-2. The

corpus luteum contains human relaxin-2 mRNA (258), and

human relaxin-2 has been isolated from plasma of pregnant

women (324, 566, 567). Consistent with this being the ma-

jor source of relaxin in pregnancy, in women with ovarian

failure and those lacking corpora lutea who become preg-

nant by in vitro fertilization, circulating levels of relaxin are

undetectable (148, 269). Comparison of the pattern of re-

laxin secretion in human pregnancy with that in other spe-

cies reveals major differences. In pregnant women, relaxin

levels peak in early pregnancy at fairly modest levels but

then fall, whereas in other mammals such as pigs, rats, and

mice relaxin levels are much higher and rise throughout

pregnancy, falling just prior to parturition (47).

During pregnancy there is also expression of relaxin in the

placenta (191), with Northern blotting detecting relaxin

mRNA in the amnion, chorion, decidua parietalis, basal

plate, and placental trophoblast (439). Relaxin immunore-

activity was found in the amnion (70, 287, 439), chorion

(70, 287, 439), decidua (287, 579) (70, 439), basal plate

(166) (287, 439), and placental trophoblast (439, 579).

Relaxin has also been puriﬁed from the placenta (166, 570).

In other tissues, such as breast tissue, relaxin mRNA was

detected by RT-PCR and subsequent Southern analysis,

with increased expression in neoplastic samples (522). Re-

laxin immunoreactivity is present in lobular and ductal ep-

ithelium and myoepithelium of the normal and neoplastic

breast, with secretion detected from metaplastic epithelium

(355). The peptide is also present in milk (141).

In the male, similar approaches have identiﬁed human re-

laxin-1 and human relaxin-2 mRNA (191, 258) and relaxin

immunoreactivity in the prostate (489), speciﬁcally within

the glandular epithelium of the prostate, the glandular epi-

thelium of the seminal vesicles and the ampulla of the vas

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

425Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

deferens (576), and immunoreactive human relaxin-2 is

also secreted into seminal plasma (117, 320, 557, 567).

2. Detection of relaxin mRNA and protein in other

mammalian reproductive tissues

In rats and mice, as in humans, the corpus luteum is a major

source of relaxin particularly during pregnancy, but there

the resemblance ends. In ovaries from nonpregnant ani-

mals, relaxin mRNA (46, 80, 108, 190, 244) and protein

levels (473) are relatively low. Speciﬁcally, relaxin immu-

noreactivity is localized to the luteal cells of the ovary (14).

These levels increase at estrus (108) and in pseudopregnant

rats (473), but major increases only occur with pregnancy

(14, 472, 473). Unlike humans, rats and mice display a

marked increase in relaxin levels which peak just before

birth and then return to low levels post-partum (47).

In the nonpregnant mouse, relaxin mRNA expression has

been detected by Southern blotting of RT-PCR products in

the testis, epididymis, prostate, mammary gland, endome-

trium, and myometrium (46). Similarly in the rat, relaxin

mRNA expression has been detected by RT-PCR and

Northern blot analysis in the placenta, uterus, mammary

gland, testis, and prostate (190).

The source and function of relaxin in reproductive tissues

from other mammalian species have been described in detail

elsewhere (47). However, irrespective of the source, relaxin

levels are greatly increased during pregnancy in all mam-

mals that have been studied. In pigs, as in rats and mice, the

corpus luteum is the main source of relaxin in pregnancy,

but in the horse, rabbit, hamster, cat, and dog, the placenta

appears to be the main site of synthesis (see Ref. 47 for

details). The guinea pig is unusual in that the uterus is the

main site of relaxin production during pregnancy (47).

3. Relaxin as a neuropeptide in the CNS

There are no studies to date detailing expression of relaxin

within human brain. However, in the rat, relaxin mRNA

expression in the brain has been detected by RT-PCR (190)

and Northern blotting (402). Speciﬁc localization of the

peptide was also determined by in situ hybridization histo-

chemistry in anterior olfactory nuclei, taenia tecta, and piri-

form cortex (81, 333, 402), the orbital cortex (333, 402),

the ﬁelds CA1–2 of Ammon’s horn, the dentate gyrus of the

hippocampus and the neocortex (402), and the anterior

cingulated cortex and the arcuate nucleus (333) (FIGURE 6).

Immunohistochemistry has identiﬁed relaxin expression in

the cytoplasm and proximal processes of cell bodies in the

arcuate nucleus, anterior olfactory nucleus, taenia tecta,

and piriform cortex (333). In the mouse, relaxin expression

has been detected by Southern blotting of RT-PCR products

in the cortex, hippocampus, hypothalamus, thalamus,

pons/medulla, and cerebellum (46). Broadly the distribu-

tion of expression of relaxin in mouse brain resembles that

of the rat (306).

4. Distribution of relaxin in other tissues

In the human, relaxin has been detected in the circulation

using ELISA (497) and radioimmunoassay (141, 269, 391,

393, 426, 498) techniques. Cultures of human luteinizing

granulosa cells also secrete relaxin, as determined using an

immunoassay (499). In mice, relaxin expression has been

detected by Southern blotting of RT-PCR products in the

thymus, heart, and kidney, with lower levels in the lung,

spleen, and skin (46). RT-PCR identiﬁed relaxin expression

speciﬁcally in the medulla and cortex of the kidney (445),

and in the atria and ventricles of the heart (136). Radioim-

munoassay identiﬁed relaxin in the peripheral circulation in

pregnant mice (393). Distribution of relaxin expression in

the rat is similar to mice, with mRNA demonstrated by

RT-PCR in the kidney, heart, liver, and pancreas (190).

Radioimmunoassay has identiﬁed relaxin in the peripheral

circulation of pregnant rats (393, 472), and a reverse hemo-

lytic plaque assay detected relaxin that was secreted from

cultured atrial cardiomyocytes from neonatal rats (524). In

some instances, only relaxin-3 but not relaxin expression

was identiﬁed in the heart by RT-PCR (290, 443).

B. Production and Secretion of INSL3 in

Mammals

INSL3 expression occurs mainly in the reproductive system

in mammals with the highest expression in the testis. How-

ever, INSL3 is expressed at equivalent levels in the ruminant

ovary and has an expression pattern during pregnancy that

is similar to relaxin (40, 41, 432). As ruminants lack a

relaxin gene (563), it has been postulated that INSL3 may

act in lieu of relaxin in bovine and ovine species (48). As the

expression of INSL3 in all mammalian species has been

covered in detail elsewhere (47), this section will focus in

INSL3 in humans and rodents.

1. Detection of INSL3 mRNA and protein in the

mammalian reproductive system

INSL3 mRNA is highly expressed in the testis of every

mammalian species so far examined. In humans, INSL3

mRNA has been identiﬁed in the testis by Northern blot

analysis (2, 84), RT-PCR (254), and Western blotting

(227). Expression of INSL3 has been speciﬁcally localized

to the Leydig cells using in situ hybridization (254, 285) and

immunohistochemistry (24, 285) (FIGURE 4, TABLE 1).

INSL3 has also been detected by immunoassay in human

serum (8, 11, 50, 72, 172, 173), with higher levels in males

compared with females correlating with the high expression

in the Leydig cells of the testis. In the male, INSL3 levels in

the peripheral circulation decline with age (11). Immunoas-

say has also identiﬁed INSL3 expression in the amniotic

BATHGATE ET AL.

426 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

ﬂuid of male, but not female, fetuses (10) suggesting that

INSL3 is expressed in fetal testis as has been demonstrated

in other mammals (47) which also correlates with its role in

testis descent (see sect. VIIB). INSL3 mRNA and protein are

also expressed in the prostate, with strong expression in the

basal epithelium (286). In the female, INSL3 is expressed in

the ovary, speciﬁcally within the corpus luteum as deter-

mined by RT-PCR (521), Northern blotting (521), and im-

munohistochemistry (24), in addition to expression in the

trophoblast of the ovary determined using RT-PCR (521)

and the theca interna cells using immunohistochemistry

(24). Both in situ hybridization and immunostaining have

identiﬁed INSL3 expression in the endometrium, speciﬁ-

cally within the epithelial cells, endometrial gland cells, and

stromal cells (230). The peptide is expressed in human

breast tissue, determined using RT-PCR, with in situ hy-

Cells positive for relaxin

mRNA and/or immunoreactivity

Cells positive for relaxin-3

mRNA and/or immunoreactivity

Main paths of relaxin-3

immunoreactive fibers

Subsidiary paths of relaxin-3

immunoreactive fibers

CPut

Orb

AON

VTT

DTT

Acb

DBB

Amy AHi

SON

MPA

PVN DMH

PHLH

VMH

Arc

IPN

SuM

SN VR

PnR

DTg

PBN

PAG

IGL

APT

SFO

PVA

Sub

SC IC

Pir

Hpt

SHy

BST

NST ARP

Sp5

Amb

Relaxin

binding sites

RXFP3

binding sites

Regions containing both relaxin

and RXFP3 binding sites

FIGURE 6. Localization of relaxin-RXFP1 and relaxin-3-RXFP3 sites in rat brain. The ﬁgure shows a sagittal

section of rat brain with indications of the areas containing cells staining positive for relaxin mRNA or

immunoreactivity or relaxin-3 mRNA or immunoreactivity. The regions containing RXFP1 binding sites deter-

mined using radiolabeled relaxin (blue) or RXFP3 binding sites obtained using labeled

125

I-R3/I5 (yellow) are

outlined together with sites containing both receptors (green). Acb, nucleus accumbens; Ahi, amygdalohippo-

campal area; Amb, nucleus ambiguus; Amy, amygdala; AON, anterior olfactory nucleus; AP, anterior pituitary;

APT, anterior pretectal nucleus; Arc, arcuate nucleus of hypothalamus; ARP, area postrema; BST, bed

nucleus of stria terminalis; C, cerebellum; CC, corpus callosum; CM, central medial thalamic nucleus; CPut,

caudate putamen; Cx, cerebral cortex; DBB, diagonal band of Broca; DG, dentate gyrus; DMH, dorsomedial

nucleus of hypothalamus; DR, dorsal raphe nucleus; DTg, dorsal tegmental nucleus; DTT, dorsal taenia tecta;

GP, globus pallidus; Hi, hippocampus; Hpt, hypothalamus; IC, inferior colliculus; IGL, intergeniculate leaﬂet; IL,

intermediate lobe of pituitary; IO, inferior olive; IPN, interpeduncular nucleus; LH, lateral hypothalamus; LS,

lateral septum; ME, median eminence; MPA, medial preoptic area; ND, nucleus of Darkschewitsch; NI,

nucleus incertus; NL, neural lobe of pituitary; NST, nucleus of solitary tract; OB, olfactory bulb; Orb, orbital

cortex; PAG, periaqueductal gray; PBN, parabrachial nucleus; PF, posterior hypothalamus; Pir, piriform

cortex; PnR, pontine raphe; PP, peripeduncular nucleus; PVA, paraventricular thalamic area; PVN, paraven-

tricular nucleus of hypothalamus; Re, reuniens thalamic nucleus; S, septum; SC, super colliculus; SFO,

subfornical organ; SHy, septohypothalamic nucleus; SN, substantia nigra; SON, supraoptic nucleus; Sp5,

spinal trigeminal tract; Sub, subiculum; SuM, supramammillary nucleus; Th, thalamus; VMH, ventromedial

nucleus of hypothalamus; VP, ventral pallidum; VR, ventral raphe nuclei; VTT, ventral taenia tecta. Sources of

material: relaxin mRNA and IR (81, 402); piriform cortex; septohypothalamic nucleus; arcuate nucleus;

horizontal/vert diagonal band of Broca; median preoptic region; 3rd ventricle lining (not included in schematic);

paraventricular hypothalamic nucleus; paraventricular thalamic area; orbital cortex and neocortex; anterior

olfactory area; taenia tecta (dorsal and ventral); anterior olfactory nucleus; taenia tecta; piriform; hippocam-

pus; CA1–3 of Ammons horn and dentate gyrus; orbital cortex/neocortex; relaxin binding (81, 333, 402);

relaxin-3-positive IR neurons and ﬁbers (329, 508); RXFP3 binding sites and mRNA (486, 487, 507); inferior

olive; taenia tecta; ventral pallidum; diagonal band of Broca; septohypothalamic; lateral, posterior, dorsomedial

hypothalamus; intergeniculate nucleus; peripeduncular nucleus.

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

427Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

bridization showing localization of INSL3 to the tubu-

loaveolar and ductal epithelium (227). INSL3 expression

has also been detected in placental tissue using Western

blotting (227, 230), with speciﬁc expression in the tropho-

blasts of anchoring villi and fetal membranes, maternal

blood vessels, and the basal plate identiﬁed using in situ

hybridization and immunostaining (230).

In the mouse and the rat, INSL3 is expressed in the testis as

determined by Northern blotting (23, 412, 491, 595) and

RT-PCR (23, 276), with speciﬁc expression within the Ley-

dig cells recorded using Northern blotting (424), in situ

hybridization (23, 412, 424, 525), and immunohistochem-

istry (23, 184, 412). Levels of INSL3 mRNA are highest in

the fetal testis shortly before birth, after which they decrease

until puberty when they again rise.

In the rat, INSL3 has been identiﬁed in the circulation of the

male using immunoassay but levels in the female are very

low (9, 68). Plasma levels in rats are correlated with mRNA

levels being highest just before birth and during adulthood

(9, 68) and decline with age as in humans (9). There is also

evidence for the transport of INSL3 across the blood-testis

barrier into the germinal compartment (9). In the mouse,

INSL3 levels in the blood are also much higher in males

than females (9). INSL3 protein expression has also been

demonstrated in the seminiferous tubules of the (9) mouse

using immunohistochemistry (23). RT-PCR has detected

expression of INSL3 mRNA in the epididymis (23), pros-

tate (23), and the posterior part of male embryos (595). In

the female mouse and rat, INSL3 mRNA was detected in

the ovary using Northern blotting (491, 595) and RT-PCR

(23, 276), with speciﬁc expression in the mouse in the cor-

pus luteum and stromal cells determined using immunohis-

tochemistry (23). In addition, in the rat, the use of RT-PCR

shows speciﬁc expression in the thecal cells surrounding

preovulatory follicles (276).

2. Is INSL3 a neuropeptide?

Despite evidence for the expression of the INSL3 recep-

tor, RXFP2, in brain, no studies have identiﬁed INSL3

expression in the CNS of rodents or humans (189, 469).

Circulating levels of INSL3 could potentially affect some

areas of the brain such as the pituitary, hypothalamus,

and areas of the brain stem, although this remains to be

demonstrated (86).

3. Detection of INSL3 in other peripheral tissues

There is limited evidence for the expression of INSL3 in

nonreproductive tissues. In humans, INSL3 expression, as

determined using in situ hybridization, is upregulated in the

neoplastic thyroid gland (228).

C. The Location and Release of Relaxin-3 in

Mammals

1. Identiﬁcation of relaxin-3 in brain

In contrast to the widespread expression of relaxin and

INSL3, studies that have mapped the speciﬁc distribution of

relaxin-3 and RXFP3 expression have provided valuable

insights into the physiological functions of this ligand-re-

ceptor pair. Relaxin-3 expression measured by Southern

blotting of RT-PCR products from mouse tissue (46)

showed relaxin-3 mRNA in abundance in the brain (FIGURE

6); at moderate levels in the thymus, kidney, and spleen; and

at low levels in the heart and liver, with no noticeable gen-

der differences. Northern blots and in situ hybridization

studies showed that relaxin-3 is localized to cells of the pars

ventromedialis of the dorsal tegmental nucleus (46). The

expression of rat relaxin-3 was subsequently found to mir-

ror that of the mouse homolog (FIGURE 6) (80). Similarly,

relaxin-3 expression is highest in the human brain, although

substantial expression is also found in the testis, where its

role remains to be demonstrated (314). The high levels of

relaxin-3 expression in the brain of numerous species have

led to a focus on the role of the peptide in the CNS.

Most neuroanatomical studies have been conducted in ro-

dents, and in rat brain, the distribution of relaxin-3 has

been characterized with two different antibodies (329,

516). It is expressed in the nucleus incertus (NI), located in

the median dorsal pons and also referred to as the dorsal

tegmental nucleus pars ventromedialis (373), nucleus reces-

sus pontismedialis (261), or nucleus “O” (413). Neurons

expressing relaxin-3 are also present in the pontine raphe

nucleus; the anterior, lateral, and ventrolateral periaque-

ductal gray (PAG); and in an area dorsal to the lateral

substantia nigra (FIGURE 6) (329). As in the rat, the primary

source of relaxin-3 in the mouse brain is the NI, with some

relaxin-3-positive neurons also located in the pontine cen-

tral gray and adjacent to the fourth ventricle (487). Discrete

populations of relaxin-3-containing neurons were also ob-

served in the pontine raphe nucleus, anterior periaqueduc-

tal gray, and a region dorsal to the substantia nigra and

medial to the peripeduncular nucleus. A recent study of sites

of relaxin-3 expression in zebraﬁsh has revealed expression

patterns similar to those observed in rodents (125). Relax-

in-3 expression occurs in neurons located near the fourth

ventricle and in the periaqueductal gray (conﬁrmed by co-

localization with proenkephalin-like gene 1). In the ma-

caque (Macaca fascicularis) brain, relaxin-3 is expressed in

areas similar to the NI in rodents (332). In the rhesus ma-

caque (Macaca mulatta), using a different antibody, there

was a similar pattern of expression (478). This consistent,

speciﬁc and restricted expression of relaxin-3 in multiple

species suggests that relaxin-3 has quite conserved and thus

important functions in the brain.

BATHGATE ET AL.

428 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

The target nuclei of the neuronal projections from relaxin-

3-expressing cell bodies provide further insights into relax-

in-3 function. The projections of NI neurons in the rat brain

have been extensively mapped (187, 395). Ascending pro-

jections from the NI connect to the raphe, mammillary com-

plex, hippocampus, cortex, amygdala, habenula, medial

septum, and hypothalamus (FIGURE 6). Overall, the NI ap-

pears to have strong reciprocal connections with the median

raphe and the interpeduncular nuclei. The NI appears to

have important roles in behavioral arousal (187, 395).

D. Distribution of Other Relaxin Family

Peptides in Mammals

INSL4 is highly expressed in the placenta, and RT-PCR

analysis suggests that it is most highly expressed in the

human placenta during the ﬁrst trimester of pregnancy (94).

No INSL4 transcripts were identiﬁed in human ovary, pros-

tate, testis, brain, skin, esophagus, adrenal, breast, kidney,

thyroid, stomach, liver, colon, rectum, omentum, or spleen.

Immunoreactive INSL4 is detected in the plasma and amni-

otic ﬂuid during pregnancy, with higher amniotic ﬂuid lev-

els found in abnormal pregnancies (such as trisomy 21),

suggesting that INSL4 may be a marker of chromosomal

abnormalities during pregnancy (368, 369).

The INSL5 gene was identiﬁed from colon EST libraries,

and the highest expression of INSL5 mRNA is in the colon

in mice and human (100). INSL5 mRNA is also expressed

in the kidney, thymus, heart ovary, and brain (FIGURE 4),

although there are some differences in tissue expression

between the mice and humans, with mRNA also detected in

human uterus, placenta, prostate, spleen and bone marrow,

and in mouse testis (100, 236, 315). Relatively little is

known about the distribution of INSL5 protein. In the kid-

ney, INSL5 appears to be located in the cytoplasm of a

subset of epithelial cells in the loop of Henle (236). In the

mouse brain, putative INSL5 immunoreactive cells are lo-

cated in the paraventricular, supraoptic, accessory secre-

tory and supraoptic retrochiasmatic nucleus throughout the

rostrocaudal extent of the hypothalamus and pituitary

(137). In contrast, another study showed a lack of INSL5

mRNA expression in the mouse brain (507).

INSL6 mRNA was identiﬁed by Northern blotting only in

the testis of human tissues and not in heart, brain, placenta,

lung, liver, skeletal muscle, kidney, pancreas, spinal cord,

lymph node, trachea, adrenal gland, or bone marrow (317).

The distribution of rat INSL6 mRNA was similar as deter-

mined by Northern blotting, with mRNA identiﬁed in the

testis and prostate of rat tissues (317). In situ hybridization

of rat and Rhesus macaque tissues identiﬁed INSL6 mRNA

in the spermatocytes and round spermatids in the seminif-

erous tubules but not in the Leydig cells (317). Studies in

mice have also demonstrated that INSL6 is expressed in

meiotic and postmeiotic germ cells correlating with the

marked reduction in sperm numbers and motility in INSL6

knockout mice (85). A recent study has demonstrated that

INSL6 is expressed in skeletal muscle in mice and may func-

tion as a myogenic regenerative factor (584).

V. LOCALIZATION OF RELAXIN FAMILY

PEPTIDE RECEPTOR MRNA AND

PROTEIN IN MAMMALIAN TISSUES

The tissue distribution of receptors for relaxin family pep-

tides has been determined by their mRNA expression pat-

terns using RT-PCR, Northern blotting, or in situ hybrid-

ization or by protein expression using immunohistochem-

istry or receptor autoradiography (FIGURE 6;TABLE 1). The

expression patterns of all the receptors generally match

their identiﬁed roles as cognate receptors for their respective

ligands. There is much information regarding RXFP1 ex-

pression, both in terms of the variety of approaches used

and the tissues examined (TABLE 1), with receptor expres-

sion being identiﬁed in both female and male reproductive

tissues, the brain and numerous nonreproductive tissues

such as the kidney, heart, and lung. Less information is

available for RXFP2, RXFP3, and RXFP4, although more

detailed evidence is now emerging for RXFP3 expression in

the brain, suggesting that this receptor may have promise as

a therapeutic target (see sect. VIIIC).

A. The Tissue Distribution of RXFP1:

the Receptor for Relaxin

1. Reproductive tissues

Prior to the discovery of RXFP1, localization of the un-

known relaxin receptor was determined by monitoring the

binding of labeled relaxin peptides to various tissues. In this

manner, early studies demonstrated relaxin binding in ho-

mogenates of mouse pubic symphysis and uterine tissue, rat

mammary gland and guinea pig pubic symphysis and cervix

(TABLE 1) (363). Other autoradiographic studies utilised a

relaxin variant (B33 relaxin, which facilitates peptide label-

ing with

Por

P) to identify relaxin binding in rat uterus

(401, 403, 515). Following receptor deorphanization,

RXFP1 distribution was identiﬁed using immunohisto-

chemistry in both myometrial and epithelial cells of the rat

uterus (239). In humans, RXFP1 has been identiﬁed by

binding and immunohistochemistry in luminal and glandu-

lar epithelial cells, myometrium, blood vessels, and stromal

extracellular matrix (TABLE 1) (288). Similar localization

has been reported using

P relaxin (67) or immunohisto-

chemistry (297, 325). Other immunohistochemical studies

for RXFP1 have reported binding solely to endometrial

stromal cells in humans but also to glandular epithelial cells

of the marmoset endometrium (255). There is some evi-

dence to suggest that RXFP1 expression in the human en-

dometrium varies with the menstrual cycle. Autoradiogra-

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429Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

phy was used to localize speciﬁc relaxin binding to human

uterus and showed binding to the glandular epithelium and

endometrial luminal epithelium. Both binding and RXFP1

mRNA levels increased markedly in the early secretory

phase of the menstrual cycle compared with the prolifera-

tive phases (67). Weaker binding occurred in endometrial

stromal tissue and little in myometrium (67). Immunohis-

tochemical studies are more equivocal with either broad

agreement (297) with binding studies or evidence for more

intense staining during the proliferative phase of the cycle in

studies with a high degree of variability (325).

In human breast tissue, RXFP1 was detected in the stromal

mesenchyme adjacent to the glandular epithelia and in epi-

thelial-derived cells in some breast tumors (255). Cultured

slices of ovarian cortical tissue displayed RXFP1 mRNA

and protein in ﬂat follicular cells of primordial follicles and

granulosa cells of primary and secondary follicles (474).

The distribution of RXFP1 in rodent reproductive tissues

is similar to that in humans. RXFP1 mRNA has been

detected by RT-PCR in the mouse uterus and testis (274).

Incorporation of the LacZ reporter gene into the RXFP1

knockout mouse allowed receptor expression to be dem-

onstrated near the spermatids and in the Leydig cells of

the testis, the prostate, the nipple, the circular layer of the

myometrium, the lamina propria under the luminal epi-

thelium and the longitudinal layer of the myometrium,

the basal layer of the vaginal epithelium and vaginal

smooth muscle cells, as well as vascular supporting tissue

and vascular epithelium of the oviduct (293). In rats,

antibodies directed against the ectodomain of RXFP1

identiﬁed receptor protein in the myometrial and the ep-

ithelial layer of the endometrium of the uterus, the mus-

cularis layer of the vagina, and the myometrial layer of

the cervix (239). RXFP1 has also been detected in rat

nipple and mammary gland (300).

2. RXFP1 receptors in the CNS

RXFP1 receptors identiﬁed by receptor autoradiography

are widely distributed in the brain localized to discrete re-

gions of the olfactory system, neocortex, hypothalamus,

hippocampus, thalamus, amygdala, midbrain, and medulla

(400, 404, 515) (FIGURE 6). High levels of RXFP1 binding

in the subfornical organ (SFO), organum vasculosum of the

lamina terminalis (OVLT), and the paraventricular and su-

praoptic hypothalamic nuclei (400, 404, 515) provide the

anatomical and biochemical basis for the control of plasma

osmolality by relaxin (361, 362, 483, 504, 505). RXFP1

mRNA has also been demonstrated in rat brain by North-

ern blotting (237, 455) and by RT-PCR (167, 290, 298,

443), in mouse brain using a reporter gene approach (293)

and by Northern blotting (455), and in human brain using

RT-PCR (239).

3. Localization of RXFP1 in the cardiovascular and

renal systems

The important roles of relaxin in the cardiovascular adap-

tive changes associated with pregnancy are reﬂected in the

presence of RXFP1 mRNA in the rat (237) and human

kidney (239). In rodents, the heart is also clearly inﬂuenced

by relaxin. Atria of both male and female rats contain a high

density of relaxin binding sites (401, 402, 515), and RXFP1

mRNA has been detected in the rat (237, 290) and mouse

(293) heart. However, lower levels of RXFP1 mRNA have

also been detected in the human heart (TABLE 1) (239).

More recent studies using Western blotting show RXFP1

expression in left atrium but not left ventricle in both failing

and nonfailing human heart (127). Relaxin is a potent chro-

notropic and inotropic agent in the rat heart (271, 290, 417,

418) and also has inotropic effects on the human heart

(127) (see sect. VIIA). It produces its effects by acting di-

rectly on RXFP1 receptors located mainly in the atria.

B. Tissue Distribution of RXFP2:

the Receptor for INSL3

1. Localization of RXFP2 in reproductive tissues

Although fewer studies have examined the localization of

RXFP2 compared with RXFP1, it is clear that the RXFP2/

INSL3 receptor/ligand pairing has highly specialized roles

in reproduction. Studies of the distribution of RXFP2 show

mRNA in rat ovary and in rat and mouse testis and guber-

naculum by Northern analysis, RT-PCR, and in situ hybrid-

ization (12, 276, 298, 301, 453) (FIGURE 4;TABLE 1). In the

rat, RXFP2 receptor mRNA has been localized to the

oocyte in the ovary and the germ cells of the seminiferous

tubules associated with germ cell function (276) (See sect.

VIIB). A more recent study has conﬁrmed the expression of

RXFP2 mRNA in rat postmeiotic germ cells and also local-

ized RXFP2 immunoreactivity to human postmeiotic germ

cells (12). This study also demonstrated RXFP2 mRNA in

rat Leydig cell, immunoreactive RXFP2 in human Leydig

cells, and INSL3 binding in a Leydig cell line. In human,

RXFP2 mRNA is found in uterus and testis (239, 354). The

pattern of localization of RXFP2 thus correlates with the

known roles of INSL3/RXFP2 in gubernacular develop-

ment and reproductive physiology (see TABLE 1 and sect.

VIIB).

2. Is there a role for RXFP2 in the CNS?

RXFP2 mRNA is expressed in the rat brain with high den-

sities in the thalamus where it is present in the intralaminar,

parafascicular, dorsolateral, ventrolateral, and posterior

thalamic nuclei (460). Mapping of the RXFP2 protein using

125

I INSL3 revealed high peptide binding in thalamic re-

gions but also in the striatum and certain layers of the

cortex, suggesting axonal transport of receptors to terminal

BATHGATE ET AL.

430 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

sites (460). However, in situ hybridization studies of the

distribution of INSL3 mRNA using oligo probes failed to

provide evidence for INSL3 expression in rat brain (469),

perhaps suggesting that central RXFP2 receptors respond

to circulating INSL3 (see sect. IVB). Comparison between

RXFP2 expression patterns in the rat and mouse revealed

detailed differences, most notably the presence of both

RXFP2 mRNA and protein in the cortex of the mouse with

mRNA found in layer V and layer VIb and

125

I INSL3

binding present in deeper layers and also layer I (189).

However, as in the rat, there was no evidence for INSL3

mRNA expression in mouse brain. (189).

3. Evidence for RXFP2 in other peripheral tissues

In the initial study that described the deorphanization of

RXFP1 (LGR7) (239), examination of the tissue distribu-

tion of the related RXFP2 receptor by RT-PCR provided

evidence for mRNA expression in brain, kidney, muscle,

testis, thyroid, uterus, bone marrow, and peripheral lym-

phocytes (FIGURE 4;TABLE 1). Signal strength was strong in

testis; weaker in lymphocytes, bone marrow, and brain; and

weak in the remaining tissues (239). In a more detailed

study in rat kidney, both RXFP2 mRNA and

125

I INSL3

binding were found in renal glomeruli and mesangial cells,

with particularly high levels observed in late stage gestation.

RXFP2 receptor-mediated responses are present in human

and mouse osteoblasts related to roles for INSL3 in bone

function (162) (see sect. VIIB). RXFP2 mRNA is also ex-

pressed in human thyroid tissues and in mouse follicular

thyroid epithelial cells (225).

C. Localization of RXFP3 and RXFP4: The

Receptors for Relaxin-3 and INSL5

1. RXFP3 has a widespread distribution in the CNS

Following deorphanization, RXFP3 was found to be highly

expressed in the human brain as determined by RT-PCR

(352). Subsequent studies identiﬁed the brain as the pre-

dominant site of RXFP3 expression in the mouse by North-

ern blotting and in both mouse and rat by in situ hybridiza-

tion (62, 314). In rat brain, high levels of mRNA were

found in the olfactory bulb, paraventricular and supraoptic

nuclei, and preoptic and posterior areas of the hypothala-

mus, hippocampus, septum, and amygdala with lower lev-

els in cortex, peraqueductal grey, nucleus incertus, and ar-

eas of brain stem (FIGURE 6) (314, 508). The chimeric pep-

tide

125

I-R3/I5, a selective, high-afﬁnity ligand for RXFP3

and RXFP4, has been used to identify chimeric receptor

binding sites in the cerebral cortex, olfactory bulb, and

superior colliculus in the rat (RXFP4 is a pseudogene in the

rat) (FIGURE 6) (312).

Recent studies provide a comprehensive description of the

distribution of RXFP3 in mouse brain (487) by both in situ

hybridization and peptide binding. While the overall pat-

tern is very similar to that in the rat, suggesting a strongly

conserved ligand-receptor pairing, there are some detailed

differences. For example, in the mouse, both peptide and

RXFP3 are present in the substantia innominata and the

olivary and posterior pretectal nuclei, whereas in the rat

expression in the olfactory bulb, entorhinal cortex, arcuate

nucleus, and paraventricular thalamic nucleus is more dom-

inant (487). As yet, there are no systematic studies of

RXFP3 localization in human or primate brain.

2. Evidence for RXFP3 in peripheral tissues

The ﬁrst studies on the expression of RXFP3 mRNA in

human tissues indicated very low expression of RXFP3

mRNA in the adrenal gland, testis, salivary gland, and pan-

creas by RT-PCR. Subsequent studies demonstrated

RXFP3 mRNA expression in the human testis by RT-PCR

(314), although the function of the receptor in this tissue is

not known.

3. Expression of RXFP4

Studies prior to the deorphanization of RXFP4 examined

the distribution of the orphan GPCR, GPR100 using

Northern blotting of human tissues. Expression of GPR100

was identiﬁed principally in peripheral tissues including

heart, skeletal muscle, salivary gland, bladder, kidney, liver,

placenta, stomach, jejunum, thyroid, ovary, and bone mar-

row with the highest expression in the pancreas (63) (FIG-

URE 4;TABLE 1). Subsequent studies following the deorpha-

nization of RXFP3 showed high levels of receptor mRNA

expression by RT-PCR in the human colon with additional

expression in the placenta, testis, thymus, prostate, kidney,

and brain (313). The expression of RXFP4 in the colon

matches the expression of the INSL5 peptide and indicates

potential functions of INSL5 as a gut hormone (see sect.

VIID).

VI. SIGNAL TRANSDUCTION PATHWAYS

ACTIVATED BY RELAXIN FAMILY

PEPTIDE RECEPTORS

A. Pleiotropic Signaling Pathways Activated

by RXFP1

Since type A LGRs and the coevolved genes encoding gly-

coprotein hormone subunits can be traced to both nema-

todes and insects, the three subfamilies of LGRs are thought

to have evolved before the emergence of vertebrates and nem-

atodes (237). The signaling pathways activated by RXFP1 and

RXFP2 therefore represent one of the earliest forms of GPCR

signaling (42). RXFP1 and RXFP2 couple to a variety of G

proteins to inﬂuence cAMP accumulation within a number of

cell lines (198, 237, 239). In addition, there is evidence for

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431Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

activation of MAP kinases, tyrosine kinases and nitric oxide

(NO) as well as activation of signaling pathways associated

with connective tissue metabolism, much of which predates

receptor identiﬁcation (FIGURE 7A).

There is further evidence suggesting that relaxin activates

the glucocorticoid receptor (GR), a nuclear receptor that

acts as a ligand-dependent transcription factor (130). Acti-

vation of the GR by relaxin, and the subsequent changes in

gene transcription, may account for the many effects of

relaxin on the expression levels of a variety of proteins,

including those involved in connective tissue metabolism

(see sect. VIA4). Relaxin stimulation of differentiated

THP-1 cells (into a macrophage phenotype) blunts the pro-

duction of cytokines including interleukin (IL)-1, IL-6, and

tumour necrosis factor (TNF)-

␣

, and this effect is abolished

by the GR antagonist RU486 (128). Relaxin coimmunopre-

cipitates with the GR, and the amount of the GR within the

nucleus increased after 30 min stimulation with relaxin

(128). Furthermore, a modiﬁed relaxin that was unable to

activate RXFP1 could still interact with the GR, and relaxin

was found to cause phosphorylation of S211 of the GR,

which is used as a biomarker of agonist-related receptor

activation (128). Indeed relaxin, via its interaction with the

GR, is also able to autoregulate its own expression (131).

Thus, although relaxin appears to interact with the GR,

independently of its own receptor, the relative contribution

of this pathway to the physiological effects of relaxin, and

how this integrates with the more classical relaxin-stimu-

lated signaling pathways, remains to be determined.

1. Increases in cAMP levels mediated by RXFP1:

a second messenger system inﬂuenced by many

interacting mechanisms

In studies predating receptor identiﬁcation, treatment with

relaxin was found to cause an increase in cAMP accumula-

tion in THP-1 cells (411), MCF-7 cells (57), the mouse

pubic symphysis (69), uterine strips (446), uterine longitu-

dinal muscle (399) from estrogen-primed rats, and in cul-

tures of human endometrial cells (156), human endometrial

glandular epithelial cells (96), newborn rhesus monkey

RX F P 2 AC

ATP

cAMP

βγ

CRE Transcription

INSL3

RXFP2 Signalling

RXFP4

RXFP4 Signalling

PLC

DAG

INSL5

RXFP3

RXFP3 Signalling

PLC

PI3K

AP1 Transcription

Src

Shc

SOS

Transactivation

Grb

EGFR

Ras

Raf

MEK1/2

ERK1/2

NFκB Transcription

PKC IκB

Relaxin-3

RXFP1

AC5

Gαi3

ATP

cAMP

ATP

cAMP

βγ

PI3K

PKCζ

NFκB Transcription CRE Transcription

Relaxin

RXFP1 Signalling

βγ

Akt

ERK1/2

PIP2

InsP3

Ca2+

Gαs

GαoB

Gαs

GαoB

Gαi2 Gα16 GαoB

Gαi2

FIGURE 7. Signal transduction mechanisms activated by interaction of relaxin family peptides with their

cognate RXFP receptors. Relaxin interacts with RXFP1 (A) to cause coupling of the receptor with G

␣

sto

activate adenylyl cyclase (AC) and G

␣

to modulate this effect (198, 236, 237). The receptor also couples to

␣

and

␤␥

subunits from this interaction to activate PI-3-kinase to cause translocation of PKC-

␨

, which in turn

activates AC5 (198, 204, 382, 383). cAMP resulting from these interactions appears to be compartmen-

talized and inﬂuences different signaling outcomes (197). INSL3 activates RXFP2 (B) to couple to G

␣

and G

␣

and inﬂuence cAMP production (198, 301). Relaxin-3 binds to RXFP3 (C) causing coupling to G

␣

and G

␣

and inhibition of AC (314). Release of

␤␥

subunits from these G proteins activates MAPK signaling and NF

␬

transcription (545, 546). Relatively little is known of INSL5/RXFP4 signaling (D), but the receptor couples to

␣

and G

␣

in a similar fashion to RXFP3 (313) but also couples to G

␣

and Ca

2⫹

when this promiscuous

G protein has been transfected into cells (315).

BATHGATE ET AL.

432 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

uterine cells (294), rat myometrial cells (235), and rat ante-

rior pituitary cells (109). The physiological relevance of the

cAMP response to relaxin is particularly well demonstrated

in human endometrial stromal cells, where basal and relaxin-

stimulated cAMP levels are enhanced by inhibition of phos-

phodiesterase (PDE) 4 (FIGURE 7A), and relaxin stimulation

and PDE4 inhibition act synergistically to induce decidual-

ization (37). Increases in cAMP in response to relaxin (243,

512) or addition of a cell-permeable cAMP analog (517)

induce decidualization, which is an important process re-

quired to support implantation of the developing embryo.

Increases in cAMP are also linked to the physiological ef-

fects of relaxin upon angiogenesis; treatment with human

relaxin in a murine model increased the degree of angiogen-

esis at wound sites, an effect that was associated with an

increased expression of vascular endothelial growth factor

(VEGF), an important proangiogenic protein (see sect.

VIIA4) (541). Interestingly, in cultures of normal human

endometrial cells (NHE cells), human relaxin increased

VEGF expression, and these effects are prevented by AC

inhibition and mimicked by either the AC activator for-

skolin or a PDE inhibitor (540). This suggests that relax-

in-stimulated cAMP production also mediates increased

VEGF transcription, and thus angiogenesis (see sect.

VIIA4).

Following receptor characterization, the importance of

cAMP as a signaling pathway for relaxin was cemented by

the observation that constitutively active mutants of both

RXFP1 and RXFP2 (transmembrane helix 6: D637Y) in-

creased cAMP accumulation in a ligand-independent man-

ner (FIGURE 3) (237, 239). Many subsequent studies have

focused on cAMP accumulation generated by activation of

these two receptors. It is now well recognized that both

RXFP1 and RXFP2 couple to G

␣

to increase cAMP (198,

237, 239), an effect that is negatively modulated by cou-

pling to G

␣

(198). Only RXFP1 can couple to G

␣

activate a further surge of cAMP accumulation via a G

␤␥

phosphatidylinositol 3-kinase (PI3K)-protein kinase C

(PKC)-

␨

pathway to stimulate adenylyl cyclase 5 (AC5) (FIG-

URE 7A)(198, 204, 382, 383).

Activation of the unique G

␣

pathway by RXFP1 was initially

identiﬁed in THP-1 cells that endogenously express the recep-

tor. In this cell line, relaxin causes a biphasic increase in cAMP

accumulation over time, with the later phase partially suscep-

tible to the PI3K inhibitors LY294002 and wortmannin;

subsequently, relaxin stimulation of RXFP1 was also

shown to increase PI3K activity (383). PKC-

␨

was proposed

as the candidate protein that linked PI3K activation to

cAMP formation. Indeed, studies that predated receptor

identiﬁcation in human cultured secretory endometrial

stromal cells showed that human relaxin treatment over 4

days increased the amount of PKC activity in membranes

versus cytosolic fractions of the cell (272); these observa-

tions support the concept of relaxin-stimulated transloca-

tion of PKC to the cell membrane.

The atypical PKC isoforms, including PKC-

␨

, are insensitive

to diacylglycerol and Ca

2⫹

but are either directly or indi-

rectly activated by phosphatidylinositol 3,4,5-trisphos-

phate (PIP

) and other lipids (378, 397, 493). Furthermore,

some general PKC inhibitors (including bisindoylmal-

eamide I) have very low activity for the atypical isoforms

(341, 536). Porcine relaxin was found to cause a concentra-

tion-dependent translocation of PKC-

␨

to the cell mem-

brane in a number of cell lines: MCF-7 (human breast can-

cer), PHM1–31 (pregnant human myometrial), MMC

(mouse mesangial), and human monocytic (THP-1) cells

(382). PKC-

␨

translocation was dependent on PI3K activa-

tion by relaxin, but independent of cAMP accumulation

(FIGURE 7A); furthermore, PI3K-dependent cAMP in-

creases mediated by relaxin were dependent on PKC

␨

expression (382). In MCF-7 cells, relaxin stimulation ac-

tivates PI3K and causes translocation of PKC-

␨

without

increasing cAMP accumulation; however, subsequent

transfection of AC5, but not AC2 or AC4, into these cells

produced a cAMP response to relaxin (381). Thus

RXFP1 activates PI3K, precipitating the translocation of

PKC

␨

to the cell membrane, whereby the protein stimu-

lates AC5 (FIGURE 7A).

This pathway has also been demonstrated in HEK293 cells

transiently or stably expressing RXFP1 (198, 204), and was

found to be downstream of G

␣

and G

␤␥

subunits. Experi-

ments utilizing G

␣

i/o

mutants that are insensitive to ADP

ribosylation by pertussis toxin (PTX) revealed initial cou-

pling of RXFP1 to inhibitory G

␣

(in addition to G

␣

) and

identiﬁed G

␣

as the mediator of the G

␤␥

-PI3K-PKC-

␨

AC5 pathway. Interestingly, a PTX-sensitive stimulation of

cAMP had been previously demonstrated in an endogenous

setting: in rat left atria, relaxin induced a small increase in

cAMP accumulation that was reduced by PTX pretreat-

ment, and PTX pretreatment also reduced the inotropic and

chronotropic responses to relaxin (290), indicating the

physiological relevance of this signaling pathway. This

pathway has recently been shown to be of greater impor-

tance in the failing heart (typically characterized by in-

creased G

␣

i/o

expression) (66, 150), allowing preservation

of the positive inotropic effects of relaxin in humans (127).

Site-directed mutagenesis identiﬁed the ﬁnal 10 amino acids

of the RXFP1 COOH terminus, and in particular R752 as

essential residues for activation of the G

␣

pathway (FIG-

URE 3) (204). The activation of this pathway is also depen-

dent on the presence of lipid-rich membrane domains, sug-

gesting a degree of speciﬁc compartmentalization of the

RXFP1-stimulated cAMP response (204). Furthermore,

GTP

␥

S-immunoprecipitation assays identiﬁed activation of

␣

immediately following stimulation of RXFP1 by re-

laxin, thus suggesting the delay in activation of the G

␣

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

433Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

␤␥

-PI3K-PKC

␨

-AC5 pathway is downstream of the G

protein itself (204). In HEK293 cells, only activation of G

␣

and G

␣

-dependent cAMP signaling pathways increases

CRE-mediated gene transcription, whereas G

␣

-mediated

signaling appears to selectively regulate NF

␬

B-dependent gene

transcription (FIGURE 7A)(197). This observation again sug-

gests compartmentalization of relaxin-RXFP1 signaling

events and infers that distinct physiological outcomes could be

anticipated downstream of different cAMP signaling path-

ways (197).

Recently, a constitutive RXFP1-dependent cAMP response

has been identiﬁed using FRET-based cAMP biosensors in

single rat cardiac ﬁbroblasts, HeLa cells, and HEK293 cells

expressing RXFP1 (201). The response is dependent on a

protein complex, or signalosome, centered upon the relaxin

receptor, and the signalosome is highly sensitive to attomo-

lar concentrations of relaxin. Importantly, this may provide

the basis for cellular responses to low levels of relaxin pres-

ent in the circulation. The signalosome consists of RXFP1

that is scaffolded to AC2 by AKAP79, facilitating efﬁcient

activation of the AC by G

␣

and G

␤␥

subunits. The cAMP

produced is tightly regulated by the activity of protein ki-

nase A-activated PDE4D3, that is in turn scaffolded to the

receptor COOH terminus (speciﬁcally requiring S704) by

␤

-arrestin 2 (FIGURE 8A)(201). The stimulatory (AKAP79

and AC2) and regulatory (

␤

-arrestin 2, PKA, and PDE4D3)

arms of the signalosome are both spatially and functionally

distinct. Knockdown of AKAP79 does not affect interaction

of regulatory components with the receptor, and knock-

down of

␤

-arrestin 2 does not inﬂuence interactions be-

tween RXFP1 and the stimulatory components. Targeted

protein knockdown or overexpression of dominant nega-

tive mutants (201) demonstrates that the complex is iso-

form speciﬁc (i.e., interacting with PDE4D3 but not

PDE4D5) (234) and that assembly of the regulatory com-

ponents is dependent on constitutive association between

the receptor and

␤

-arrestin 2, but not

␤

-arrestin 1 (FIGURE

8A)(120). cAMP production by the RXFP1-signalosome is

dependent on constitutive association between helix 8 of

the receptor and AKAP79, but not gravin (AKAP250) or

AKAP149 (21, 122, 484). Importantly, this signaling mech-

anism is distinct from those activated by higher concentra-

tions of relaxin, and the complex dissociates following ac-

RXFP3

Relaxin-3 signalling bias

PLC

PI3K

AP1 Transcription

Src

Shc

SOS Ras

Raf

MEK1/2

ERK1/2

NFκB Transcription

PKC IκB

Relaxin-3

Internalization

p38

JNK

MKK4/7

RXFP3

Relaxin signalling bias

PLC

AP1 Transcription

Ras

Raf

MEK1/2

ERK1/2

PKC

Relaxin

βγ

p38 JNK

MKK4/7

PDE4D3

cAMP

PKA

AKAP79

Basal

PDE4D3

ATP

cAMP

βγ

PKA

AKAP79

Sub-picomolar relaxin

Signalosome activation

CNanomolar relaxin

Signalosome dissociation

PDE4D3

PKA

AKAP79

Activation of cannonical

cAMP signalling pathways

Gαs

β-arrestin 2

S704

AMP

ATP

RXFP1 AC2 RXFP1 AC2

Gαs

β-arrestin 2

S704

AMP

GαoB Gαs

Gαi3

RXFP1 AC2

β-arrestin 2

GαoB

Gαi2

GαoBGαi2

FIGURE 8. Atypical signaling mechanisms utilized by RXFP1 and RXFP3. Signaling complexes termed

signalosomes are formed between RXFP1, AKAP79, AC2, PKA, PDE4D3, and

␤

-arrestin-2 (A) (196, 201).

These complexes are exquisitely sensitive and respond to attomolar concentrations of relaxin with increases in

cAMP (B). This signaling mechanism is distinct from those utilized by higher concentrations of relaxin, and

exposure of the receptor to nanomolar concentrations causes dissociation of the signalosome (C) and cAMP

production by the canonical pathways (see FIGURE 7). RXFP3 displays signaling bias with respect to the

cognate peptide human relaxin-3 and human relaxin-2 (545). Interaction of relaxin-3 with RXFP3 leads to

inhibition of adenylyl cyclase and activation of MAP kinases, NF

␬

B and AP-1 transcription (D), whereas human

relaxin-2 causes only a subset of these pathways to be activated (E).

BATHGATE ET AL.

434 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

tivation of RXFP1 with nanomolar concentrations of pep-

tide that increase cAMP by previously described mechanisms

(FIGURES 7AAND 8C) (198). The high degree of sensitivity

exhibited by the RXFP1 signalosome has been demon-

strated previously in a few other physiological systems, in-

cluding suppression of pro-inﬂammatory cytokine produc-

tion by interleukin-15 (6), proliferation of helper T-cells by

interleukin-1 (398), the effects of neuropeptides and neuro-

steroids in nociception (447, 537), and the long-term effects

of transforming growth factor-

␤

on basal FSH levels (575).

However, although signalosome formation and activity has

also been demonstrated in primary cells endogenously ex-

pressing RXFP1, the exact physiological role for the mech-

anism has yet to be determined.

Relaxin-induced cAMP accumulation may also occur via a

G protein-independent mechanism, and in some cells in-

creased cAMP accumulation may be downstream of a ty-

rosine kinase. In THP-1 cells and cultures of primary

human myometrial or endometrial stromal cells, porcine

relaxin induced an increase in cAMP accumulation that

was blocked by inhibition of tyrosine kinase activity (13,

38, 216, 302). This same response was potentiated by the

phosphotyrosine phosphatase inhibitors [bpV(phen) and

mpV(pic)] that mimic the effect of tyrosine kinase activa-

tion (38). Indeed, in studies predating receptor identiﬁca-

tion in human lower uterine segment ﬁbroblasts, relaxin

stimulation resulted in tyrosine phosphorylation of cellular

extracts, with no effect on cAMP accumulation (407). Re-

laxin-mediated increases in cAMP accumulation in this

context are thought to occur via a tyrosine kinase-mediated

inhibition of a PDE, thereby preventing cAMP hydrolysis

and thus increasing cAMP levels. Interestingly, studies have

also shown that the same tyrosine kinase inhibitors did not

affect relaxin-stimulated cAMP accumulation in HEK293

cells expressing RXFP1 (13), emphasizing the variation in

cellular responses that can be observed between different

cell types. In those cells utilizing the tyrosine kinase path-

way however, there was also some degree of cAMP inhibi-

tion in the presence of the PI3K inhibitor, LY294002 (13,

216), and evidence of a negative feedback loop involving

PKA (13).

A number of other studies in an endogenous setting have

also demonstrated activation of PKA downstream of

cAMP. Increased cAMP accumulation by relaxin results in

PKA activation in a human myometrial cell line (124). In

this case, PKA (via its interaction with AKAP79) can inhibit

the phosphoinositide turnover that is stimulated by oxyto-

cin and is required for smooth muscle contraction (124,

592). In the same cell line, human relaxin can also stimulate

aCa

2⫹

-sensitive K

⫹

channel independently of increases in

2⫹

; a PKA inhibitor, Rp-cAMPS, prevented the effects of

relaxin, and the stimulation was mimicked by the addition

of PKA

␣

-catalytic subunits to the preparation (364). Fur-

thermore, the inhibition of oxytocin-induced events by re-

laxin is attributed to activation of PKA, and is prevented

following the use of a PKA inhibitor (16, 247). Thus relaxin

stimulation of RXFP1 absolutely results in the activation of

cAMP-PKA pathways, although the precise mechanism

whereby RXFP1 initiates cAMP accumulation appears to

vary somewhat with cell type.

2. Activation of RXFP1 causes increased

phosphorylation of mitogen-activated protein

kinases

In addition to increased cAMP accumulation, a number of

cell types that express RXFP1 including human endometrial

stromal cells (586), THP-1 cells and primary cultures of

human coronary artery cells, pulmonary artery smooth

muscle cells, renal myoﬁbroblasts (370), and ﬁbrochondro-

cytes (4) respond to relaxin with a rapid activation of

p42/44 MAPK (extracellular signal regulated kinase 1/2,

ERK1/2) (FIGURE 9).

In normal human endometrial cells (NHE cells), relaxin

stimulation causes rapid and transient phosphorylation of

ERK1/2, with a peak response between 5–10 min (586).

The same time course proﬁle following relaxin stimulation

was also recorded for phosphorylation of MEK and CREB,

but in these cells there was no effect of relaxin treatment on

Akt or JNK phosphorylation; furthermore, an inibitor of

MEK abolished phosphorylation of ERK1/2 in response to

relaxin, suggesting that MEK is activated upstream of

ERK1/2 (586). Increased phosphorylation of ERK1/2 fol-

lowing relaxin stimulation was also observed in THP-1 cells

and cultures of human coronary artery and pulmonary ar-

tery smooth muscle cells and the MEK-ERK1/2 pathway

was also found to control relaxin-stimulated transcription

of VEGF in these cells (FIGURE 9) (586).

In contrast to the brief increase in ERK1/2 phosphorylation

described above, relaxin stimulation caused a more prolonged

increase in ERK1/2 phosphorylation in HeLa cells and pri-

mary human umbilical vein endothelial cells (HUVECs) (129).

Furthermore, in HeLa, EAhy926 (an endothelial cell line),

HT-29 (a colonic cell line), and primary ﬁbrochondrocyte

cells, relaxin increased the phosphorylation of both ERK1/2

and Akt after 30 min (FIGURE 9) (4, 128). In primary ﬁbro-

chondrocytes, it has also been shown that in addition to

ERK1/2 and Akt, treatment with relaxin also activates PI-

3-kinase, PKC-

␨

,NF

␬

B, c-fos, and Elk-1 all of which inﬂu-

ence the expression of MMP-9 (4) (see sect. VIIA5). Simi-

larly, relaxin induces a sustained increase in ERK1/2 phos-

phorylation in rat renal myoﬁbroblasts, and this was

potentiated by inhibition of G

i/o

by PTX, suggesting that

phosphorylation of ERK1/2 may be downstream of G pro-

tein coupling (370). In contrast, in human vascular smooth

muscle cells (hVSMC), relaxin stimulation did not affect

ERK1/2 phosphorylation but instead increased the phos-

phorylation of p38 MAPK (129). Thus, although relaxin

increases phosphorylation of a number of kinases in multi-

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

435Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

ple cell types, the precise isoform and mode of activation

appears to vary, and the physiological consequences of ac-

tivation of these pathways are as yet unclear.

3. Role of NO signaling in RXFP1-mediated

responses

There is a large volume of evidence that shows relaxin treat-

ment increases NO synthesis both acutely and chronically,

although the exact mechanism and whether this is directly

or indirectly linked to RXFP1 is still unclear (104, 385).

Several physiological effects of relaxin within the cardiovas-

cular system are mediated by NO, including inhibition of

lipopolysaccharide (LPS)-induced neutrophil adhesion in

coronary endothelial cells (386), inhibition of the activation

of neutrophils by proinﬂammatory agents via increased in-

ducible NO synthase (36) expression (345), increased cor-

onary blood ﬂow in rat and guinea pig hearts (36), and the

increased renal vasodilation and hyperﬁltration in rats via

the ET

receptor (FIGURE 9) (113). Relaxin also increases

CREB

VEGF

ERK

Gαi3 Gαo Gαi3 GαoB

GαS

ETB

ET-1

RXFP1

Relaxin

ET-1-32

VSM

bigET-1

AKT

PDE

PKCζ

Adenylate

cyclase

PKA

cAMP

ATP AMP

IκB

NFκB

eNOS

nNOS

iNOS

Nucleus

Altered gene transcription

MMPs

2, 9

βγ βγ

PI3K

FIGURE 9. Vasodilator mechanisms activated by relaxin acting at RXFP1 receptors. Relaxin induces vaso-

dilatation by activation of multiple mechanisms. Vasodilation occurs both acutely and chronically, and the action

of relaxin differs in different blood vessels and in humans even with medication being administered (171). Acute

actions that likely inﬂuence vascular tone include cAMP generation and increases in NO generation (104, 385).

Chronic actions include the induction of eNOS, nNOS, and iNOS that in addition to the direct effects of NO cause

increased expression of MMPs that cleave big ET-1 to release ET(1–32) that acts on ET

receptors to cause

NO-dependent vasorelaxation (265).

BATHGATE ET AL.

436 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

the activity and expression of the three types of NOS: en-

dothelial NOS (eNOS; NOS III) (19, 20, 27, 28), inducible

NOS (iNOS; NOS II) (20, 28, 30, 31, 89, 154, 345, 346,

427), and neuronal NOS (nNOS; NOS I) (19) (FIGURE 9).

Furthermore, activation of NOS by relaxin results in down-

stream increases in cGMP, due to production of NO and

activation of guanylyl cyclase (30–32, 346). Based on acti-

vation of NOS and cGMP, there are a number of pathways

by which relaxin could increase NO production. First,

eNOS activity can be increased by Akt phosphorylation;

thus coupling of RXFP1 to G

␣

and the associated release

of G

␤␥

subunits can activate PI3K, that could potentially

also activate Akt, allowing phosphorylation of eNOS at

S1172 (385) (FIGURE 9). Second, iNOS activity can be up-

regulated following stimulation of NF

␬

B-controlled tran-

scription; thus coupling of RXFP1 to G

␣

increases cAMP

and activates PKA, which can then potentially phosphory-

late and inactivate I

␬

B, thereby stimulating NF

␬

B-con-

trolled transcription and increasing iNOS-mediated NO

production (31, 154). Indeed, the increase in iNOS expres-

sion mediated by relaxin in HUVECs (427) and rat coro-

nary endothelial cells (154) was abolished by NF

␬

B inhib-

itors (FIGURE 9). More evidence supporting this possibility

comes from studies that demonstrate stimulation of NF

␬

transcription by relaxin over short time periods (129, 130,

224), suggesting that this pathway may represent a mecha-

nism for more transient increases in NO (FIGURE 9). Finally,

the pathway whereby relaxin stimulation of RXFP1 could

upregulate eNOS in the kidney is better deﬁned: it is hy-

pothesized that this physiological effect of relaxin is due to

increased activity of matrix metalloproteinase (MMP) 2

(445) or MMP9 (215, 224), which can process big endothe-

lin (ET) to ET

1–32

,ET

1–32

then activates the ET

receptor,

increasing the activity of eNOS, and thus producing NO

(FIGURE 9) (263).

4. RXFP1 signaling inﬂuences connective tissue

metabolism

Relaxin can modulate connective tissue metabolism at a

number of levels, including the inhibition of proﬁbrotic fac-

tors (such as TGF-

␤

), the inhibition of ﬁbroblast prolifera-

tion and differentiation, and the activation of MMP-medi-

ated extracellular matrix degradation. Identiﬁcation of the

signaling pathways that lead to these end points are now

gaining attention, and greater knowledge of these processes

should allow more targeted efforts in relaxin-related drug

discovery.

TGF-

␤

is one of the most important cytokines in the pro-

gression of ﬁbrosis; binding of TGF-

␤

to its type II receptor

leads to formation of a receptor complex with the type I

receptor, receptor phosphorylation, activation of a receptor

(R)-Smad (Smad1, 2, 3, 5 or 8), and cotransport of R-Smad

with a common (Co)-Smad (Smad4) into the nucleus to

control gene expression (reviewed in Ref. 347). The antiﬁ-

brotic effects of relaxin are thought to result from inhibition

of the action of TGF-

␤

(FIGURE 10) (349, 443, 539, 542). In

human renal ﬁbroblasts, TGF-

␤

increases the expression of

␣

-SMA (a marker of ﬁbroblast differentiation), type I col-

lagen, and ﬁbronectin, and these effects are reversed by

relaxin. The inhibitory effects of relaxin are due to inhibi-

tion of Smad2 (an R-Smad) (215, 370) but relaxin treat-

ment also decreases TGF-

␤

-induced phosphorylation of

Smad2 (but not Smad3), and knockdown of Smad2 alone

had an effect similar to treating the cells with relaxin (215).

Subsequent studies in rat renal myoﬁbroblasts conﬁrmed

inhibition of Smad2 phosphorylation by relaxin and

identiﬁed the upstream mediators of this inhibition:

nNOS-NO-cGMP (370). Thus relaxin inhibits TGF-

␤

and thus ﬁbroblast differentiation by a NO-dependent

pathway (FIGURE 10).

Relaxin also affects connective tissue metabolism by in-

creasing the production of MMPs including MMP1 in hu-

man lung ﬁbroblasts (542), human dermal ﬁbroblasts (539)

and rat renal cortical ﬁbroblasts (349), MMP2 in rat car-

diac ﬁbroblasts (443) and renal ﬁbroblast cell lines (215),

MMP9 in renal ﬁbroblast cell lines (215) and THP-1 cells

(224), and MMP13 in rat hepatic stellate cells (FIGURE 10)

(55). Increased MMP9 activity in THP-1 cells (that endog-

enously express relaxin receptors) was due to changes in

transcriptional regulation of the MMP gene mediated by

␬

B, as inhibition of NF

␬

B reduced the relaxin-dependent

increase in MMP9 expression and activity (FIGURE 10)

(224). This may suggest an interesting link between the

effects of relaxin upon MMPs and NO, both of which ap-

pear to be mediated by increased NF

␬

B transcription. To

complement the increased production of MMPs in response

to relaxin, the peptide can also decrease the expression of

tissue inhibitors of MMPs (TIMPs) in human dermal ﬁbro-

blasts (539), with speciﬁc inhibition of TIMP1 and TIMP2

in cultured rat hepatic stellate cells (55, 564).

A recent report indicates that relaxin increases the activity

of the peroxisome proliferator-activated receptor (PPAR)-

␥

(482), a nuclear receptor that heterodimerizes with the ret-

inoid X receptor to increase the transcription of target genes

(reviewed in Ref. 153). PPAR

␥

has similar inhibitory effects

on TGF-

␤

signaling and similar end-point physiological ef-

fects upon ﬁbrosis to relaxin itself (482). Thus this obser-

vation may provide another missing link in the signaling

pathways associated with connective tissue metabolism

that are activated by relaxin. However, any physiological

relevance of this activation, and indeed the relevance of this

to relaxin signaling pathways, remains to be shown.

B. Signaling Pathways Activated by RXFP2

Although relaxin and INSL3 are closely related peptides

and RXFP1 and RXFP2 have similar structures, the intra-

cellular signaling pathways that are initiated following

INSL3 stimulation of RXFP2 are less elaborate. INSL3 (or

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

437Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

relaxin) stimulation of RXFP2 expressed in HEK293T cells

results in coupling to G

␣

to increase cAMP accumulation,

and to G

␣

to negatively modulate increases in cAMP

(FIGURE 7B)(198, 301). RXFP2 signaling therefore closely

resembles the ﬁrst stage of RXFP1 signaling in response to

relaxin (198). There is also evidence for an inhibitory effect

of G

␤␥

subunits upon AC; the G

␤␥

subunits are likely de-

rived from G

␣

, as although removal of G

␤␥

using

␤

ARK-ct or inhibition of G

␣

i/o

with PTX both increased

cAMP the effects were not additive (198). Unlike RXFP1,

for RXFP2 there is no evidence for constitutive activity or a

high sensitivity response to INSL3 (201), and in addition,

RXFP2 does not activate the G

␣

-cAMP signaling pathway

that is unique to RXFP1 (198, 204). Downstream of cAMP

production, activation of RXFP2 (by either relaxin or

INSL3) induces increased CRE-dependent gene transcrip-

tion (FIGURE 7B)(197) in a manner similar to the relaxin

response through RXFP1.

The effect of both G

␣

and G

␣

upon cAMP accumulation

has also been shown in cells that endogenously express

RXFP2. INSL3 stimulation of RXFP2 in rat gubernacular

cells (301) and in a human osteoblast cell line (MG-63)

(162) leads to increased cAMP accumulation, probably by a

␣

-mediated interaction with AC, similar to the response

in HEK293T cells expressing RXFP2. However, in con-

ERK

Gαi3 GαoB

GαS

TGFβ

RXFP1

Relaxin

AKT

PDE

PKCζ

Adenylate

cyclase

PKA

cAMP

ATP AMP

IκB

NFκB

eNOS

nNOS

iNOS

Nucleus

Altered gene transcription

MMPs

1, 2, 9, 13

Fibrosis

Collagen

αSMA

βγ

PI3K

pSmad2

FIGURE 10. The effects of relaxin acting on RXFP1 to inﬂuence connective tissue metabolism. The antiﬁ-

brotic actions of relaxin are mediated through increases of expression of eNOS, nNOS, and iNOS and increased

synthesis of NO. This in turn increases expression of MMPs to reduce ﬁbrosis and also inhibits pSmad2, a

mediator involved in the proﬁbrotic effects of TGF-

␤

BATHGATE ET AL.

438 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

trast, primary cultures of testicular germ cells and oocytes

respond to INSL3 activation of RXFP2 with a PTX-sensi-

tive inhibition of cAMP accumulation (276), consistent

with RXFP2 coupling to G

␣

. Thus, as for RXFP1, the net

signaling outcome of RXFP2 stimulation will depend on

which signaling components (especially G protein isoforms)

are expressed in a particular cellular background.

C. Signaling Pathways Activated Following

Stimulation of RXFP3

Activation of RXFP3, by human relaxin-3, causes PTX-

sensitive inhibition of forskolin-stimulated cAMP accumu-

lation, suggesting that the receptor is coupled to inhibitory

␣

i/o

proteins. RXFP3 activation by human relaxin-3 also

induces phosphorylation of ERK1/2 and other MAPKs

through G

␣

i/o

via either a PI3K-dependent or PKC-depen-

dent mechanism (FIGURE 7C)(545, 546). Although initial

studies on RXFP3 suggested human relaxin-3 or the

〉-chain of human relaxin-3 were the only relaxin family

peptides that could compete for

125

I-human relaxin-3 bind-

ing or inhibit AC activity (314), more recent work has dem-

onstrated that human relaxin-2 also has agonist actions at

RXFP3 albeit activating different signaling pathways (see

below) (545). Thus human relaxin-2 acts as a biased ligand

relative to human relaxin-3 at RXFP3, which may be sig-

niﬁcant for the development of compounds with novel

modes of action and speciﬁcity at this receptor (FIGURE 8, D

AND E).

1. G protein coupling and second messenger

signaling at RXFP3

Whole cell functional responses downstream of RXFP3 ac-

tivation have been examined using the cytosensor micro-

physiometer that detects changes in whole cell metabolism

by measuring ﬂuctuations in extracellular pH, that occur

due to the extrusion of H

⫹

ions as a by-product of cell

signaling. In CHO-K1 cells stably expressing the human

RXFP3 receptor, this approach provided more evidence

that RXFP3 signaling occurred mainly through the engage-

ment of G

␣

i/o

proteins, as pretreatment with PTX blocked

human relaxin-3-stimulated increases in the extracellular

acidiﬁcation response (543). This was consistent with pre-

vious observations that human relaxin-3 inhibited forsko-

lin-stimulated cAMP accumulation, presumably by engage-

ment of G

␣

i/o

proteins (314). The use of inhibitors of PI3K

and MEK 1/2 also indicated that signaling pathways down-

stream of these two kinases were activated by human relax-

in-3 (see sect. VIB3). In addition, a second generalized cell

signaling screen using eight different reporter gene con-

structs in CHO-K1 cells (transiently expressing human

RXFP3 receptors) showed that following stimulation with

human relaxin-3, RXFP3 activates signaling upstream of

activator protein 1 (AP-1) promoters (see sect. VIB4) and

␬

B promoters (see sect. VIB5), but not serum response

element, CRE, heat shock element, nuclear factor of acti-

vated T-cells element, E-box DNA binding element, or glu-

cocorticoid response element (545).

2. RXFP3 is coupled to the inhibition of cAMP

accumulation in recombinant and endogenously

expressing systems

Following stimulation with human relaxin-3, RXFP3 inhib-

its forskolin-stimulated cAMP accumulation in CHO-K1 or

HEK293 cells stably expressing RXFP3 receptors and in

mouse SN56 cells that endogenously express the receptor

(FIGURE 7C)(314, 546). Human relaxin-3-mediated inhibi-

tion of AC is completely prevented in cells pretreated with

PTX, suggesting that this response is G

␣

i/o

dependent. In

CHO-K1 cells transiently transfected with PTX-insensitive

(C351I mutation) variants of G

␣

i/o

proteins, and treated

with PTX (to remove the inﬂuence of endogenous G

␣

i/o

proteins), G

␣

was the major G protein involved in the

inhibition of forskolin-stimulated cAMP accumulation,

whereas in HEK293 cells, G

␣

, and G

␣

were all

important (544). Together, these observations indicate that

although the same downstream signal output may be ob-

served, the signaling partners involved may vary with the

different cellular context.

Forskolin-stimulated cAMP accumulation is also inhibited

in RXFP3-expressing cells by human relaxin-3 B-chain pep-

tides or to a lesser degree by human relaxin-2 and porcine

relaxin in the CHO-K1, HEK293, and SN56 cell back-

grounds (314, 545, 546). These responses likely also occur

via RXFP3 engagement of G

␣

i/o

proteins, although this re-

mains to be determined.

3. Activation of RXFP3 increases phosphorylation

of ERK1/2

Human relaxin-3 activation of RXFP3 causes a rapid and

transient increase in ERK1/2 phosphorylation (peak re-

sponse 2–5 min) in CHO and HEK293 cells, stably express-

ing human RXFP3 (FIGURE 7C)(545, 546). Examination of

a variety of relaxin family peptides revealed that in addition

to human relaxin-3, the human relaxin-3 B-chain dimer

also activates ERK1/2 albeit with low potency and efﬁcacy

(546). There was also a weak response to human relaxin-2

but no detectable response to porcine relaxin or INSL3

(545). Pretreatment with PTX caused an ⬃90% inhibition

of human relaxin-3 stimulated ERK1/2 phosphorylation in

CHO-RXFP3 cells, whereas in HEK-RXFP3 cells the signal

was completely abolished (546). This suggests that the cou-

pling of RXFP3 to PTX-sensitive G

␣

i/o

proteins mediates

ERK1/2 phosphorylation, in both recombinant and endog-

enous systems (546).

RXFP3-mediated ERK1/2 phosphorylation occurs via two

parallel pathways, both of which are downstream of G

␣

i/o

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

439Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

in both CHO-K1 and HEK293 cells stably expressing hu-

man RXFP3 receptors, and in SN56 cells endogenously ex-

pressing mouse RXFP3 receptors. The ﬁrst pathway is de-

pendent on PI3K activation (⬃50% blockade of human

relaxin-3 stimulated ERK1/2 phosphorylation by the PI3K

inhibitors LY294002 or wortmannin), while the other

pathway requires PKC (partial blockade of ERK1/2 phos-

phorylation by both general and isoform-selective PKC in-

hibitors) (FIGURE 7C)(546). Interestingly, many MAPKs,

including ERK1/2, are implicated in central feeding re-

sponses in rats (374, 448, 468), which may suggest that

activation of ERK1/2 by RXFP3 is a physiologically rele-

vant signaling pathway, although direct links between

RXFP3, ERK1/2 activation, and increases in feeding remain

to be shown.

4. RXFP3 activates AP-1-linked reporter genes

Since multiple MAPK signaling pathways [p38 MAPK

(436), JNK (116), and ERK1/2 (423, 558)] converge on

AP-1 elements to increase gene transcription, and are likely

involved in activation of the AP-1-linked reporter genes, it

was postulated that RXFP3 may also couple to other

MAPK pathways (FIGURE 8, DAND E) (see sect. VIB3).

Indeed, human relaxin-3-mediated AP-1 reporter gene acti-

vation was completely blocked in CHO-RXFP3 cells pre-

treated with the p38 MAPK inhibitor (RWJ67657),

whereas a MEK inhibitor (PD98059) or a JNK inhibitor

(SP600125) shifted the human relaxin-3 concentration-re-

sponse curve to the right. In contrast, in HEK-RXFP3 cells,

JNK inhibition abolished human relaxin-3-stimulated AP-1

reporter activation, whereas p38 MAPK or MEK inhibition

shifted concentration-response curves to the right. In SN56

cells (as in CHO-K1 cells), p38 MAPK inhibition com-

pletely abolished AP-1 reporter gene activation and JNK

and MEK inhibition partially blocked AP-1 activation

(545). This suggests that all three MAPKs are involved in

human relaxin-3-mediated AP-1 activation, and that the

hierarchy of the different signaling pathways varies with the

cell background.

Pretreating the cells with PTX blocked the human relaxin-

3-stimulated AP-1 reporter activation in SN56 cells but not

in CHO-RXFP3 and HEK-RXFP3 cells (545). This suggests

that while AP-1 reporter gene activation was downstream

of G

␣

i/o

in a mouse-derived cell line, this same pathway was

activated by a G

␣

i/o

-independent pathway downstream of

the human RXFP3 receptor (545).

These MAPK signaling pathways may have physiological

signiﬁcance since in forced swim tests in rats, there are

dramatic increases in phosphorylated MEK1/2, ERK1/2,

and JNK1/2/3 immediately following the swim test (468),

suggesting that both ERK1/2 and JNK1/2/3 are important

in mediating central stress responses. In addition, human

relaxin-3 mRNA was increased in the nucleus incertus

(516), which may correlate with the increase in MAPK acti-

vation, although the direct links between human relaxin-3,

RXFP3, MAPK phosphorylation, and stress responses remain

to be demonstrated in brain.

5. RXFP3 is coupled to NF

␬

B signaling

Activation of RXFP3 by human relaxin-3 increased NF

␬

reporter gene activation in CHO and HEK293 cells tran-

siently expressing human RXFP3, and also in SN56 cells

endogenously expressing mouse RXFP3, and this activation

was blocked by PTX pretreatment (545). This again sug-

gests that NF

␬

B activation occurs downstream of G

␣

i/o

pro-

tein engagement by the receptor; however, the precise

mechanisms and physiological signiﬁcance of this pathway

remain to be determined (FIGURE 8D).

6. Ligand-directed signaling bias at RXFP3

Ligand-directed signaling bias is gaining increasing cre-

dence in GPCR pharmacology. It describes the selective

stabilization of particular receptor conﬁrmations by ligands

resulting in selective activation of downstream signal trans-

duction pathways (22, 151, 277). There is now evidence

that several relaxin peptides interact with RXFP3 to acti-

vate distinct signaling proﬁles through different, although

sometimes overlapping pathways (FIGURE 8, DAND E). Al-

though the original description of RXFP3 indicated that

this receptor showed a high selectivity for human relaxin-3

in both binding and AC inhibition assays (314), cross-reac-

tivity with other relaxin peptides was not explored over a

wider range of signal transduction pathways. However,

monitoring the whole cell effects of relaxin family peptides,

using the cytosensor microphysiometer and reporter gene

assays, provided the ﬁrst indication that other relaxin fam-

ily peptides may elicit responses through RXFP3. Examina-

tion of the metabolic responses recorded by microphysiom-

etry indicated that human relaxin-2 caused a small change

in the extracellular acidiﬁcation rate in CHO cells stably

expressing RXFP3 (543). In addition, when human relax-

in-2, porcine relaxin, and human INSL3 were tested in the

reporter gene paradigm, human relaxin-2 was identiﬁed to

have greater potency and efﬁcacy at the AP-1 reporter gene

pathway than the cognate ligand human relaxin-3; this is a

hallmark of ligand-biased signaling, and suggests that this

phenomenon also occurred downstream of the RXFP3 re-

ceptor (FIGURE 8, DAND E)(545).

Reexamination of cAMP signaling in three distinct cellular

backgrounds (CHO, HEK293, and SN56) revealed strong

inhibition of forskolin-stimulated cAMP accumulation by

human relaxin-3. However, human relaxin-2, porcine re-

laxin, and human INSL3 also inhibited the forskolin-stim-

ulated cAMP accumulation albeit with much lower potency

than human relaxin-3 (545). Interestingly, some species-

speciﬁc inhibition of forskolin-stimulated cAMP accumula-

BATHGATE ET AL.

440 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

tion was noted, with INSL3 weakly inhibiting cAMP accu-

mulation downstream of the human RXFP3 receptor, but

not downstream of the mouse RXFP3 receptor (545). This

is in contrast to a previous study, which reported no inhi-

bition of forskolin-stimulated cAMP accumulation by ei-

ther porcine relaxin or human INSL3 (314). Since the sen-

sitivity of inhibitory cAMP assays is highly dependent on

both the degree of activation of AC by forskolin and the

time of stimulation, the differences observed between the

two studies most likely result from different experimental

paradigms.

Addition of human relaxin-2, porcine relaxin, and INSL3 to

RXFP3-expressing cells caused AP-1 reporter gene activa-

tion in a concentration-dependent and cell-type-speciﬁc

manner (545). AP-1 was activated in CHO-RXFP3 and

HEK-RXFP3 cells with an order of potency of human re-

laxin-2 ⬎human relaxin-3 ⬎porcine relaxin. Human re-

laxin-2 also activated AP-1 in SN56 cells with a similar

pattern to that in CHO-RXFP3 and HEK-RXFP3 cells, al-

though the pEC

was shifted to the right (545). Given the

differences observed between different species relaxins at

RXFP1 (see sect. IIC1), this shift in potency order may be

reversed with the use of mouse relaxin instead of human

relaxin-2. In CHO-RXFP3 cells, human relaxin-2-mediated

AP-1 reporter gene activation was strongly inhibited by the

p38 MAPK inhibitor (RWJ67657) or the JNK inhibitor

(SP600125); however, the MEK inhibitor (PD98059)

shifted the human relaxin-2 concentration-response curve

to the right without decreasing the maximum response, im-

plicating p38 MAPK and JNK as the major MAPKs in-

volved in mediating this response downstream of human

RXFP3. Contrastingly, when the same response was exam-

ined in HEK293 cells expressing human RXFP3, human

relaxin-2-stimulated AP-1 reporter gene activation was

most strongly inﬂuenced by p38 MAPK or MEK inhibition

and was not affected by JNK inhibition, suggesting that p38

MAPK and ERK were not the major MAPKs involved in

mediating this response downstream of human RXFP3. In

the SN56 cell line, expressing mouse RXFP3, the p38

MAPK, JNK, and MEK inhibitors all equally blocked the

human relaxin-2-stimulated AP-1 reporter gene activation,

suggesting that all three kinases were equally important in

this cellular context (545). Together these results suggest

that the cellular background, in addition to the species ho-

molog of the receptor, play important roles in the pattern of

activation of AP-1 reporter genes. Direct measurement of

ERK1/2, p38 MAPK, and JNK phosphorylation following

stimulation with the relaxin family peptides has conﬁrmed

the ﬁndings of these inhibitor-based studies. Treating

CHO-RXFP3 or HEK-RXFP3 cells with human relaxin-2

increased ERK1/2 phosphorylation in a rapid and transient

manner with a peak response at 2–5 min; however, this

response was not observed in the SN56 cell background

(545) (Kocan et al., unpublished data).

Pretreatment with PTX failed to block either human relax-

in-3-stimulated AP-1 activation in CHO-RXFP3 or HEK-

RXFP3 cells, or porcine relaxin-stimulated AP-1 reporter

gene activation in CHO-RXFP3 cells, suggesting that hu-

man relaxin-3 and porcine relaxin activate AP-1 reporter

genes by a G protein-independent mechanism, again sug-

gesting ligand-directed signaling bias (545).

Although G protein-dependent activation of ERK1/2, p38

MAPK, and JNK by human relaxin-2 and porcine relaxin

were suggested initially by inhibitor studies, later conﬁrmed

by direct MAPK phosphorylation assays, the G

␣

i/o

-inde-

pendent pathway remains to be identiﬁed for human relax-

in-3 and porcine relaxin downstream of the human RXFP3

receptor. All of the AP-1 reporter gene responses arising

following stimulation of RXFP3 in SN56 cells were blocked

by PTX, suggesting that different signaling effectors were

involved in mediating the AP-1 reporter gene activation

downstream of the mouse RXFP3 receptor (545).

D. Signaling Pathways Activated by RXFP4

Little information currently exists about signaling path-

ways activated downstream of RXFP4. Stimulation of cells

recombinantly expressing human RXFP4 by INSL5 or hu-

man relaxin-3 increases GTP

␥

S binding and inhibits fors-

kolin-stimulated cAMP accumulation, suggesting that

RXFP4 like RXFP3 is G

␣

i/o

coupled (FIGURE 7D)(313,

315). When RXFP4 was coexpressed with G

␣

in HEK293

cells, both INSL5 and human relaxin-3 stimulated a strong

2⫹

signal in a concentration-dependent manner (313,

315), which may suggest that RXFP4 can couple to addi-

tional G proteins; however, direct evidence for RXFP4-G

protein coupling is lacking.

E. Identiﬁcation of G Proteins That Couple

to RXFP Receptors

1. G proteins that couple to RXFP1 and RXFP2

The speciﬁcity of RXFP1 and RXFP2 for particular G

␣

i/o

isoforms was identiﬁed using PTX-insensitive G protein

mutants (198). PTX inactivates G

␣

i/o

proteins by ADP-ri-

bosylation of C351 so that mutation of this residue to I

renders the isoform of interest insensitive to inactivation by

PTX. Sequential expression of the three G

␣

and two G

␣

isoforms in HEK293T cells stably expressing either RXFP1

or RXFP2 caused the restoration of the expected cAMP

signaling proﬁles (following PTX pretreatment) by expres-

sion of only G

␣

for RXFP2 and following expression of

both G

␣

and G

␣

for RXFP1. Activation of G

␣

and

␣

by RXFP1, but the selective activation of G

␣

RXFP2 was subsequently conﬁrmed using [

S]GTP

␥

S im-

munoprecipitation; importantly, activation of G

␣

was ev-

ident within 3 min of relaxin stimulation, suggesting that

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

441Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

the observed delay in activation of the G

␣

-cAMP pathway

occurs downstream of receptor coupling to this G protein

isoform (204). Studies using receptor mutants further dem-

onstrated that activation of the G

␣

-cAMP pathway in-

volves the ﬁnal 10 amino acids of the RXFP1 COOH ter-

minus and requires R752; interestingly, these 10 amino ac-

ids are missing in RXFP2 (the COOH terminus is 10

residues shorter), perhaps explaining the inability of this

highly similar receptor to activate G

␣

(204).

Due to the classiﬁcation of both RXFP1 and RXFP2 as

GPCRs, and their ability to increase cAMP in recombi-

nant receptor expression systems, it has been assumed

that the receptors couple to G

␣

. The ability of PTX to

enhance the cAMP generated following stimulation of

RXFP2 or in the early phase (⬍10 min) following stimula-

tion of RXFP1 tends to support this view. More direct evi-

dence has been generated using peptide fragments of the

third intracellular loop of RXFP1 (minimum length 615–

629; ICL3) and G

␣

; addition of the RXFP1-ICL3 alone to

rat tissues (striatum, cardiac and skeletal muscle membrane

preparations) increased cAMP and “antagonized” the re-

sponse to relaxin. Furthermore, addition of peptide frag-

ments of G

␣

“antagonized” both relaxin stimulation and

RXFP1-ICL3 peptide stimulation of rat tissues (475). A

functional interaction between RXFP1 and G

␣

has also

been demonstrated using pharmacological inhibition; ap-

plication of NF449 to HEK293 cells expressing RXFP1

signiﬁcantly decreased receptor-stimulated cAMP accumu-

lation, whereas a low concentration of cholera toxin (a G

␣

activator) substantially enhanced the cAMP response to re-

laxin (201). Taken together, this suggests that G

␣

(and prob-

ably G

␣

) couples to RXFP1 within the ICL3, while coupling

to G

␣

is dependent on the ﬁnal 10 amino acids of the receptor

COOH terminus.

2. G proteins that couple to RXFP3 and RXFP4

Functional assays suggest that RXFP3 and RXFP4 are G

␣

i/o

coupled receptors that can inhibit forskolin-stimulated

cAMP accumulation in cells expressing the receptors (313,

314, 545). G

␣

i/o

coupling to RXFP3 is further supported by

studies in the cytosensor microphysiometer, where pretreat-

ment of CHO-RXFP3 cells with the G

␣

i/o

protein inhibitor

PTX strongly inhibited the extracellular acidiﬁcation rate

response of these cells, suggesting that most of the signaling

pathways activated by RXFP3 occur downstream of G

␣

i/o

(543). RXFP3 and RXFP4 do not activate calcium signaling

and are therefore presumed not to couple to G

␣

proteins

(313, 314). This pattern of G protein coupling is supported

by more direct approaches. To examine the G protein sub-

units important for RXFP3 signaling, CHO-RXFP3 and

HEK-RXFP3 cells were transfected with PTX-insensitive G

proteins (C351I mutation), treated with PTX and then

ERK1/2 activation measured to determine which G proteins

could recapitulate the functional response. PTX pretreat-

ment completely abrogated the ERK1/2 response in both

CHO-RXFP3 and HEK-RXFP3 cells. In CHO-RXFP3

cells, the ERK1/2 response was partially restored by trans-

fection of G

␣

or G

␣

but not by mutant G

␣

. In HEK-RXFP3 cells, the pattern differed somewhat

in that signaling was partially restored by expression of

mutant G

␣

or G

␣

but also by mutant G

␣

. These dif-

ferences may relate to different colocalization of receptors

and G proteins in lipid rafts in particular cell types.

F. Trafﬁcking of RXFP Receptors

1. Trafﬁcking of RXFP1 and RXFP2 to the cell

surface

Asparagine (N)-linked glycosylation is an important

posttranslational modiﬁcation for cell surface expression

of GPCRs (430). RXFP1 has six predicted N-linked glyco-

sylation sites within the NH

-terminal tail, leucine-rich re-

peat, and LDLa modules, and RXFP2 has ﬁve predicted

N-linked glycosylation sites within the same region (203).

Western blot analysis of a hemagglutanin (HA)-tagged

RXFP1 showed multiple receptor bands, with a high-mo-

lecular-mass (95 kDa) band, representing the mature, gly-

cosylated form of the receptor and a lower molecular mass

(80 kDa) band, representing the immature form of the re-

ceptor (280). Digestion of RXFP1 with endoglycosidase H

or PNGase F removes the N-linked glycosylation from the

receptor, and only the lower molecular weight band is then

observable by Western blot (280). Similar results have been

achieved using a FLAG-tagged RXFP1 receptor where mu-

tagenesis of each of the predicted N-linked glycosylation

sites demonstrates that all such sites are utilized in RXFP1

(572). Additionally, glycosylation within the leucine-rich

repeats was shown to be important for both cell-surface

localization of the receptor and full cAMP signaling efﬁ-

cacy, but has no role in ligand binding (572). As yet, the

glycosylation status of RXFP2 has not been studied in de-

tail.

In addition to glycosylation, the LDLa modules of RXFP1

and RXFP2 are important for receptor trafﬁcking from the

ER to the plasma membrane. Expression of mutant RXFP1

receptors at the cell surface was not changed (compared to

native RXFP1), by deletion of the LDLa module, or by

mutation of the conserved LDLa residues, even though

these receptors could no longer activate cAMP signaling in

the cells (231, 280, 456). In one study, mutation of the

putative N-linked glycosylation site (N36Q) decreased both

cAMP production and cell surface expression (decreased to

37% compared with native RXFP1), suggesting that glyco-

sylation of RXFP1 in the LDLa module plays a role in cell

surface expression. However, an independent study on the

same RXFP1 (N36Q) mutant showed only slight decreases

in cell surface expression that was actually correlated with

decreases in total cell expression. In RXFP2, mutation of

the conserved cysteine residue (C71Y) or the conserved as-

BATHGATE ET AL.

442 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

partic acid residue (D70Y) (which destabilizes the LDLa

module) (231) decreased the expression of receptors at the

cell surface and compromised cAMP signaling (compared

with native RXFP2), also suggesting that the LDLa module

is important in cell surface expression of RXFP2 (64). In

addition, swapping the LDLa modules of RXFP1 and RXFP2

to produce a chimeric receptor improved cell surface delivery

of RXFP1, compared with native RXFP1 (280), suggesting

that the LDLa module of RXFP2 is also important for delivery

of the receptor to the plasma membrane.

RXFP1 receptors also form functional homodimers within

the ER, which are maintained while the receptors are traf-

ﬁcked from the ER to the plasma membrane (282, 510,

511). Together these observations suggest that RXFP1 ex-

pression at the cell surface is regulated at a number of stages

during receptor synthesis and maturation, to ensure deliv-

ery of matured, functional receptors to the cell surface.

2. Limited evidence for trafﬁcking of RXFP3 and

RXFP4

There are two conserved putative N-glycosylation sites in

the NH

terminus of both RXFP3 and RXFP4 and an ad-

ditional nonconserved site in ECL3 of human RXFP4; how-

ever, there is currently no information as to whether these

sites are utilized or are important for receptor trafﬁcking.

Cell surface expression of RXFP3 and RXFP4 has only been

demonstrated by whole cell radioligand binding assays

(213, 545).

G. Regulation of RXFP Receptors

1. RXFP1 and RXFP2 are weakly regulated by

phosphorylation and internalization

A consistent feature associated with biological effects

mediated by RXFP1 is sustained signaling (see sect. IXA),

which is unusual for a GPCR. This is also observed when

RXFP1 and RXFP2 are expressed in HEK293T cells

where cAMP levels remain elevated for up to 6 h after

ligand exposure (87). RXFP1 and RXFP2 also display a

lack of internalization following exposure to ligand when

expressed in HEK-293T cells and Cos-7 cells (FIGURE 11)

(87). Both receptors are only weakly phosphorylated and

internalized as measured by whole cell radioligand bind-

ing, and internalization is unaffected by overexpression

of G protein-regulated kinase (GRK) 2/3, suggesting that

any internalization is not regulated by these isoforms.

However, GRK4, -5, and -6 were not tested, and it re-

mains to be determined whether RXFP1 and RXFP2 in-

ternalization is regulated by other GRKs. Confocal im-

aging of GFP-tagged

␤

-arrestin also failed to show cell

surface localization of

␤

-arrestin in cells cotransfected

with either RXFP1 or RXFP2 (FIGURE 11) (87), suggest-

ing that the receptors do not recruit either of the nonvi-

sual

␤

-arrestin isoforms. In primary human decidual cells

and HEK293T cells stably transfected with RXFP1, weak

internalization occurs upon agonist stimulation as mea-

sured by cell surface ELISA, although this may also re-

ﬂect the constitutive activity of the receptor (281). Fur-

thermore, it was suggested that overexpression of

␤

-ar-

restin 2 in HEK-RXFP1 cells further reduced the number

of cell surface receptors (281). Interestingly, a recent

study provided evidence for constitutive association be-

tween RXFP1 (but not RXFP2) and

␤

-arrestin 2, which

controls the endogenous constitutive activity of the re-

ceptor by scaffolding PDE4D3 (201). Thus

␤

-arrestin

may be involved in RXFP1 signaling but is not recruited

following receptor stimulation nor is it associated with

the internalization of either RXFP1 or RXFP2.

2. RXFP3 internalization in response to agonist

exposure

Internalization of RXFP3 occurs following 10 min of

stimulation with human relaxin-3 in CHO-K1, HEK293,

and SN56 cell backgrounds, as assessed by radioligand

internalization assays (70–90% of receptors internal-

ized) (545). RXFP3 internalization was also observed by

confocal microscopy of a GFP-tagged RXFP3 following

stimulation with human relaxin-3 but not human relaxin-2,

porcine relaxin, or INSL3 in CHO-RXFP3 or HEK-

RXFP3 cells (545). Studies using BRET to detect interac-

tions between RXFP3 and

␤

-arrestins in CHO cells

showed that human relaxin-3 but not human relaxin-2

promotes RXFP3/

␤

-arrestin interaction (FIGURE 12).

This suggests that treatment with the cognate ligand hu-

man relaxin-3 causes RXFP3 to undergo

␤

-arrestin-de-

pendent internalization. Pretreatment of cells with PTX

prevented ⬃50% of these interactions, suggesting that

they are partially mediated by G

i/o

(Kocan et al., unpub-

lished data). The detailed mechanism of RXFP3 internal-

ization, phosphorylation, and recycling/degradation all

remain to be determined. To date, there are no data on

the regulation of RXFP4.

VII. PHYSIOLOGICAL ROLES OF PEPTIDE-

RECEPTOR PAIRS

A. Relaxin-RXFP1

Although RLN1 was the ﬁrst human relaxin gene to be

cloned (245) and is expressed in decidua, trophoblasts, and

prostate (208, 439), its physiological role is unclear. A na-

tive human relaxin-1 has not been isolated, although a syn-

thetic human relaxin-1 peptide based on the structure of

human relaxin-2 does have similar biological properties

and potency to human relaxin-2 at RXFP1 receptors (514).

The RLN1 gene is only found in humans and the great apes,

but in some of these species, it is doubtful that a functional

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

443Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

peptide is produced. Even in humans where mRNA expres-

sion is detected in multiple tissues, there is no evidence for

functional peptide production. It is likely that RLN1 has

arisen as a result of a gene duplication event.

The RLN2 gene on the other hand (246) produces human

relaxin-2 in the corpus luteum of the ovary and is released to

circulate in the blood during pregnancy (47, 470), although it

is also produced by the prostate in males (259). RLN2 mRNA

is expressed in the corpus luteum, endometrium, decidua, pla-

centa, and mammary gland as well as in the heart and brain

(42). The RLN2 gene is equivalent to the RLN1 gene found in

nonprimate species, and it encodes the relaxin peptide that

was ﬁrst discovered due to its roles in reproduction. The lo-

cations of sites of relaxin expression (see sect. V, FIGURE 4)

provide a clue to the widespread roles of the relaxin-RXFP1

system extending well beyond its original roles in reproduc-

tion. This section will outline the physiological roles of

relaxin acting through its receptor RXFP1 focusing pre-

dominantly on studies in rodents and correlating this with

proposed physiological roles in humans. It should be noted

that the physiological roles of relaxin outlined in this sec-

tion might not be relevant to other species. A detailed out-

line of the physiological roles of relaxin in all mammalian

species can be found here (47).

1. Reproductive physiology in the female

Relaxin produced by the corpus luteum and/or placenta has

important roles in pregnancy and parturition and is a major

circulating hormone during pregnancy in all mammalian

species. Relaxin has actions on the pubic symphysis, cervix,

uterus, vagina, and mammary glands. It also has important

roles in the cardiovascular changes that occur during preg-

nancy.

Modiﬁcation to the pelvic girdle involving growth of the

interpubic ligament is essential for successful birth in many

species (47, 470, 471). Relaxin is believed to mediate the

increased ﬂexibility and elasticity of the interpubic ligament

that is associated with pregnancy in several species (394,

496). In the relaxin knockout mouse, the interpubic liga-

GFP-βarr1 GFP-βarr2

0 min 60 min 0 min 60 min

RXFP1

RXFP2

AT1R

FIGURE 11. RXFP1 and RXFP2 receptors do not internalize following prolonged exposure to relaxin. HEK-

293 cells transiently expressing RXFP1 (Top panels), RXFP2 (Middle panels), AT1R (Bottom panels), and

green ﬂuorescent protein (GFP)-labeled

␤

-arrestin-1 (GFP-

␤

arr1; left) or GFP-

␤

-arrestin-2 (GFP-

␤

arr2; right).

Cells were stimulated with 1

␮

M agonist [relaxin (A); INSL3 (B); ANG II (C)] and imaged before stimulation and

60 min after stimulation. Bar indicates 5

␮

m. Many G protein-coupled receptors such as the AT1 receptor

interact with

␤

-arrestins and are internalized (bottom panels). RXFP1 (top panels) and RXFP2 (middle panels)

did not interact with

␤

-arrestins 1 or 2 or internalize. [Modiﬁed from Callander et al. (87).]

BATHGATE ET AL.

444 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

ment fails to develop, suggesting a role for relaxin (588).

The mechanisms likely involve actions on collagen remod-

eling as in other reproductive organs (see below). Although

such a role is not clear in humans or other primates (339), it

has been postulated that relaxin levels correlate with severe

pelvic pain and excessive joint laxity seen in some women

during pregnancy (335, 340).

Relaxin is also responsible for the softening and hypertro-

phy of the cervix during the second half of pregnancy in

mammals, which involves effects on collagen, elastin, pro-

teoglycans, and glycosaminoglycans (47). Cervical develop-

ment is impaired in both relaxin-deﬁcient rats (83) and

relaxin knockout mice (588), highlighting a role of relaxin

in this process. The mechanisms involve interactions with

steroid hormones and prostaglandins (47). Studies in rats

demonstrate that relaxin promotes growth of epithelial and

stromal cells in the cervix (82, 83) by stimulating cell pro-

liferation (305) and inhibiting apoptosis (590). In late preg-

nant relaxin-deﬁcient rats, pigs, and relaxin knockout mice,

there is no dispersion or disorganization of collagen ﬁber

bundles in the cervix as occurs in control animals (304, 327,

589). In humans, relaxin levels increase during cervical rip-

ening, but ripening still occurs following embryo transfer

where circulating relaxin levels are undetectable (139). Al-

though direct application of porcine relaxin to the cervix

appeared in early studies to assist in ripening (152, 339),

clinical trials with recombinant human relaxin-2 failed to

conﬁrm this ﬁnding (52).

Relaxin inﬂuences uterine contractility (295) and uterine

growth (495) during pregnancy, but this role is highly species

dependent. In humans, relaxin has little effect on uterine tone

(337, 338), but in rat, mouse, guinea pig, hamster, and pig, the

uterus is relaxed by relaxin (for review, see Ref. 47). In rat and

mouse, the uterus contains relaxin binding sites and mRNA

(47) in the myometrium (see sects. IVA1 and VA1).

In nonpregnant rats, the effect of relaxin on the growth and

development of the uterus is associated with vascular dila-

tation (547) and in rhesus and marmoset monkeys with

endometrial angiogenesis (145, 185). In primates and rats,

pretreatment with estrogens enhances the effects of relaxin

(1, 145), and in pigs, estrogens are obligatory for the

growth-promoting effects of relaxin (242, 581). The mech-

anism involved in the interaction between relaxin and es-

trogen is not well understood, although in rats estrogen

pretreatment dramatically increases relaxin receptor bind-

ing sites in the uterus (400, 401, 515). Although these ﬁnd-

ings largely support a uterotrophic effect of relaxin, this

0.02

0.01

0.00

-0.01

0.02

0.01

0.00

-0.01

6810

-2 0

-4

6810

-2 0

-4

time (min)

Ligand-induced BRET ratioLigand-induced BRET ratio

relaxin

relaxin-3

Ligand or vehicle

addition

Ligand or vehicle

addition

Interactions with β-arrestin 1

Interactions with β-arrestin 2

Unstimulated Vehicle

relaxin-3 relaxin

relaxin

relaxin-3

FIGURE 12. RXFP3 receptors interact with

␤

-arrestins and undergo internalization. The panels show

HEK293 cells transfected with GFP2-labeled RXFP3 receptors. Unstimulated cells or cells incubated with

vehicle show RXFP3 receptors localized to the plasma membrane (top panels). Exposure of cells to human

relaxin-3 but not human relaxin-2 caused internalization of RXFP3 receptors (bottom panels) (545). Examina-

tion of interactions between RXFP3-Rluc8 and one of the

␤

-arrestin fusion proteins (

␤

-arrestin 1-Venus or

␤

-arrestin 2-Venus) cotransfected into Flp-In CHO cells using BRET showed that interactions occurred between

RXFP3 and both

␤

-arrestin 1 and

␤

-arrestin 2 following human relaxin-3 but not human relaxin-2. Scale bar ⫽

␮

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

445Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

does not play a role in pregnancy in mice and rats (252) but

is important in pigs (365). In humans it is likely that utero-

trophic effects of relaxin are associated only with implan-

tation (see sect. VIIIA2) and not with the later stages of

pregnancy.

Relaxin also inﬂuences the growth of the vagina during

pregnancy in mice (194, 450) and rats (82, 590, 591). In

relaxin knockout mice, this growth does not occur, high-

lighting the essential role of relaxin (589). Relaxin binding

is present in the vaginal luminal epithelial cells as well as

circular and longitudinal smooth muscle cells in the rat

(591) and human (288). The effect of relaxin on vaginal

development during pregnancy together with its actions on

the cervix and pubic symphysis are essential for normal

delivery in some species.

Relaxin has trophic effects on the mammary gland. In

mice transgenically overexpressing mouse relaxin, there

is a 20-fold increase in serum levels that is associated

with hypertrophic nipple development in virgin females

(158). This phenotype is absent in mice that also had

deletion of RXFP1 (158). In rats (205) relaxin in combi-

nation with estrogen and progestagen promotes mam-

mary gland development in ovarectomized immature an-

imals. The major effect in rodents is on nipple develop-

ment, whereas in pigs it is mainly on the mammary gland

(248). In rats, administration of a monoclonal antibody

to relaxin during the second half of pregnancy prevents

nipple development (251), and an identical phenotype

occurs in relaxin knockout mice (588). Pups born to

relaxin knockout mice die within 24 h unless cross-fos-

tered to wild-type mothers. The effects are entirely due to

the poor nipple development as female knockout mice are

able to produce milk normally (588). The same pheno-

type is displayed by RXFP1 knockout mice (293) and is

not rescued by administration of INSL3 (274). In con-

trast, relaxin-deﬁcient pigs show normal nipple develop-

ment and function (248, 582). Mammary gland develop-

ment in mice and rats does not require relaxin, although

it may affect tissue differentiation, but in pigs the devel-

opment of the mammary gland parenchyma requires re-

laxin. Relaxin binding sites indicative of RXFP1 recep-

tors are present in the mammary glands of pigs, rats, and

humans. In humans, RXFP1 receptors are localized to the

nipple, epithelial cells (288), and stromal tissue (255).

Major cardiovascular changes are associated with preg-

nancy in most mammals. Relaxin directly acts on the

blood vessels, kidney, and heart to contribute to the car-

diovascular changes that occur during pregnancy (see

sect. VIIA4). In rodents, relaxin causes a decrease in

plasma osmolality during pregnancy associated with

changes in thresholds for thirst and AVP secretion (311,

387). In relaxin-deﬁcient rats (387) and relaxin knockout

mice (588), the decrease in plasma osmolality does not

occur. Although plasma osmolality is reduced in human

pregnancy, there is currently no evidence that relaxin is

involved. Relaxin may also inﬂuence water drinking dur-

ing pregnancy in rodents.

Relaxin has many well-deﬁned reproductive roles in many

species, but in humans these effects are often absent or

ill-deﬁned. Relaxin is produced in the human ovary during

the follicular phase of the menstrual cycle and is secreted

into the circulation (99, 140, 497). It has been suggested

that this relaxin facilitates implantation and maintenance of

pregnancy in the ﬁrst trimester. Early studies showed that

administration of relaxin to monkeys caused growth of, and

increased angiogenesis in, the uterus (145, 185, 214, 222).

It is likely that similar effects occur in the human endome-

trium, since in the clinical trial of relaxin for the treatment

of scleroderma (149), women who received 24-wk subcu-

taneous infusion of relaxin reported heavy, irregular, or

prolonged menstrual bleeding (540). However, in humans

and other primates, the peak of relaxin secretion in the ﬁrst

trimester of pregnancy coincides with embryo implanta-

tion, suggesting that relaxin is involved in implantation (see

sect. VIIIA2). Although there is an association between re-

laxin secretion and the success of implantation in primates,

it is clearly not mandatory since women without ovaries can

become pregnant by ovum donation even though they have

undetectable levels of circulating relaxin (267). Relaxin secre-

tion is correlated with increased expression of RXFP1 mRNA

and relaxin binding in the human endometrium in the secre-

tory phase of the menstrual cycle (67, 88). RXFP1 immunore-

activity is also shown in endometrial stromal cells (255) in

humans and monkeys (145). Relaxin also induces decidualiza-

tion of endometrial stromal cells in culture (529). In contrast,

relaxin does not appear to be important for the decidualiza-

tion of the endometrium in mice (168).

In humans and other primates, increasing evidence points to a

role for relaxin in implantation. Relaxin treatment is associ-

ated with increased endometrial angiogenesis, thickening, and

bleeding (145, 185). In macaques, relaxin treatment of cycling

females in the peri-implantation period increases endometrial

thickness and implantation-related bleeding. Relaxin treat-

ment also increased rates of implantation and multiple preg-

nancies (214). In addition, plasma levels of relaxin peak in the

ﬁrst trimester, coincident with embryo implantation. Rises in

relaxin levels are associated with chorionic gonadotrophin

(497, 498), are altered in patients with early pregnancy loss

(498), and are predictive of in vitro fertilization success in

granulosa cell cultures (499). However, relaxin probably only

facilitates implantation since this still occurs in humans and

other primates that lack ovaries (182, 267). The relaxin that is

produced in the male reproductive tract appears in semen and

increases sperm motility and facilitates penetration into

oocytes (557). A potential role for this relaxin may be to act on

the female reproductive tract to prepare the endometrium for

implantation.

BATHGATE ET AL.

446 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

2. Reproductive physiology in the male

Relaxin is found in the male reproductive tract in most

mammals. Human relaxin-2 has been puriﬁed from human

male seminal plasma and is identical to luteal relaxin (567).

The source of this relaxin appears to be the prostate (489,

576), but its function in the male is unknown, although as

discussed above seminal relaxin may be involved in implan-

tation. RXFP1 has been localized to sperm in both mice

(293) and humans (91, 160), and treatment of human

sperm with relaxin increases motility (91) and induces hy-

peractivation, intracellular calcium and cAMP, and acro-

some reaction in human sperm (160).

Prostate-derived relaxin may have local actions in the male

reproductive tract, since the testis and prostate in mice (442),

rats (237), and humans (239) express RXFP1 mRNA. Inter-

estingly, studies in one strain of the relaxin and RXFP1 knock-

out mouse suggested a role for relaxin in male reproduction.

The mice displayed poor growth of the reproductive tract with

the relaxin knockout, demonstrating effects on the epididymis,

seminal vesicles, and prostate (442), whereas the RXFP1

knockout mice displayed a smaller epididymis (293). Reduced

growth was associated with increased collagen as demon-

strated in the reproductive organs of the female relaxin knock-

out mouse (589). Examination of sperm maturation in the

relaxin and RXFP1 knockout animals suggested that there

might be apoptosis in early stages of spermatogenesis (293,

442). In this relaxin knockout mouse strain (442) and one

strain of RXFP1 knockout mice (293), the changes in the male

reproductive tract are associated with reduced fertility. How-

ever, independent strains of Rln1 ⫺/⫺and RXFP1 knockout

mice (180) showed no prostate phenotype, and another strain

of RXFP1 knockout mice had no male reproductive tract phe-

notype or association with reduced fertility (274). There are

also reports that the phenotypes seen in early generations of

relaxin and RXFP1 knockout are lost in later generations

(257), a similar phenomenon to that seen in the INSL6 knock-

out (85). The authors suggest that the phenotype may “be

taken as evidence of a genuine physiology, but one which

because of redundancy, genetic modiﬁers, and possibly trans-

generational epigenetic modiﬁcation can be masked by in-

breeding” (257). However, the contradictory results from

other strains of relaxin and RXFP1 knockout mice suggest

that a clear role for relaxin in the male reproductive tract

awaits further studies.

3. Central actions of relaxin on RXFP1

In mammals, relaxin acts directly on receptors located in the

SFO and OVLT to cause a reduction in plasma osmolality

(504). In rats, the decline in plasma osmolality during the

second half of pregnancy is associated with increased serum

relaxin levels (311, 472) and is absent in pregnant relaxin-

deﬁcient rats that have undergone ovariectomy or been

treated with relaxin antibodies (387). Likewise, in wild-

type mice, the decrease in plasma osmolality that occurs in

late pregnancy is not observed in relaxin knockout mice

(588). In humans, there is a decrease in plasma osmolality

with pregnancy, but this may not be an effect of relaxin,

since women that become pregnant following ovum dona-

tion have undetectable relaxin levels yet still show this effect

(267, 268). However, the apparent lack of relaxin in these

conditions may have to be reassessed using more sensitive

methods of detection since the discovery of responses to

relaxin (201) at plasma levels well below the level of detec-

tion using current ELISA techniques. In rats, water con-

sumption is strongly stimulated by relaxin and is also in-

creased in the second half of pregnancy (396, 502). Intracere-

broventricular (ICV) or intravenous relaxin also promotes

drinking in nonpregnant rats (502). In rats given relaxin

monoclonal antibodies in the second half of pregnancy, there

is a reduction in water consumption. These actions are pro-

duced by relaxin acting on RXFP1 receptors located in the

SFO and OVLT (FIGURE 6). Administration of porcine or hu-

man relaxin-2 (intravenously) causes increased c-fos expres-

sion in neurons of the peripheral and dorsal segments of the

SFO and in the dorsal cap region of the OVLT, as well as in the

supraoptic and paraventricular nuclei of the hypothalamus

(361, 362, 504, 505), all sites of localization of RXFP1 (FIG-

URE 6). Circulating relaxin penetrates these brain regions and

may be responsible for the plasma osmolality changes that

occur during pregnancy (556).

Other central actions of relaxin have been studied in

much less detail. RXFP1 receptors in the circumventricu-

lar organs and hypothalamic nuclei may have a role in the

timing of parturition in rats since this is disrupted by

central administration of a relaxin monoclonal antibody

(503). RXFP1 receptors are highly expressed in the ba-

solateral amygdala and administration of relaxin to this

region impairs fear related memory consolidation in rats

(331). However, no speciﬁc agonist or antagonist studies

have been carried out to determine whether these effects

are mediated by RXFP1 or to determine the source of

endogenous relaxin that activates these receptors (86).

Although RXFP1 is also highly expressed in other regions

associated with memory formation such as the neocor-

tex, thalamic nuclei, hippocampus, and supramammil-

lary nucleus (FIGURE 6), there are no studies to date that

have examined possible effects on memory. RXFP1 re-

ceptors are also present in the oxytocin-containing cells

of the paraventricular and supraoptic hypothalamic nu-

clei (81), and relaxin administration increases oxytocin

neuron activity and oxytocin release (555).

4. Effects of relaxin on the cardiovascular system

Relaxin has an important role in many of the adaptive car-

diovascular changes that occur in pregnancy (101). These

include increases in plasma volume, cardiac output, and

heart rate, as well as decreased blood pressure and vascular

resistance (103, 104, 118, 119). In both female and male

rats, relaxin administration increases renal plasma ﬂow and

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447Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

glomerular ﬁltration rate (114) and in female rats causes the

reduction in plasma osmolality associated with pregnancy

(387). In rats, ovariectomy or passive immunization with

monoclonal antibodies for rat relaxin prevent these adap-

tive changes (387). In humans, relatively few studies have

been conducted; however, they do suggest similar effects of

relaxin on the cardiovascular system. In the clinical trial for

scleroderma, long-term (6 mo) infusion of relaxin increased

creatinine clearance and produced a modest decrease in

blood pressure (149, 527). More recent trials of relaxin for

the treatment of cardiac failure have shown that a short

(24 h) infusion of relaxin is associated with decreased sys-

temic vascular resistance, serum creatinine, pulmonary

wedge pressure, and a small decrease in systolic blood pres-

sure (133, 134, 527). Comparison of the cardiovascular

changes that occur in pregnancy with those seen following

administration of relaxin shows marked similarity in both

rats and humans (105).

Vasodilation in arterioles, capillaries, and venules is a com-

mon response to relaxin in reproductive tissues (35, 304,

547), heart (33, 36, 343), liver (34), and cecum (58). Re-

laxin is a potent vasodilator in arteries (102, 360), although

the effect is vessel speciﬁc (360). Relaxin is a physiological

antagonist of vasoconstrictors in mesenteric arteries (348,

492), primary bovine aortic smooth muscle cells (31), and

uterine artery (318). Relaxin produces its vasodilator ef-

fects in guinea pig and rat coronary arteries by increasing

NO synthesis (36), later conﬁrmed in bovine cultured

smooth muscle cells (31). The hormone also reduces the rise

in intracellular Ca

2⫹

produced by

␣

-thrombin or angioten-

sin II (31, 154). In humans, relatively few studies have been

conducted, but those performed to date show vasodilator

effects in gluteal resistance or subcutaneous arteries but

little or no effect in pulmonary, myometrial, or placental

vessels (170, 360, 415). In gluteal arteries, the vasodilator

responses likely involve NO and interestingly were inﬂu-

enced by the medication being administered to patients.

Arteries obtained from patients on ACE inhibitors showed

marked attenuation of the vasodilator response to relaxin,

effects that appeared to be further enhanced by inhibition of

cyclooxygenase (170). However, in patients not receiving

ACE inhibitors, indomethacin had little effect (170). The

vasodilator mechanisms suggested for relaxin in both hu-

mans and in animal models involve activation of NOS (104,

385), VEGF, matrix metalloproteinases, ET

receptors

(129, 389), and modiﬁcation of the extracellular matrix of

the vessel walls (FIGURE 9) (263, 307, 360, 568).

The molecular mechanisms of relaxin-induced vasodilation

depend on the duration of hormone exposure, that is, there

are rapid and sustained vasodilatory responses. Our current

understanding is that the vasodilatory responses to relaxin

are mediated by actions at its major receptor RXFP1. Rapid

relaxation responses to relaxin have been recorded in hu-

man gluteal arteries (170) and rat and mouse renal but not

rat mesenteric arteries (360). The responses are endothe-

lium dependent and blocked by NOS inhibitors, the PI3K

inhibitors wortmannin and LY294002 and by PTX pre-

treatment but not by the VEGF receptor antagonist SU5416

(FIGURE 9) (360). The current data suggest that the re-

sponses involve

␤␥

activation of PI3K, Akt phosphoryla-

tion, and eNOS. Sustained responses to relaxin also involve

NOS, since blockade with L-NMMA prevents the renal he-

modynamic and hyperﬁltration responses to relaxin (114),

and these effects can be recapitulated in vitro (389). Treat-

ment with NOS inhibitors or removal of the endothelium

also enhances contractions of vascular smooth muscle to

agonists in both pregnant rats (111, 179) and in rats treated

chronically with relaxin (389). The mechanism involved

does not appear to be attributable to changes in NOS ex-

pression, since the alterations in renal haemodynamics as-

sociated with pregnancy are not accompanied by large

changes in NOS expression. In pregnant rats, eNOS expres-

sion in the renal artery fell by 39%, whereas iNOS and

nNOS expression increased by 31 and 25%, respectively

(5). Rather, it is likely that the vasodilator response involves

endothelin acting at endothelial ET

receptors to release

NO (FIGURE 9). This could be further enhanced as a result

of increased expression of the ET

receptor (129), although

other studies have failed to ﬁnd support for this mechanism

(279). What is clear, however, is that the ET

receptor

antagonist RES-701–1 blocks the renal hemodynamic

changes produced by relaxin (113) in rats and antagonizes

inhibition of renal artery smooth muscle produced by re-

laxin or in pregnancy (389). Similar effects are produced in

vitro by the ET receptor antagonist SB209670 but not by

the ET

selective BQ123 (179).

There is substantial evidence to suggest that the ligand that

interacts with ET

receptors to activate NOS is ET

1–32

produced by the action of MMP2 and MMP9 on big ET

(163). The link to relaxin is that in blood vessels from

pregnant or relaxin-treated nonpregnant rats, pro-MMP2

and MMP2 activity and pro-MMP2 protein and mRNA are

increased (262, 263). MMP9 activity also appears to be

increased somewhat, although more recent studies suggest

that MMP9 is more important in relatively short-term re-

sponses to relaxin (4–6 h) with reversal of the effects being

produced by MMP9 rather than MMP2 neutralizing anti-

body (264). The involvement of MMP2 and MMP9 in the

vasodilator responses to relaxin or pregnancy is supported

by studies with MMP inhibitors. The selective MMP2 in-

hibitor cyclic CTTHWGFTLC, the MMP inhibitor

GM6001, TIMP-2, and MMP-2 neutralizing antibody all

inhibit the vasodilator actions of relaxin in renal arteries

(263), whereas inhibition of the formation of ET

1–21

phosphoramidon had no effect. These studies clearly impli-

cate MMPs in the vasodilator actions of relaxin, although it

should be borne in mind that MMPs also inﬂuence vasore-

activity by interacting with other systems such as cleavage

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448 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

of calcitonin gene-related peptide to promote vasoconstric-

tion (164).

While a case has been made for a role of VEGF in the

vasodilator actions of relaxin, it is not yet precisely clear

what that role is. In nonvascular tissues, treatment with a

range of compounds that increase cAMP levels also in-

creases VEGF expression. So, in human endometrial cells,

relaxin increases VEGF expression, and these effects are

prevented by AC inhibition, and mimicked by either the AC

activator forskolin or a PDE inhibitor (540), suggesting that

increased cAMP production mediates increased VEGF tran-

scription, and thus angiogenesis (FIGURE 9). However, in

vascular tissues, the effects are less clear. Preincubation of

rat or mouse renal arteries or human subcutaneous arteries

with the VEGF receptor antagonist SU5416 blocks the va-

sorelaxant effects of relaxin (359), although the effects of

SU5416 alone and its speciﬁcity can be questioned (17,

316). In other studies using rat renal arteries, SU5416 po-

tentiated the vasodilator effects of relaxin (360). VEGF neu-

tralizing antibodies also block the effects of relaxin, but this

can be mimicked using placental growth factor antibodies

(359), again making interpretation of the results difﬁcult. It

is possible that the antibodies by blocking the vasodilator

effects of VEGF and PGF may produce physiological antag-

onism of the response to relaxin without necessarily being

directly related to the primary mechanism of action.

Relaxin also acts directly on the heart. The presence of

relaxin receptors (RXFP1) in the heart was ﬁrst suggested

by the demonstration of high-afﬁnity binding sites for re-

laxin in rat atria (401, 402). Subsequently, in the same

species, relaxin was shown to be one of the most powerful

inotropic and chronotropic agents known (271). The posi-

tive chronotropic effects of relaxin occur in perfused intact

hearts (36, 107, 531, 534) and isolated right atria (271,

350, 514, 553, 554), and the positive inotropic effects occur

in left atria (271, 350, 514, 553, 554). The chronotropic

effects of relaxin occur together with the secretion of atrial

natriuretic peptide in isolated perfused rat hearts (534). In

rat atrial myocytes, relaxin inhibits outward potassium cur-

rents, increases action potential duration, and enhances

2⫹

entry (417, 418). In rabbit sinoatrial node cells, re-

laxin caused increases in the rate of spontaneous action

potentials and increased the L-type Ca

2⫹

current (206) by a

PKA-dependent mechanism. Until recently, these actions of

relaxin on the heart were thought to be largely conﬁned to

rodents. However, a recent study (127) shows that relaxin

has similar inotropic effects in human atria that are pre-

served in failing hearts and involves PKA, outward K

⫹

cur-

rents, and PI3K. Interestingly, the effects of relaxin in the

cardiovascular system, unlike the effects on reproduction,

are not gender speciﬁc and are observed in both males and

females. There is also evidence that relaxin protects against

myocardial injury caused by ischemia and reperfusion (33).

Pretreatment of rats with relaxin 30 min before 30 min of

cardiac ischemia produced by ligating the left anterior de-

scending coronary artery markedly reduced the size of the

penumbra, and reduced cardiac arrhythmias, mortality as

well as myeloperoxidase activity, malonyldialdehyde pro-

duction, Ca

2⫹

content as well as causing an improved mor-

phology (33). Relaxin may be a naturally occurring cardio-

protective agent, since in congestive heart failure the expres-

sion of human relaxin-1 and human relaxin-2 is increased in

both atria and ventricles and the level of expression follows

the degree of failure (132, 169) but is not a predictor of

clinical outcomes (169).

5. Effects of relaxin-RXFP1 on connective tissue

metabolism

The effects of relaxin on collagen synthesis and breakdown

in reproductive tissues were the ﬁrst biological effects of

relaxin to be recorded (220). Since then it has become evi-

dent that relaxin has more general antiﬁbrotic properties

(47, 181), and a number of attempts have been made to put

these to therapeutic use (462, 526–528, 538). One of the

interesting aspects that has emerged is that the antiﬁbrotic

properties of relaxin are clearly seen only in disease condi-

tions associated with excessive collagen deposition. Several

studies have examined relaxin as a possible treatment for

the connective tissue disease scleroderma. Although relaxin

was shown to be safe and well-tolerated in clinical trials,

and even effective in some patients in a phase II trial (462),

it failed to show clinical efﬁcacy in a larger scale phase III

trial (149). In spite of these disappointing ﬁndings, there has

been increasing evidence from animal studies that relaxin

has a role in controlling collagen turnover. In relaxin

knockout mice, there is a progressive increase in tissue ﬁ-

brosis with age in male mice that is reversed in lung (444),

kidney (445), and heart (443) by the administration of re-

laxin.

In the lung, TGF-

␤

causes detrimental changes associated

with ﬁbrosis that are reversed by relaxin. Treatment with

relaxin reduces expression of collagen types I and III and

increases levels of MMPs (542). Antiﬁbrotic effects were

also produced in mice treated with bleomycin (542). In

kidney-derived ﬁbroblasts, relaxin displays similar effects

on proﬁbrotic changes induced by TGF-

␤

and was shown to

mediate these effects through the NO/guanylyl cyclase

pathway that caused decreases in Smad2 phosphorylation

and nuclear localization (FIGURE 10) (370). Relaxin also

displays antiﬁbrotic effects in a variety of rat renal models

including ﬁbrosis produced by bromoethylamine treatment

(181), in an anti-glomerular basement membrane model

(356), and in spontaneously hypertensive rats (307). In car-

diac ﬁbroblasts, relaxin reduces collagen type I and III ex-

pression and increases MMPs (443). In cardiac ﬁbrosis pro-

duced by chronic stimulation of

␤

-adrenoceptors by isopren-

aline (585) or by cardiac speciﬁc transgenic overexpression of

␤

-adrenoceptors (44), relaxin administration or delivery

via an adenovirus vector markedly reduced cardiac ﬁbrosis.

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

449Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

In streptozotocin-treated mRen-2 rats (a model for diabetic

cardiomyopathy), relaxin treatment reduced left ventricular

collagen, myocardial stiffness, and diastolic dysfunction

(440). This was associated with a signiﬁcant decrease in

TIMP-1 expression and an increase in extracellular matrix-

degrading MMP-13 (440). However, in a chronic pressure

overload model in mice, relaxin was ineffective (569), pos-

sibly because cardiac RXFP1 may be downregulated in this

model or the serum concentrations of relaxin are insufﬁ-

cient to offset the extensive ﬁbrosis. There is also good

evidence supporting antiﬁbrotic actions of relaxin in a num-

ber of models of liver ﬁbrosis (54). Overall, the evidence

suggests that relaxin may have useful antiﬁbotic properties

in a number of pathological conditions.

In a recent study it has been shown that relaxin treatment of

primary ﬁbrochondrocytes increases the expression of

mRNA for MMP-9 and MMP-13 and that the effect is

mediated by RXFP1 and not RXFP2 receptors (4). Similar

treatment caused activation of PI3K, Akt, PKC-

␨

, and

ERK1/2 that could be blocked by the use of appropriate

inhibitors that also blocked the induction of MMP-9. The

effect of relaxin on MMP-9 expression was also blocked by

prior transfection of a dominant negative form of Akt or by

siRNA knockdown of ERK1/2, PKC-

␨

, Elk-1, c-fos, and to

a minor extent NF

␬

B (4). This is one of the ﬁrst studies that

connects many of the known pathways of relaxin/RXFP1

signaling to a recognized response to relaxin treatment and

provides insights that could be useful in translating the an-

tiﬁbrotic effects of relaxin into a clinical setting.

Relaxin also has beneﬁcial effects in wound healing (92). This

may involve the vasodilator effects mentioned above, but in

addition, relaxin may hasten the synthesis of new blood vessels

by enhancing the local production of VEGF (541).

6. Role of relaxin in the formation and spread of

tumors

There is also now increasing evidence that relaxin is pro-

duced by cancer cells and can act in an autocrine manner

on RXFP1 receptors expressed on these cells. To date,

relaxin has been shown to be expressed by endometrial

(273), mammary (522), thyroid (226), and prostate tu-

mors (157, 532). There has long been an association of

relaxin with breast cancer (reviewed in Refs. 26, 479),

and relaxin treatment of breast cancer cells increases

their invasive potential (59). Furthermore, elevated se-

rum relaxin levels have been reported in breast cancer

patients and in patients with metastases (60). It is possi-

ble that the relaxin produced by breast cancer cells is

involved in tissue remodeling during breast cancer pro-

gression (60). Relaxin has also been associated with pros-

tate cancer progression, and blocking the actions of re-

laxin or RXFP1 in rodent models of prostate cancer re-

sults in decreased cancer growth (see sect. VIIIA7).

B. INSL3-RXFP2

INSL3 was isolated from a porcine testis cDNA library and

designated Leydig insulin-like peptide (2). Murine INSL3

was independently cloned from a cDNA library of clones

preferentially expressed in testicular tissue (424). INSL3 is a

major secretory product of the prenatal and postnatal tes-

ticular Leydig cells in all species tested (2, 424). Detection of

INSL3 expression in human cyclic corpus lutea put to rest

suggestions that INSL3 expression is restricted to testis

(521). Transcripts were subsequently detected in other tis-

sues and species including ruminant ovary, uterus, and pla-

centa (40, 432) as well as mouse (595) and marmoset ovary

(583).

1. Reproductive physiology: male

The development of INSL3 knockout mice enabled physio-

logical functions to be attributed to the peptide (379, 596).

INSL3 knockout mice develop normally, but male knock-

out mice are infertile and bilaterally cryptorchid, with the

testis located high in the abdominal cavity adjacent to the

kidney. There may also be torsion of the vas deferens and

testicular artery and localization of the right testis in the

contralateral position (379) likely due to a lack of attach-

ment of the testis to the inguinal region by the gubernacu-

lum and regression of the suspensory ligament. At birth, the

size and histology of testes from knockout animals was

normal but subsequently degenerated, and spermatids,

spermatozoa, and mature sperm were absent in adults. This

is likely to be a temperature effect, since surgical reversal of

cryptorchidism led to normal spermatogenesis in seminifer-

ous tubules (384, 596).

The cryptorchid phenotype is not due to androgen deﬁ-

ciency, and INSL3 knockout males have serum testosterone

levels similar to wild-type and heterozygous males (379)

and apart from cryptorchidism have normal genitalia (596)

and androgen-dependent behaviors (379). The gubernacu-

lum in knockout mice is similar to that found in wild-type

females. In vitro, coculture of explant gubernaculum from

either wild-type or knockout mice, with the testis, induced

growth of the gubernaculum (147), suggesting that INSL3

secreted by the testes is important for testicular descent.

Similarly cryptorchidism is the phenotype displayed in the

white spotting (crsp) transgenic mouse which has a 550-bp

deletion affecting the G protein-coupled receptor affecting

testis descent (GREAT) (186, 406). This receptor (formerly

LGR8) is now termed RXFP2, the cognate receptor for

INSL3 (42). Since gubernacular cells undergo cell division

in response to INSL3, both in vitro and in vivo studies

suggest that the INSL3-RXFP2 system in male urogenital

structures is essential for development of the gubernaculum

during embryogenesis and for normal transabdominal tes-

ticular descent. A recent study has demonstrated that

INSL3 signaling in the fetal gubernaculum both in vitro and

BATHGATE ET AL.

450 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

in vivo involves genes that induce morphogenetic programs,

including BMP and WNT signaling (266). Additionally,

another study demonstrated that INSL3 signaling is essen-

tial for myogenic differentiation in the gubernaculum and

involved genes in the Wnt/

␤

-catenin and NOTCH path-

ways (270).

Transgenic mice overexpressing INSL3 were generated to

further investigate the actions of INSL3 on the gubernacu-

lum (3, 292). Females display ovaries positioned over the

bladder, attached to the abdominal wall by well-developed

craniosuspensory ligament (CSL) and gubernacula. Al-

though this supported the idea that gubernacular develop-

ment is an androgen-independent process, subsequent stud-

ies suggested otherwise (see below). In transgenic males, the

pancreatic expression of INSL3 rescued the cryptorchid

phenotype resulting from the deletion of the endogenous

testicular INSL3 gene. Finally, in female INSL3 transgenic

RXFP2 knockout mice, there is a wild-type phenotype, con-

ﬁrming that the actions of INSL3 on the gubernaculum are

mediated by RXFP2 (65).

However, there is a suggestion that INSL3 alone is not

sufﬁcient for normal testicular descent. Normal testicular

descent appears to rely on both INSL3-induced develop-

ment of the gubernaculum and androgen-mediated cranial

suspensory ligament regression (3, 375). Androgens have

been shown to modulate RXFP2 expression in the guber-

naculum. Androgens alone are insufﬁcient to stimulate gu-

bernacular cell proliferation but in combination with

INSL3 have a proliferative effect, which is blocked by an

RXFP2 antagonist or siRNA (580). In vivo,the RXFP2

antagonist blocked testosterone replacement therapy-in-

duced testis descent in 50% of LH receptor knockout mice.

Thus RXFP2 signaling appears to mediate the effects of

androgens on the gubernaculum, which is important for the

inguinoscrotal phase of testicular descent. This is particu-

larly important as most human defects in testicular descent

are associated with the inguinoscrotal phase (250).

Many early papers investigating the role of INSL3-RXFP2

in cryptorchidism emphasized the relevance of this ligand-

receptor pair to cryptorchidism in humans. Cryptorchidism

is the most common birth defect of the male genitalia, af-

fecting 1–4% of live male births, with a greater incidence in

premature infants (257). While the phenotype of INSL3 or

RXFP2 knockout mice suggested that mutations in these

genes could account for cryptorchidism in some infants, this

is in fact highly unlikely. Some cryptorchid patients display

polymorphisms in either INSL3 or RXFP2, but very few of

these mutations confer a functional change in either the

peptide or the receptor. Mutation detection analysis in

genomic DNA samples from formerly cryptorchid patients

identiﬁed two mutations in INSL3 (533). The ﬁrst a non-

sense mutation in the C-peptide region of pro-INSL3

(R49X) results in a nonfunctional peptide, and the second,

in the B chain (P69L) of the pro-INSL3 sequence results in a

peptide with reduced activity (65). Similarly, of the four

RXFP2 mutations identiﬁed in humans, only one is ex-

pected to alter receptor function. The T222P mutation in

the fourth leucine-rich repeat is thought to result in a con-

formational change that interferes with ligand action (186),

probably by inhibiting expression of mutant RXFP2 recep-

tors at the cell surface (64). Recently, an N-ethyl-N-nitro-

sourea-induced mutation in mice that impaired testicular

descent and reduced testis weight was found to be due to a

mutation in leucine-rich repeat 8 (D294G) (209). Recom-

binant expression of the RXFP2 D294G mutant showed

reduced cell surface expression and a corresponding reduc-

tion in maximal cAMP response compared with wild type.

Patients with the T222P mutation displayed heterogeneous

phenotypes such as unilateral and bilateral cryptorchidism,

retractile testis, normozoospermia, and complete azoosper-

mia (186). All mutations of INSL3 and RXFP2 identiﬁed in

cryptorchid patients have only been found in the heterozy-

gous state. As the expression of INSL3 in testicular Leydig

cells is exceptionally high, it is unlikely that a mutation in a

single allele would be sufﬁcient to induce bilateral cryp-

torchidism.

Nonetheless, there is evidence that the INSL3-RXFP2 sys-

tem is affected in cryptorchid patients that have been ex-

posed to environmental toxins. The use of diethylstilbestrol

for hormonal support in pregnant women was terminated

due to a high incidence of genital defects such as cryp-

torchidism in offspring (183). In mice, maternal exposure to

estrogens, including 17

␣

- and

␤

-estradiol and diethylstil-

bestrol, downregulates expression of INSL3 in embryonic

Leydig cells, and the gubernacula fail to develop which

results in cryptorchidism (380). Hence, it is possible that

environmentally derived estrogens could cause cryptorchid-

ism in humans by suppression of INSL3 expression in the

fetal testis. Similarly, treatment of pregnant rats with phtha-

late esters reduces testicular INSL3 expression as a conse-

quence of Leydig cell toxicity (97). As phthalates are used in

a variety of products as plasticisers, humans in developed

countries are substantially exposed, although, due to health

concerns, their use is decreasing.

2. Reproductive physiology: female

INSL3 is produced in the ovary of all mammals predomi-

nantly in the thecal cells of the follicle (256). Female INSL3

knockout mice demonstrate impaired fertility associated

with longer estrous cycle length, smaller litter sizes, accel-

erated follicular atresia and luteolysis, and premature loss

of corpora lutea (379, 490). These phenotypes suggest that

INSL3 inhibits follicular and luteal cells from entering the

apoptotic pathway. In support of this, INSL3 expression in

vivo varies with follicular development with levels greatest

at the early antral stages and declining prior to expression

of cholesterol side-chain cleavage cytochrome P-450 or 3

␤

hydroxysteroid dehydrogenase expression in the theca in-

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

451Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

terna as follicles enlarge or enter atresia (253). Thus thecal

cell INSL3 expression may be important in follicle selection

in addition to being antiapoptotic.

In mouse ovaries, INSL3 expression coincides with the ap-

pearance of growing follicles, and expression is higher in

the follicular than the luteal phase (595). During pregnancy,

INSL3 mRNA is greatest during phases 1–3 of follicle de-

velopment and decreases to its lowest levels between day 8

and 17 of pregnancy (595). Together, these results suggest

that INSL3 expression is correlated with follicular matura-

tion.

In rats, INSL3 may mediate the actions of gonadotropins in

both female and male germ cells. In the mammalian ovary,

oocytes show prolonged arrest at the prophase of meiosis I.

The preovulatory surge in luteinizing hormone induces the

resumption of meiosis in the oocyte, reprogramming of mu-

ral granulosa cells, expression of new mRNAs and proteins,

and changes to the secretory properties of the cells sur-

rounding the oocyte. Control of these factors is associated

with the levels of cAMP in the oocyte (535). In rat ovary,

expression of RXFP2 is restricted to the oocyte, suggesting

a similar paracrine function for the INSL3-RXFP2 system.

LH also increases transcription of INSL3 in ovarian theca

cells, and binding of INSL3 to RXFP2 expressed in the

oocyte leads to activation of G

and decreases in cAMP

production (276). Studies in cultured preovulatory follicles

from gonadotropin-treated female rats further indicated

that INSL3 causes meiotic progression of the arrested

oocytes.

The ability of LH to regulate INSL3 levels is supported by

data from several other species and models, including hu-

mans (50), mice (159), and roe deer (229). INSL3 may also

be involved in polycystic ovary syndrome (PCOS), a com-

mon female endocrine disorder characterized by obesity,

anovulation or amenorrhea, acne, and excessive production

or enhanced effects of androgens. In normal women, INSL3

levels are positively correlated with total testosterone, free

androgen index, basal LH concentrations and hirsuitism

score, and negatively correlated with frequency of menstru-

ation (178). In patients with PCOS, circulating INSL3 levels

are associated with higher luteinizing hormone levels, in-

creased basal and ovarian hyperandrogenemia and hirsuit-

ism score, worsened menstrual pattern, and higher mean

ovarian follicle number. It is likely that in normal-weight

women with PCOS, increased LH may be responsible for

increased androgen secretion from ovarian tissues mediated

by INSL3 (178).

3. Neurophysiology

RT-PCR was used to demonstrate RXFP2 expression in

human brain (239), and in rats, in situ hybridization histo-

chemistry showed RXFP2 mRNA in numerous thalamic

nuclei (469), including the parafascicular nucleus; the dor-

solateral, ventrolateral, and posterior thalamic nuclei; and

the medial habenula (460). These sites correspond to the

distribution of RXFP2 determined by

125

I-INSL3 binding

(460).

In contrast, INSL3 mRNA has only been detected in bovine

hypothalamus by RT-PCR (40) and Northern blotting (41)

and is not present in rodent brain (469). This may reﬂect the

greater importance of INSL3 in ruminants (40), which do

not express relaxin (560). The ligand-receptor mismatch

may support the proposal that RXFP1 and RXFP2 evolved

as receptors for relaxin family peptides during mammalian

evolution (561) and that prior to the emergence of INSL3,

there was a now defunct ligand-receptor pairing of which

only RXFP2 remains.

It is also possible that INSL3 derived from testicular or

ovarian cells crosses the blood-brain barrier to activate

RXFP2. Circulating INSL3 levels are relatively high (9,

172) at ⬃1% of testosterone levels. However, INSL3 infu-

sions into rat brain suggest that RXFP2 inﬂuences sensori-

motor rather than gonadal function (460). It remains to be

determined whether INSL3 crosses the blood-brain barrier

to activate RXFP2.

4. Physiological functions of INSL3-RXFP2 in other

tissues

Knowledge of other physiological functions of INSL3 is

based largely on the developmental pattern of INSL3 and

RXFP2 expression in bone, tumors, and kidney.

INSL3 levels vary throughout life and often closely mirror

testosterone, which is also produced by the Leydig cells of

the testis. During adulthood, Leydig cell function may be

altered and INSL3 levels decline in late-onset hypogonad-

ism. In 25 young adult men with cryptorchid hypogonad-

ism caused by the T222P mutation of RXFP2, 64% had

reduced bone density but normal plasma testosterone levels

(162). RXFP2 mRNA and protein are present in human

osteoblast cells, and an osteoblast cell line responded to

INSL3 with a dose-dependent increase in cAMP. Like hu-

man, mouse osteoblasts express RXFP2, but not INSL3,

and RXFP2 knockout mice are osteopenic and have func-

tional osteoblast impairment (162). This suggests that

INSL3 may have an endocrine role in bone physiology and

that RXFP2 mutations may be linked with osteoporosis in

men. In cultured human osteoblasts (414), INSL3 regulates

the expression of genes necessary for proliferation and dif-

ferentiation, matrix deposition, and osteoclastogenesis, in

addition to dose-dependent proliferation. This correlates

well with deﬁcits in INSL3-RXFP2 signaling that are corre-

lated with reduced bone mass (161, 162).

A role for INSL3 in tumor biology has been suggested on

the basis of expression of INSL3 and a splice variant in

human hyperplastic thyroid adenoma and thyroid cancer

BATHGATE ET AL.

452 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

(228). INSL3 is also present in human prostate carcinomas

and increases motility in a human androgen-insensitive

prostate carcinoma cell line PC-3 (286). More recently it

has been demonstrated that all human thyroid adenomas,

three types of human thyroid carcinoma tissues and mouse

non-cancerous follicular epithelial cells of the thyroid ex-

press RXFP2 mRNA (225). In addition, thyroid cancer cells

expressing INSL3 demonstrate enhanced motility and in-

creased colony formation in vitro and enhanced tumour

growth in vivo. Finally, the Ca

2⫹

binding protein S100A4,

which increases cancer cell mobility and enhances tumour

tissue vascularization, is increased by INSL3, suggesting

that S100A4 is a downstream target of RXFP2 signaling in

thyroid cells (225).

RXFP2 mRNA expression is highest in rat kidney at late-

stage gestation and decreases dramatically at birth with the

lowest levels in adulthood (175). RXFP2 is expressed in

mesangial cells in mature glomeruli and inhibits prolifera-

tion of cultured primary glomerular cells, suggesting that

INSL3 and RXFP2 inﬂuence the genesis or maturation of

renal glomeruli and regulate mesangial cell density (175).

Pod1 is one of the main transcription factors involved in

glomerulogenesis (425), and an E-box consensus sequence

capable of binding Pod1 in conjunction with other helix

loop helix transcription factors is located upstream of the

gene for RXFP2 (176). Therefore, Pod1 may regulate ex-

pression of RXFP2 in the kidney during development to

facilitate glomerular cell proliferation (155). RXFP2 ex-

pression levels in the embryonic kidney were signiﬁcantly

greater in Pod1 knockout mice than in heterozygous or

wild-type controls, indicating that RXFP2 is downstream of

Pod1 and Pod1 negatively regulates expression of RXFP2

in the glomeruli (155).

C. The Neuropeptide Relaxin-3 and Its

Cognate Receptor RXFP3

1. The relaxin-3 and RXFP3 system

The endogenous receptor for relaxin-3 is believed to be

RXFP3 on the basis of the coevolution of the ligand-recep-

tor pair (561), the overlap of relaxin-3 ﬁbers, and RXFP3

expression in the brain (FIGURE 6) (46, 80, 314, 332, 334,

486, 487, 516) and the pharmacological proﬁle of RXFP3

which shows high afﬁnity for relaxin-3 (314, 545, 546).

However, at least pharmacologically, relaxin-3 may also

interact with RXFP1 (501) and RXFP4 (313) and may also

interact with RXFP2 in some species (453).

Relaxin-3 and RXFP3 expression is highest in the brain,

leading to a focus on roles of this ligand-receptor pair in the

CNS. Functional studies of relaxin-3-RXFP3 have been fa-

cilitated by the development of a chimeric peptide R3/I5

that activates RXFP3 and RXFP4 but not RXFP1 (312),

and R3(B⌬23–27)R/I5, an antagonist with selectivity simi-

lar to the chimeric R3/I5 peptide (299) (see sect. IIC).

2. Role of the relaxin-3-RXFP3 system in stress

Relaxin-3 and RXFP3 are present in hypothalamic and ex-

trahypothalamic regions involved in the hypothalamic-pi-

tuitary-adrenal axis (312, 329, 487, 508). Corticotropin

releasing factor (CRF) is synthesized and released from the

paraventricular nucleus of the hypothalamus (PVN) in re-

sponse to stress. Interestingly, RXFP3 is abundantly ex-

pressed in the PVN, in addition to other regions commonly

associated with stress and anxiety, including the bed nu-

cleus of the stria terminalis, lateral septum, periaqueductal

gray, and the dorsal raphe (FIGURE 6) (312). The CRF

receptor mediates neural responses to CRF and most re-

laxin 3-containing neurons in the NI coexpress this receptor

(506, 516). The concept that relaxin-3 modulates stress

responses through interactions with CRF was investigated

by ICV administration of CRF to rats. This caused activa-

tion of relaxin-3 containing neurons, and neurogenic stres-

sors increased relaxin-3 mRNA in the NI (516). Further-

more, in NI neurons, relaxin-3 occurs in presynaptic vesi-

cles. This is in accord with the concept that relaxin-3

released from nerve endings in the NI is involved in regula-

tion of stress responses. Stress also transiently increases re-

laxin-3 mRNA in the rat brain following a forced swim

stress paradigm (25). These effects were partly blocked by

pretreatment with the CRF

antagonist antalarmin. While

these studies demonstrate the inﬂuence of CRF and stress on

relaxin-3, it is not yet clear whether relaxin-3 acting via

RXFP3 inﬂuences the CRF system.

3. Role of the relaxin-3-RXFP3 system in feeding and

metabolism

In the rodent, RXFP3 is expressed in the periventricular

hypothalamic nucleus, PVN, and supraoptic nucleus (SON)

(FIGURE 6) (486, 487), suggesting that the relaxin-3-RXFP3

system has a role in neuroendocrine and metabolic control.

Human relaxin-3 given ICV to rats either in the early light

or early dark phase, transiently but signiﬁcantly increased

food intake (358). This effect did not result from increased

spontaneous activity or arousal. In addition, the orexigenic

effects of relaxin-3 are likely mediated by RXFP3, and not

RXFP1, since human relaxin-2 administration had no effect

(358).

The role of the PVN in this response was investigated using

acute and chronic human relaxin-3 infusions into the PVN,

both of which increased food intake (357). Human relax-

in-3 injection into the SON, arcuate nucleus (ARC), and

anterior preoptic area (APOA) also transiently increased

food intake. However, given that RXFP3 is not present in

the ARC or APOA, it is unclear how relaxin-3 exerts its

effects in these nuclei. Finally, ICV infusion of R3/I5 also

increased food intake, and this effect was blocked by pre-

administration of the antagonist R3(B⌬23–27)R/I5 (299).

ICV administration of R3(B⌬23–27)R/I5 alone has no ef-

fect on food intake (509), suggesting that under resting

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

453Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

conditions there is minimal tone in the relaxin-3-RXFP3

system.

Although relaxin-3 inﬂuences food intake, there is little ev-

idence to suggest that infusion of the peptide increases body

weight. In one study, chronic ICV human relaxin-3 did

increase body weight (219), but other studies have failed to

show weight changes following ICV or intrahypothalamic

injection of relaxin-3 (357, 358). In rats, chronic ICV

R3(B⌬23–27)R/I5 did not affect body weight (509). There

is also limited evidence to suggest that body weight is inﬂu-

enced by endogenous relaxin-3. Although relaxin-3 knock-

out mice were initially reported to have lower body weights,

this has not been replicated by other investigators (488,

509), suggesting that the decreased body weight may have

been due to differences in control animals, housing, or diet.

Alternatively, the chronic absence of relaxin-3 may be as-

sociated with compensation by other systems, whereas

acute infusions of relaxin-3 could transiently increase food

intake.

Several studies that investigated the effects of relaxin-3 in-

fusion on feeding also measured blood hormone levels.

Chronic ICV infusion of human relaxin-3 increased plasma

leptin and insulin levels (219). Similarly, increases in leptin

in ad libitum fed animals and TSH in ad libitum and pair fed

animals were observed with chronic infusion of human re-

laxin-3 into the PVN (357). These effects occurred in the

absence of changes in energy expenditure.

Studies of the roles of relaxin-3 in neuroendocrine function

and metabolism currently lack supporting mechanisms,

which prevents the generation of concrete conclusions.

More detailed studies are needed that examine the neural,

signaling, and gene transcription changes that lead to the

relaxin-3-induced feeding response. This may help to tease

out confounding effects on reward, motivation, and arousal

and determine whether there is a true feeding response to

relaxin-3 stimulation.

4. The relaxin-3-RXFP3 system in behavioral

activation and arousal

Much of the evidence for a role for relaxin-3 in behavioral

activation and arousal comes from the parallel study of the

neuroanatomy of relaxin-3 projections and sites of RXFP3

expression. Structures in the septohippocampal pathway of

rodents are heavily innervated by relaxin-3-positive projec-

tions from the NI (FIGURE 6). One of the major functions of

this pathway is the generation of hippocampal theta

rhythm, which has oscillations at 4–12 Hz. Theta rhythm is

controlled by pacemaker neurons of the medial septum

(MS) and is involved in behaviors such as vigilance, explo-

ration, orientation, navigation, locomotor control, and

working memory. The NI has an important role in theta

rhythm, and electrical stimulation of the nucleus causes

theta rhythm in the hippocampus. On the other hand, le-

sions of the NI disrupt theta rhythm initiated by stimulation

of the reticularis pontine oralis (390). RXFP3 signaling also

inﬂuences theta rhythm as in both anesthetized and con-

scious rats, RXFP3 modulates neuronal activity in the hip-

pocampus and MS to promote hippocampal theta rhythm

(330). In behavioral studies, blockade of RXFP3 in the MS

with R3(B⌬23–27)R/I5 caused impaired performance in a

paradigm that investigated theta rhythm-dependent spatial

working memory. R3(B⌬23–27)R/I5 produced dose-de-

pendent decreases in performance, which could be over-

come by coadministration of R3/I5 (330).

The relaxin-3-RXFP3 system also inﬂuences locomotor ac-

tivity in some models of rodent behavior. Chronic ICV in-

fusion of relaxin-3 had no effect on locomotor activity in

male Wistar rats (219), whereas ICV infusions of R3/I5

increased locomotor activity, and R3(B⌬23–27)R/I5 had

no effect (509). However, female relaxin-3 knockout mice

were hypoactive in several paradigms including the locomo-

tor cell, large open ﬁeld, Y maze, and novel object tests

(485). Whether these differences are due to gender or spe-

cies remains to be determined.

Serotonin (5-hydroxytryptamine, 5-HT) has well-estab-

lished roles in cognitive, emotional, and behavioral control

(reviewed in Ref. 106). Since the NI is located close to the

dorsal raphé, a region enriched in 5-HT neurons, studies

have been carried out examining the effects of 5-HT on

relaxin-3 expression (367) and showed that most relaxin-

3-containing neurons of the NI coexpress 5-HT

recep-

tors. Inhibition of 5-HT synthesis for 3 days increased re-

laxin-3 mRNA in the NI. More studies are needed to deter-

mine the relationship between changes in 5-HT levels and

relaxin-3 expression.

5. Role of relaxin-3-RXFP3 in ﬂuid homeostasis

Unlike the relaxin-RXFP1 system, the relaxin-3-RXFP3

system is not expected to inﬂuence ﬂuid homeostasis. How-

ever, administration of human relaxin-3 into the lateral

ventricle causes drinking in rats, probably mediated by ac-

tions on RXFP1 in the SFO (45). Recently, it was demon-

strated that ICV relaxin-3 causes dose-dependent neuronal

activation in the hypothalamus and several circumventricu-

lar organs including the SFO, SON, and PVN (405). While

there is evidence for an interaction between relaxin-3 and

RXFP1 both in vitro and in vivo, there is no evidence that

this occurs physiologically, or that relaxin-3 has actions on

RXFP3 located in the circumventricular organs.

Thus the relaxin-3-RXFP3 system has a role in feeding,

metabolism, stress, arousal, learning, and memory. While

many of the hypotheses that have been developed on the

basis of neuroanatomical data have been tested in rodent

models, it is possible that the observed phenotypes do not

reﬂect the primary functions of the relaxin-3-RXFP3 system

in humans. Identiﬁcation of the effector molecules down-

BATHGATE ET AL.

454 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

stream of RXFP3 and the development of alternative in vivo

models are important future steps in the validation of pu-

tative relaxin-3-RXFP3 functions.

D. INSL5 and RXFP4

RXFP4 (formerly GPCR142) was originally identiﬁed as a

receptor for relaxin-3 (313). However, the dramatically dif-

ferent expression patterns of relaxin-3 and RXFP4 sug-

gested that relaxin-3 was not the cognate ligand for RXFP4.

Furthermore, unlike relaxin-3, RXFP4 is less well con-

served and appears as a pseudogene in both rats and dogs,

suggesting that RXFP4 may be the receptor for a ligand

other than relaxin-3 in mammals. A related peptide, INSL5,

is also a pseudogene in rats and dogs, and its expression

pattern overlaps with RXFP4 leading to the suggestion that

INSL5 was the ligand for RXFP4. Subsequently, INSL5 was

demonstrated to be a high-afﬁnity ligand for RXFP4 (315),

and mRNA is present in a variety of human tissues in-

cluding brain, kidney, testis, thymus, placenta, prostate,

salivary gland, thyroid, and colon (315), many of which

also coexpress INSL5. Overall, the receptor-ligand co-

evolution, pharmacology, and similarities in expression

proﬁles strongly indicate that RXFP4 is the endogenous

receptor for INSL5.

Transgenic INSL5 knockout/LacZ knockout mice devel-

oped to investigate the physiology of INSL5 (260) showed

␤

-galactosidase staining of tissues that conﬁrmed the distri-

bution of INSL5, with staining in the colon to a single

dispersed cell population. Mice overexpressing the INSL5

gene under control of the metallothionein or insulin pro-

moters have been produced, but there are no reports of any

physiological or behavioral ﬁndings related to INSL5 func-

tion.

E. Cross-reactivity Between Relaxin Family

Peptides and RXFP Receptors

1. Relaxin activates RXFP2 and RXFP3 in addition to

RXFP1

While RXFP1 is the cognate receptor for relaxin, some species

relaxins (including human relaxin-2) can also bind to and ac-

tivate the INSL3 receptor RXFP2. Human relaxin-2 and por-

cine relaxin bind to RXFP2 with nanomolar afﬁnity and cause

cAMP accumulation (198, 199, 239, 301, 501). However,

while these relaxins bind to RXFP2, they do so with an

afﬁnity lower than INSL3, and lower than that observed for

their interaction with RXFP1, consistent with the desig-

nated relaxin-RXFP1 and INSL3-RXFP2 ligand-receptor

pairings. Importantly both mouse and rat relaxin are unable

to bind to or activate RXFP2 in vitro (45, 454), and this

ﬁnding has been conﬁrmed in vivo using transgenic animals

(65, 158, 274). This suggests that the human relaxin-2 in-

teraction with RXFP2 may be species speciﬁc (199).

While the original characterization of RXFP3 suggested a

unique relaxin-3-selective binding and activation proﬁle

(314), a recent study also identiﬁes binding and activation

of RXFP3 by relaxin (545). This may reﬂect the fact that

relaxin and INSL3 both retain homology within the domain

of relaxin-3 that dictates RXFP3 binding (299, 545) (see

sect. IIC). By examining a number of functional outputs

(ERK1/2 phosphorylation, cAMP inhibition, and NF

␬

and AP-1 reporter gene activation), distinct patterns of cel-

lular responses to relaxin-3, relaxin, and INSL3 were iden-

tiﬁed (545). Relaxin-3 activated the widest range of signal-

ing pathways whereas relaxin and INSL3 activated only

subsets of these (see sect. VIC). Detailed binding studies

subsequently revealed that relaxin and INSL3 bind to a

distinct site on RXFP3, whereas relaxin-3 competes for

binding to RXFP3 with the

125

I-R3/I5 chimeric peptide,

relaxin-3, relaxin, and INSL3 could all compete for RXFP3

binding with

125

I-relaxin (545). Thus not only does relaxin

bind to and activate RXFP3 (in addition to RXFP1 and

RXFP2), but RXFP3 displays ligand-biased signaling, with

the pattern of cellular responses generated by this receptor

being highly dependent upon the ligand and on the cellular

background.

2. Relaxin-3 activates RXFP1, RXFP2, and RXFP4 in

addition to RXFP3

Prior to the discovery of a unique receptor for relaxin-3, it

was deﬁned by its ability to bind to and activate RXFP1, but

with a lower afﬁnity than relaxin (46). This property, in

conjunction with the inability of relaxin-3 to bind human

RXFP2, was subsequently exploited to describe a second

binding site for relaxin-3 within the second extracellular

loop of RXFP1 (501). This two-site binding model has since

been shown to be a general characteristic of relaxin family

peptide binding to both RXFP1 and RXFP2 (199, 210) (see

sect. IIIB). Although relaxin-3 does not bind and activate

human RXFP2, it has been demonstrated to weakly bind to

and activate rat RXFP2 (453).

The identiﬁcation of RXFP3 as the cognate receptor for

relaxin-3 (314) also led to the subsequent deorphanization

of RXFP4 (the INSL5 receptor) (313), based on the high

degree of similarity between the two receptors and the abil-

ity of relaxin-3 to bind to and activate both receptors.

RXFP4 (human, monkey, bovine, porcine, and mouse) was

found to bind relaxin-3 with high afﬁnity (98). Binding of

relaxin-3 to RXFP4 also caused cellular responses similar to

RXFP3, with inhibition of AC, increased GTP

␥

S binding,

and increased intracellular calcium when RXFP4 is coex-

pressed with G

␣

(98, 313, 315).

3. Interaction between INSL5 and RXFP3

Although RXFP4 was initially deﬁned by its ability to bind

relaxin-3 (313), it was subsequently reclassiﬁed as the cog-

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455Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

nate receptor for INSL5 (315). Based on the existing cross-

reactivity between many of the relaxin family peptides and

their receptors, the activity of INSL5 at all the relaxin fam-

ily receptors was subsequently assessed. Although INSL5

does not bind to RXFP1 or RXFP2, it does bind to RXFP3,

albeit with lower afﬁnity than relaxin-3 (315). However,

INSL5 is an antagonist of responses to relaxin-3 (315). It is

not currently known if the peptide is a neutral antagonist,

inverse agonist, or biased-ligand. The lack of binding afﬁn-

ity of INSL5 at RXFP1 or RXFP2 has been exploited in the

generation of the chimeric peptide (R3/I5) that is routinely

used to distinguish RXFP3 and RXFP4 binding sites from

those for RXFP1.

VIII. POTENTIAL THERAPEUTIC

APPLICATIONS FOR DRUGS

TARGETING RXFP RECEPTORS

A. RXFP1: A Promising Therapeutic Target

The long-established effects of relaxin on the adaptive

changes to pregnancy in women, in addition to its emerging

role in cardiovascular homeostasis, are the main targets of

current efforts in relaxin-RXFP1-based therapeutics. In ad-

dition, other physiological consequences of relaxin-RXFP1

interaction constitute additional, appealing therapeutic tar-

gets for future investigation.

1. Promotion of cervical ripening by relaxin

There is substantial experimental evidence for a role for

relaxin and RXFP1 in cervical ripening and parturition

(47). Based on this evidence, relaxin has been tested for its

ability to cause similar changes in women. Despite evidence

for cervical ripening in humans following application of

porcine relaxin (152, 339), there has been no demonstra-

tion of efﬁcacy for human relaxin-2. Studies failed to show

efﬁcacy of topically applied human relaxin-2, although this

may have been confounded by its low penetration into cer-

vical tissue (52). Thus, despite demonstrated efﬁcacy in pro-

moting cervical ripening for human relaxin-2 application to

the common marmoset monkey (481), a similar role in

women is still uncertain. Despite this uncertainty, relaxin

may yet prove to be a useful adjunct in parturition, with

studies suggesting that both maternal and fetal relaxin may

contribute to rupture of the fetal membrane, particularly in

instances of premature birth (523).

2. Facilitation of implantation by relaxin

A principal role of relaxin, initially described in pregnant

guinea pigs (220), involves the preparation of the birth ca-

nal for parturition. Although the evidence for the hormonal

actions of relaxin is clear in rodents and some mammals, it

is of reduced importance in higher species. In women, max-

imum circulating levels of relaxin occur during the ﬁrst

trimester of pregnancy (51, 138, 497), which is in direct

contrast to other species, such as rats (14, 108, 472) and

pigs (15, 53), where maximum circulating levels of relaxin

occur during the third trimester (393).

Due to the timing of this peak of circulating relaxin in

humans, the role of relaxin and RXFP1 in pregnancy is

more likely associated with ﬁrst trimester physiological

events such as embryo implantation. Indeed, relaxin can

interact with RXFP1 expressed on endometrial and stromal

cells, inducing cAMP accumulation and the production of

vascular endothelial growth factor (VEGF) to cause decidu-

alization and preparation of the endometrium for implan-

tation (529, 540). More speciﬁcally, the identiﬁcation of the

location and regulation of RXFP1 expression in the human

endometrium has supported a role for relaxin in the implan-

tation process: relaxin receptor binding in the uterus is dra-

matically increased just prior to, and peaks immediately

after, ovulation (67). This mirrored earlier studies monitor-

ing plasma relaxin levels in conceptive and nonconceptive

female cycles (497). Relaxin also promotes follicle develop-

ment in the human, as relaxin treatment of human ovarian

cortical tissue caused a signiﬁcant increase in secondary

follicles and a decrease in primordial follicles, in addition to

causing increased vascularization of endometrial tissue and

thickening of the endometrium in preparation for implan-

tation (474). In the macaque, relaxin treatment has been

shown to assist in vitro fertilization due to improvements in

endometrial thickening and embryo implantation (214).

Thus the relaxin-RXFP1 system is an attractive candidate to

assist in implantation.

3. Relaxin treatment for the prevention of

preeclampsia

Preeclampsia commonly occurs during pregnancy and de-

scribes the reduced blood ﬂow to the placenta leading to

systemic vasoconstriction, hypertension, renal dysfunction,

and ultimately multiple organ failure. Currently, symptoms

are usually relieved by preterm delivery of the fetus. Recent

studies suggest that the vasoconstriction that deﬁnes pre-

eclampsia may result from inhibition of factors such as

VEGF (308, 353, 593). There is preclinical evidence that

suggests that relaxin may be a useful treatment for pre-

eclampsia. In the clinical trials for the treatment of sclero-

derma, relaxin administration was associated with menor-

rhagia and metrorrhagia in a large number of women (540),

consistent with angiogenic and vasodilatory effects, and

likely due to increased VEGF expression and secretion in

human endometrial cells (408, 540). In the same study,

relaxin administration also decreased systolic and diastolic

blood pressure, consistent with a vasodilatory effect, and

improved renal function, suggesting that relaxin treatment

may relieve maternal symptoms of preeclampsia (538). Re-

laxin is currently being assessed as a treatment for pre-

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456 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

eclampsia in a randomized, double-blind, placebo-con-

trolled clinical trial (538).

4. Relaxin as a novel treatment for congestive heart

failure

Relaxin is now emerging as a potential novel treatment for

acute congestive heart failure (526). The well-established

effects associated with relaxin during pregnancy including

increased cardiac output, blood vessel compliance, and re-

nal blood ﬂow (265), together with studies that show pos-

itive inotropic and chronotropic effects on the heart (127,

271) and the observation that relaxin plasma levels increase

in heart failure (132), suggested that it may have beneﬁcial

effects (reviewed in Ref. 135). In congestive heart failure,

the heart fails to pump enough blood to perfuse vital or-

gans. It is often associated with increased blood pressure,

and mortality is highly correlated with declining renal func-

tion and increased activity of the sympathetic nervous sys-

tem. Thus the ability of relaxin to induce vasodilation and

improve renal function in pregnancy, which was reﬂected in

the previous clinical trials for scleroderma (149, 527), has

suggested that the peptide may prove useful in the treatment

of heart failure.

In a phase I clinical trial examining the safety and dose-

response relationship of short-term infusion of relaxin on

hemodynamics in patients with congestive heart failure, ad-

ministration resulted in increased cardiac output and de-

creased systemic vascular resistance and pulmonary capil-

lary wedge pressure (133). Phase II clinical trials have since

been completed; the multicenter, placebo-controlled, dou-

ble-blind, randomized, international trial (Pre-RELAX-

AHF) assessed the effects of intravenous relaxin adminis-

tration in patients with acute heart failure (526). Despite no

signiﬁcant improvement in renal function, relaxin was as-

sociated with a small but signiﬁcant improvement in self-

reported breathlessness, and there was improvement in the

incidence of cardiovascular death and readmission (526).

Administration of the peptide was also associated with a

trend towards clinical improvement of heart failure and a

reduced requirement for diuretics. These studies again sup-

ported the safety of relaxin administration as a therapeutic

and conﬁrmed the vasodilatory effects of relaxin in patients

with heart failure (133, 526). Thus there is promising scope

for the use of relaxin to improve patient outcomes following

congestive heart failure, especially in conditions of cardiac

ﬁbrosis. The peptide is currently undergoing phase III clin-

ical trials (RELAX-AHF-1) (421).

5. Antihypertensive effects of relaxin

Relaxin also exerts a local inﬂuence on the circulation by

increasing vasodilation and passive compliance: both re-

laxin and RXFP1 mRNA have been identiﬁed in thoracic

aortas, small renal arteries, and mesenteric arteries in both

rats and mice (388). Several studies suggest that relaxin

exerts these local effects through the synthesis and produc-

tion of NO, particularly in situations of natural or induced

myocardial infarction (33, 154, 343, 346). This is sup-

ported by studies in the relaxin-knockout mouse, which

demonstrated blunted NO-dependent myogenic reactivity

(388). Relaxin also induces vasodilation in human systemic

resistance arteries in vitro (170). The vasodilation of gluteal

resistance arteries was endothelium dependent (170) and

substantially inhibited in arteries from patients being

treated with ACE inhibitors. Inhibition of cyclooxygenase

(indomethacin) or soluble guanylyl cyclase (ODQ-4M) also

greatly reduced the vasodilator response to relaxin (171). In

addition to these direct vasodilator effects of relaxin, recent

evidence also suggests that relaxin treatment reverses the

arterial remodeling that is a characteristic feature in spon-

taneously hypertensive rats with age and improves large

artery compliance (568). Thus the actions of relaxin on

connective tissue metabolism may also contribute to the

long-lasting effects of a relatively short period of relaxin

treatment.

In clinical studies assessing the potential efﬁcacy of relaxin

in the treatment of scleroderma, the peptide caused a signif-

icant decrease in both systolic and diastolic blood pressure,

that was consistent with vasodilation (528). Clinical trials

investigating relaxin as a treatment for congestive heart

failure also demonstrated decreased blood pressure (134,

526).

6. Antiﬁbrotic effects of relaxin

One of the physiological effects of relaxin with broad ther-

apeutic potential is its effects on connective tissue regula-

tion and ﬁbrosis. From a therapeutic viewpoint, relaxin

decreases excess collagen deposition in ﬁbrotic lesions, with

a conservation of endogenous connective tissue structure

(181, 542). Relaxin also exerts anti-inﬂammatory effects,

can inhibit the activation of human neutrophils by pro-

inﬂammatory agents (345), and prevents histamine and

granule release by activated basophils (29) and mast cells

(344). In an animal model of allergic airway disease, there

was increased collagen deposition and decreased expression

of MMP9 in relaxin-knockout animals compared with con-

trols (371), although these studies demonstrated receptor

involvement only in airway ﬁbrosis homeostasis and not

during the inﬂammation-induced ﬁbrosis associated with

chronic allergic airway disease (441). Taken together, re-

laxin is protective during the allergic inﬂammatory re-

sponse. In rodent models of angiogenesis and wound heal-

ing, relaxin caused increased vascularization and neoangio-

genesis of ischemic wound sites (309, 541) but had no effect

on cytokine expression in cells taken from nonwound sites

(541), indicating promising speciﬁcity for therapeutic appli-

cations.

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457Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

7. Roles of relaxin in cancer

As discussed in the previous section, relaxin appears to be

recruited in many types of cancer cells as an endogenous

factor for tissue remodeling. In particular, relaxin is associ-

ated with prostate cancer progression, and increased ex-

pression of relaxin (but not RXFP1) occurs in prostate car-

cinomas and prostate cancer cell lines (157, 532, 549). Im-

portantly, downregulation of either relaxin or RXFP1

caused signiﬁcant inhibition of growth (549) and invasive-

ness, in addition to increased apoptosis (157) in rodent

prostate cancer models. Furthermore, lentiviral driven ex-

pression of an RXPF1 peptide antagonist within prostate

tumors in a mouse model of human prostate cancer sup-

pressed tumour growth, whereas control lentiviruses had

no effect (480). Hence, drugs that block relaxin action in

prostate cancer may be an effective means to slow prostate

cancer progression. Furthermore, relaxin is associated with

increased invasiveness of endometrial (273), breast (59,

60), and thyroid (226) carcinomas, suggesting that agents

blocking relaxin action may be used for the possible treat-

ment of multiple cancers.

B. RXFP2 and Its Potential as a Therapeutic

Target

INSL3 and its cognate receptor RXFP2 are clearly key reg-

ulators of the reproductive system in both male and female

mammals. Apart from reproductive tissues, thyroid and

bone are the only other major tissues to express the peptide

and receptor. There is also good evidence for RXFP2 ex-

pression in brain, but no studies to date have provided

evidence for the expression of INSL3 in either laboratory

animals or humans.

1. RXFP2 as a target for modulation of fertility

The major physiological effects of the INSL3/RXFP2 sys-

tem are on the reproductive system: lack of either INSL3 or

RXFP2 in males results in cryptorchidism (379, 596), and

overexpression of INSL3 in females causes ovarian descent

and bilateral inguinal hernia (490). INSL3 stimulation of

RXFP2 in germ cells causes meiotic progression of arrested

oocytes in preovulatory follicles and suppresses male germ

cell apoptosis (276). However, it is likely that targeting

RXFP2 in the gubernaculum in utero would not be a useful

therapeutic strategy, and indeed, most male babies with

cryptorchidism are treated surgically. However, modula-

tion of fertility is an attractive therapeutic option for the

INSL3-RXFP2 system. Activation of RXFP2 expressed on

germ cells suppresses male germ cell apoptosis (276),

whereas injection into rat testis of an INSL3-based antago-

nist targeting RXFP2 led to a signiﬁcant decrease in testis

weight, consistent with inhibition of spermatogenesis

(121). Furthermore, in a study examining a male hormonal

contraceptive regime (involving administration of testoster-

one with a progestagen over 6 mo), those patients with

persistent sperm production had higher serum INSL3 levels,

suggesting again that the INSL3-RXFP2 system plays a role

in spermatogenesis (8). Thus it is tempting to speculate that

an INSL3-based antagonist targeting RXFP2 could be an

effective male contraceptive agent. Additionally, INSL3 in

females is associated with oocyte maturation and follicular

development, and therefore, INSL3 may be effective for

certain types of female infertility.

2. Other potential therapeutic applications of RXFP2

Interestingly, in a manner similar to relaxin, the INSL3-

RXFP2 system has been shown to be present in human

prostate carcinoma cell lines and human thyroid carcino-

mas, and treatment of these cell lines with INSL3 caused

increased tumor cell motility (225, 286, 429). Another

study suggests that INSL3 and RXFP2 may also have a role

in osteoporosis (162). Thus there may be further therapeu-

tic potential for this ligand-receptor system in pathologies

including cancer and osteoporosis in the future.

C. RXFP3 as a Target for the Control of

Anxiety and Food Intake

The development of therapeutics based on the relaxin-3-

RXFP3 system is currently at the proof of concept stage.

Much of what we know about the functions of the relaxin-

3-RXFP3 system is based on a small number of anatomical,

pharmacological, and genetic interference studies in rodent

models. In addition, expression of RXFP3 has only been

detected in human tissue by RT-PCR. However, relaxin-3

has recently been detected in the macaque by both in situ

hybridization and immunohistochemistry, suggesting that

the relaxin-3-RXFP3 system may also be present in humans

(332).

Nonetheless, there is substantial interest in the therapeutic po-

tential of the relaxin-3-RXFP3 system. In general, GPCRs are

attractive drug targets due to cell surface expression and

thus good drug accessibility (particularly hydrophilic

drugs) and selectivity due to the distribution of expression

in different tissues and cell types. In addition, GPCRs are

often responsible for maintaining physiological homeosta-

sis by modulating the actions of neuropeptides and neu-

rotransmitters. As the receptor of a neuropeptide, RXFP3 is

particularly appealing. Many neuropeptides are colocated

with neurotransmitters and act synergistically with them to

modulate their actions. Thus neuropeptide receptors pro-

vide a unique opportunity to inﬂuence systems with less

dramatic consequences than interfering with amino acid

transmitters themselves. Since relaxin-3 is produced in

GABAergic neurons of the nucleus incertus, it likely acts in

concert with GABA signaling. In this way, it may be possi-

ble to ﬁne-tune the GABAergic system by targeting RXFP3.

This section focuses on the physiological functions of the

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458 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

relaxin-3 and RXFP3 system and how this may be manip-

ulated for therapeutic purposes.

1. The relaxin-3-RXFP3 system as a target for

antianxiety drugs

The relaxin-3-RXFP3 system is clearly implicated in the

regulation of the stress response. As previously mentioned

(see sect. VIIC), the great majority of relaxin 3-containing

neurons in the NI express CRF-R1 (422). Additionally, the

principal site of CRF production in the brain, the PVN, also

expresses RXFP3 (516). In rats, relaxin-3 neurons are acti-

vated by CRF administration, and expression of relaxin-3

in the NI is increased in response to swim stress (516).

Similarly, repeat forced swim has been demonstrated to

increase relaxin-3 expression, while pretreatment with the

CRF-RI antagonist antalarmin attenuated the response

(25). Currently, the physiological response to elevated re-

laxin-3 expression, the signals necessary to stimulate relax-

in-3 release, and the inﬂuence of stress on RXFP3 signaling

are unknown. From neuroanatomical data, it appears that

RXFP3 could be part of a feedback system that regulates

continued CRF release following stress; however, this has

yet to be demonstrated experimentally. While these ques-

tions remain, the potential therapeutic application of

RXFP3 modulators for the stress system remains unre-

alised.

2. Modulation of feeding by the relaxin-3-RXFP3

system

A role for the relaxin-3-RXFP3 system in energy homeosta-

sis has been suggested by studies of peptide infusion into the

brain, which demonstrate effects of relaxin-3 on body

weight, food intake, and plasma hormone levels. Chronic

ICV human relaxin-3 has been reported to increase body

weight (219), although other studies showed no change in

body weight after ICV or intrahypothalamic nuclei injec-

tion of relaxin-3 (357, 358).

Although the evidence for a role for relaxin-3 in body

weight control is equivocal, there are indications that the

peptide may inﬂuence food intake. Initially this was

prompted by the identiﬁcation of RXFP3 expression in the

hypothalamus (312, 329, 508). In this context, human re-

laxin-3 administered ICV to rats either in the early light or

early dark phase transiently increased food intake (219),

and acute or chronic human relaxin-3 infusion into the

PVN also increased food intake (357). Other hypothalamic

nuclei, such as the SON, ARC, and APOA, may also be

involved, as food intake was transiently increased by hu-

man relaxin-3 injection into these regions. However, given

that RXFP3 is not present in the ARC or APOA, it is unclear

how relaxin-3 exerts its effects at these sites. However, ICV

infusion of the RXFP3 agonist R3/I5 also increased food

intake, and this effect was blocked by preadministration of

the RXFP3 antagonist R3(B⌬23–27)R/I5 (299). Interest-

ingly, chronic ICV administration of R3(B⌬23–27)R/I5 had

no effect on feeding, but signiﬁcantly increased body weight

(509). Studies in relaxin-3 knockout mice have provided

equivocal data for a role of the relaxin-3-RXFP3 system in

body weight control, with one study describing a smaller

leaner phenotype (509) whereas another (using the same

knockout mice) failed to demonstrate any differences (485).

Several studies that investigated effects of relaxin-3 infusion

on feeding also measured blood hormone levels. Chronic

ICV human relaxin-3 increased plasma leptin and insulin

levels (219), and chronic infusion of human relaxin-3 into

the PVN increased leptin and decreased TSH in ad libitum-

fed rats (357). These effects were observed in the absence of

changes in energy expenditure. The levels of plasma endo-

crine factors were also measured in the chronic RXFP3

agonist and antagonist studies conducted in rats. Chronic

administration of the RXFP3 agonist R3/I5 signiﬁcantly

increased insulin, leptin, adiponectin, testosterone, and an-

giotensinogen levels and decreased growth hormone levels

(509). Infusion of the RXFP3 antagonist R3(B⌬23–27)R/I5

also decreased growth hormone levels. Relaxin-3 knockout

mice at 10–15 wk have reduced leptin and at 30–35 wk

reduced insulin and leptin (509).

Given the lack of consistent evidence and the numerous

hypothalamic neurotransmitter and peptidergic systems

that regulate energy balance, it is difﬁcult to assess the po-

tential of the relaxin-3-RXFP3 system as a drug target. Al-

though relaxin-3 administration increases food intake in

rodents, this effect is also seen in regions lacking RXFP3,

questioning its speciﬁcity. Infusions of R3(B⌬23–27)R/I5

are only effective in inhibiting relaxin-3 stimulated, but not

basal food intake. The results suggest that the complex

regulation of energy homeostasis by the CNS is difﬁcult to

override by manipulation of a single neuropeptide/receptor

system. Nevertheless, evidence from rodent models suggests

that RXFP3 agonists and antagonists may inﬂuence energy

homeostasis in a manner that may be useful for the treat-

ment of human metabolic conditions such as anorexia, ca-

chexia, and obesity.

IX. MAJOR UNANSWERED QUESTIONS

AND FUTURE DIRECTIONS

A. Signaling Paradigms

1. Role of signalosomes in relaxin family peptide

receptor signaling

The recent demonstration that the expression of RXFP1

receptors in cells induces the formation of signalosomes

that are constitutively active and highly sensitive to attomo-

lar concentrations of relaxin adds a new dimension to re-

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459Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

laxin signaling (see sect. VIA). A particularly interesting

feature of the RXFP1-signalosome is that attomolar con-

centrations of relaxin stimulate small increases in cAMP,

whereas nanomolar concentrations preferentially activate

the classical pathways involving G

␣

, and G

␣

(FIG-

URE 7A). Importantly, these pathways appear to act quite

independently, and there is no effect of inhibitors of classi-

cal pathway-speciﬁc proteins (including G

␣

i/o

, PI3K and

PKC) on cAMP generated in response to subpicomolar con-

centrations of relaxin. Equally there is no effect of inhibition of

signalosome-speciﬁc proteins (including AC2, AKAP79, and

␤

-arrestin 2) upon classical relaxin cAMP signaling (FIGURE

7A). Furthermore, higher concentrations of relaxin are as-

sociated with dissociation of the signalosome, which is fol-

lowed by activation of the classical signaling pathways. The

pathways also generate cAMP in quite distinct regions of

the cell. Thus signalosome-speciﬁc AC2 is known to be

preferentially excluded from lipid-rich domains (565),

whereas activation of the G

␣

pathway with nanomolar

relaxin concentrations depends on lipid-rich domains in

HEK293 cells (204). Since AC2 expression occurs predom-

inantly in brain, lung, skeletal muscle, heart, and uterine

myometrium (438, 565), it is likely that there are both tis-

sues that display RXFP1 signalosome signaling and those

that do not which may help to determine the physiological

role of signalosome-localized RXFP1. It is interesting to

note that previous studies of the regulation of RXFP1 ex-

pression by estrogens showed marked differences between

tissues, and receptor expression was markedly upregulated

in uterus but not affected in brain or heart (515). The scaf-

folding of AC2 to RXFP1 is highly dependent on AKAP79

that is known to scaffold many other proteins including

other GPCRs, PKC, several ion channels, and a number of

AC isoforms (see Ref. 196). The activity of AC isoforms

may be variably inﬂuenced by interaction with AKAP79

and AC2 activity is inhibited (142), although this appears to

be primarily offset in RXFP1 signalosomes by the scaffold-

ing of RXFP1 and AC2. The regulatory complex of RXFP1,

␤

-arrestin-2, and PDE4D3 also has some unusual features

that call into question some of the current paradigms con-

cerning the role of

␤

-arrestins in receptor desensitization

and internalization. While the

␤

-adrenoceptor is phos-

phorylated by GRK-2 following receptor activation and

subsequently becomes a substrate for

␤

-arrestin-2 which

triggers formation of clathrin-coated vesicles and internal-

ization (for review, see Ref. 328),

␤

-arrestin-2 clearly has a

different role with RXFP1. The interaction between RXFP1

and

␤

-arrestin-2 is constitutive and does not involve recep-

tor activation or phosphorylation and does not appear to be

involved in desensitization (201). Although this interaction

involves S704 of RXFP1 (201), this residue does not appear

to be phosphorylated, and indeed, exposure of RXFP1 to

higher concentrations of relaxin fails to cause phosphory-

lation, desensitization, or internalization of the receptor

(87, 514). It seems likely that in this case

␤

-arrestin-2 acts

solely as a scaffold for the formation of the regulatory com-

plex and has no role in receptor desensitization or internal-

ization.

It is interesting to speculate whether the constitutive activity

of the RXFP1 signalosome complex provides signaling that

regulates expression of proteins necessary for signalosome

formation or indeed the conventional relaxin signaling

pathways. It will also be of interest to determine whether it

is the speciﬁc activation of lipid raft-based signaling path-

ways that provides the stimulus for the dissociation of the

RXFP1 signalosomes. More overt physiological applica-

tions of this highly sensitive, concentration-dependent sig-

nalosome are not yet clear. However, it is well recognized in

humans that relaxin has a role during embryo implantation

where it is required to exert its effects over an extended

period of time, exempliﬁed by endometrial decidualization

which is crucial for embryo implantation and the mainte-

nance of pregnancy. It has been demonstrated that endome-

trial decidualization is dependent on both circulating and

locally produced relaxin (146, 214, 409, 540) and requires

the maintained elevation of cAMP (reviewed in Ref. 529).

Thus signalosome relaxin signaling may provide a mecha-

nism that provides a physiological, perhaps homeostatic,

role for the low concentrations of peptide present in the

circulation, and thus may be linked to some of the long-

term physiological effects. Future research, perhaps utiliz-

ing relaxin knockout models or single-cell models of phys-

iologically relevant targets (i.e., atrial cardiomyocytes, en-

dometrial cells, ﬁbroblasts, or neurons), will be required to

fully elucidate the downstream consequences and physio-

logical signiﬁcance of this ultrasensitive cAMP signaling

pathway.

2. Role of LDLa modules in RXFP1 and RXFP2

signaling

Relaxin family peptides interact with RXFP1 and RXFP2 in

a complex manner (see sect. VI, Aand 〉). Not only do the

peptides bind to the receptors in the LRR region and in

ECL2, but also activation of signaling requires the presence

of an intact LDLa module at the NH

terminus. A splice

variant of RXFP2 that lacks the LDLa module or an engi-

neered RXFP1 equivalent has binding properties similar to

the intact receptor but do not produce a cAMP response to

either INSL3 or relaxin, respectively (456). Also, the addi-

tion of a soluble form of RXFP1 LDLa to cells expressing

RXFP1 inhibits the cAMP response to relaxin. Thus the

activation of the cAMP signaling pathway by relaxin in-

volves at least three events: high-afﬁnity binding to the LRR

region, binding to a secondary site on ECL2, and the in-

volvement of the LDLa module. The precise mechanism

whereby the LDLa module activates signaling is not known,

although several features of the region are essential for ac-

tivity. The RXFP1 LDLa has three disulﬁde bonds formed

by six conserved cysteines and mutations of these bonds

cause loss of the cAMP response. These three disulﬁde

bonds are thought to provide the structural integrity neces-

BATHGATE ET AL.

460 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

sary for four acidic residues (N26, D30, D36, and E37) to

form a correctly folded cage that ligates a Ca

2⫹

essential for

activity (231). However, other speciﬁc residues are also nec-

essary for activity, since swapping of the RXFP1 LDLa for

the LB2 domain of the LDLR that has high structural and

sequence similarity does not produce a functional receptor.

Site-directed mutagenesis of RXFP1 has identiﬁed L7, Y9,

L22, and L23 as residues that inﬂuence the activity of the

LDLa region, and mutations of these residues generate re-

ceptors that display reduced potency in response to relaxin.

Some gain-of-function studies have been completed that

indicate that substitution of the ﬁrst nine residues of LB2

with those of the RXFP1 LDLa restores some cAMP re-

sponsiveness; however, the proﬁle required to restore full

activity has yet to be determined.

All of the current studies have examined whether particular

structural features of the LDLa module are associated with

cAMP production, but perhaps given the pleiotropic nature

of RXFP1 signaling, it would also be worth examining if all

signaling pathways are affected. Conceivably the confor-

mations of RXFP1 associated with cAMP signaling may

require the LDLa contribution but could differ from con-

formations that result in the activation of other pathways.

Several models have been proposed to explain the role of

the LDLa module in signaling. One involves a receptor

monomer with the LDLa module interacting with the TM

regions of the receptor to induce conformations that pro-

mote efﬁcient interaction with G proteins and signaling. In

this and alternative models, the LDLa acts as a tethered

ligand. An additional model suggests that the LDLa may

have a role in the formation of receptor dimers and in this

case interacts with the dimerization partner. Both models at

present lack substantial evidence to support or challenge

their credibility. What is known is that the LDLa modules

on their own or in combination with an LDLa-less receptor

do not signal, so the link between the LDLa module and the

receptor is essential.

3. Dimerization of relaxin family peptide receptors:

does it occur and what is the functional role?

There is evidence to suggest that both RXFP1 (282, 511)

and RXFP2 (510) form homodimers and heterodimers

(510). The BRET signal is saturable but is not affected by

the addition of the cognate ligand (human relaxin-2 for

RXFP1; INSL3 for RXFP2). It is likely that dimerization

primarily involves the 7-TM region of RXFP2 since a con-

struct (RXFP2–TM1–7–GFP

) that lacks the extracellular

domain of the receptor and hence the primary ligand bind-

ing site still forms dimers with RXFP2-RLuc. It has been

suggested that dimer formation is necessary for signal trans-

duction with ligand binding occurring at the LRR region of

one dimer partner followed by interaction of the bound

ligand with the ECL2 of the second partner and initiation of

signaling (510). However, at present, there are no experi-

ments with two inactive mutant receptor dimer partners

that show complementation and demonstrate rescue of

function by dimerization to support this concept. It has also

been suggested that dimer formation may explain apparent

negative cooperativity observed with both RXFP1 and

RXFP2. With both receptors, the rate of dissociation fol-

lowing incubation of cells expressing either receptor with

either radiolabeled human relaxin-2 or INSL3 was deter-

mined after allowing the system to equilibrate and then

undergo inﬁnite dilution in the absence or presence of un-

labeled ligand. The presence of unlabeled ligand was asso-

ciated with modest increases in the rate of dissociation,

suggesting negative cooperativity where the binding of li-

gand to each receptor is associated with a decrease in afﬁn-

ity of the remaining unoccupied receptors. It should be

borne in mind, however, that such behavior does not nec-

essarily reﬂect the formation of dimers (93), and studies are

required utilizing receptors that retain function but are un-

able to form dimers or on receptors expressed in model

phospholipid bilayers that allow examination of their func-

tional characteristics when in monomeric form (548, 559).

4. Bell-shaped concentration-response relationships:

explanation and relevance to clinical actions

A commonly observed characteristic of concentration-re-

sponse curves to relaxin mediated by RXFP1 is that they are

bell-shaped. These include cAMP responses in HEK293

cells transfected with RXFP1 (198); endometrial glandular

epithelial cells (96); increases in L-type Ca

2⫹

current in

rabbit sinoatrial node cells (206); MMP1 expression in hu-

man lung ﬁbroblasts (542); changes in glomerular ﬁltration

rate, renal plasma ﬂow, renal vascular resistance, and

plasma osmolality (112); as well as effects on dyspnea and

survival in the phase II clinical trial of relaxin in cardiac

failure (526). Similar ﬁndings have been reported for

RXFP2 for cAMP accumulation (198) and negative coop-

erativity (510).

Although the phenomenon of bell-shaped concentration-

response curves has been well described, there are no clear

explanations, even though the clinical ﬁndings suggest that

a better understanding of the process will be necessary to

gain the optimum therapeutic beneﬁt from relaxin treat-

ment. One mechanism that has been suggested to explain

the curves is negative cooperativity associated with receptor

dimerization (510, 511). This would argue that increases in

ligand concentration and receptor occupancy would reduce

the afﬁnity of the receptor for further interactions. How-

ever, one might expect in these circumstances that RXFP1

and RXFP2, receptors that both display negative coopera-

tivity, would exhibit bell-shaped concentration-response

curves. However, this does not appear to be the case as

cAMP accumulation in response to activation of RXFP1 at

3 or 30 min or RXFP2 at 30 min display bell-shaped curves,

whereas RXFP2 at 3 min does not (198). It could be argued

that the RXFP2 system at 3 min is not in equilibrium and

the effect of increasing receptor occupation balances out the

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

461Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

reduced receptor afﬁnity. However, this does not convinc-

ingly explain the difference between RXFP1 and RXFP2 at

the same time point. The different patterns are unlikely to

be related to a different time course of receptor coupling to

G proteins as this occurs rapidly after receptor activation

(198). Alternatively, the decrease in response with higher

concentrations of peptide could be due to desensitization

and/or internalization of the receptor. For RXFP1, this is an

unlikely explanation. Even in early functional experiments

examining relaxin responses in target tissues such as the

heart, the maximum responses were maintained for over 6 h

even with regular washing of the tissue (514), and more

recent studies showed no signiﬁcant receptor phosphoryla-

tion,

␤

-arrestin recruitment, or internalization following

exposure to ligand (87). Similar ﬁndings have been reported

in cell-population and single-cell studies (196). Even in

studies where desensitization of RXFP1 to overexpression

␤

-arrestin-2 has been demonstrated, the effects are rela-

tively small and associated with a small degree of internal-

ization (281). Recent studies from our own lab directly

examining the interaction of RXFP1 and

␤

-arrestin-1 or

␤

-arrestin-2 using BRET provided no support for interac-

tion between these proteins (Kocan, unpublished observa-

tions). On balance, it seems unlikely that internalization

and desensitization can account for the phenomenon. An-

other possibility reﬂects the multiple G proteins known to

interact with RXFP1 and RXFP2. Both receptors interact

with G

␣

and G

␣

, and in addition, RXFP1 also recruits

␣

. If the efﬁciency of coupling of G

␣

and G

␣

were

high, then the usual concentration-response curve would be

generated with low concentrations of ligand. If this is fol-

lowed however, by lower efﬁciency coupling to G

␣

which inhibits AC, then inhibition of cAMP production

could occur with higher concentrations of peptide. This,

however, cannot explain the lack of a bell-shaped curve for

RXFP2 stimulation at 3 min, and in addition, this type of

behavior would be unlikely for signaling pathways that do

not involve activation of AC. Finally, the effect could be due

to interaction between the numerous signaling pathways

activated by relaxin family peptides. For instance, another

G protein-coupled receptor, the

␤

-AR, shows clear evi-

dence of interaction between cAMP and MAPK signaling.

The

␤

-AR, like RXFP1 and RXFP2, activates multiple sig-

naling pathways, and one striking ﬁnding is that an antag-

onist of cAMP signaling is a strong activator of p38 MAPK

signaling (449). However, while the cAMP response to ago-

nists is increased with increased receptor expression, the

p38 MAPK response is almost completely abolished. This

suggested that increased cAMP generation associated with

increased receptor expression might be inhibiting p38

MAPK activity, and this was supported by inhibition of the

MAPK response by a cell-permeable cAMP analog (449).

As indicated earlier (see sect. VIA), both RXFP1 and

RXFP2 couple to G

␣

to increase cAMP and to G

␣

negatively modulate this effect (198). Only RXFP1 couples

to G

␣

to cause a further surge of cAMP by releasing G

␤␥

subunits that activate PI3K to promote PKC-

␨

transloca-

tion to the plasma membrane where it stimulates AC5

(198, 204, 382, 383). The translocation step is believed

to be responsible for the delay in the G

␣

-mediated surge

in cAMP accumulation. However, the G protein activa-

tion and release of G

␤␥

occurs rapidly after receptor

activation (198), and in addition to effects on PI3K, G

␤␥

subunits can also directly inﬂuence AC activity (200).

Thus the different pattern of appearance of G

␤␥

subunits

following activation of RXFP1 and RXFP2 could inﬂu-

ence the concentration-response relationships observed

at early stages following receptor activation.

5. Ligand-directed signaling bias: a common

phenomenon in relaxin family peptide receptors or

an idiosyncrasy of RXFP3?

Ligand-directed signaling bias describes the selective stabi-

lization of particular receptor conformations by ligands re-

sulting in selective activation of downstream signal trans-

duction pathways (22, 151, 277, 278). Although human

relaxin-3 or human relaxin-3 〉-chains were originally

thought to be the only ligands that interacted with RXFP3,

there is now evidence that several relaxin peptides interact

with RXFP3 to activate distinct signaling proﬁles through

different, although sometimes overlapping pathways (545).

Recently, these studies have been extended to examine the

speciﬁc signaling pathways activated by human relaxin-2

and human relaxin-3 and also to examine whether the

RXFP3 antagonist R3(B⌬23–27)R/I5 displayed signaling

bias. Human relaxin-2 was found to activate p38MAPK

and ERK1/2 with lower efﬁcacy than human relaxin-3, but

both peptides have similar efﬁcacy for JNK1/2/3 phosphor-

ylation and the responses are PTX sensitive. In contrast,

R3(B⌬23–27)R/I5 activated ERK1/2 in a PTX-sensitive

manner, p38MAPK in a PTX-insensitive manner, and did

not activate JNK. Antagonism by R3(B⌬23–27)R/I5 was

only seen for responses mediated by human relaxin-3 (Ko-

can et al., unpublished data).

More recently, we have examined a wide range of ligands

known to interact with RXFP1. These varied in potency

and efﬁcacy across signaling pathways including cAMP ac-

cumulation, ERK1/2, and p38MAPK phosphorylation, but

there was no indication of ligand-directed signaling bias

with agonists, antagonists, and related peptides such as

INSL3 and INSL5 (Siwek, Kocan, and Summers, unpub-

lished data). It is possible that the lack of signaling bias

observed with the ligands so far tested relates to the mode of

action of RXFP1 where the interaction of the peptide ligand

with the LRR region and the ECL region causes the LDLa

“tethered” ligand to interact with the receptor. Since the

ﬁnal common pathway is the same for all ligands, this may

explain the inability of the receptor to adopt alternative

signaling conformations. This may become possible if li-

gands that can be developed that target the site on the

receptor utilized by the LDLa module. Alternatively, li-

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462 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

gands with an allosteric mode of action may be able to

promote signaling bias. Likewise, no studies to date have

reported signaling bias at RXFP2 and RXFP4. However, it

should be borne in mind that studies to date with relaxin

family peptide receptors have largely been carried out with

a comparatively restricted range of peptides. Only recently

has evidence emerged for short linear peptides that can

activate RXFP1 and RXFP2 (see sect. IXD) (466, 467).

B. Receptor Roles and Potential

1. RXFP2 but not INSL3 in brain

One apparent anomaly that has been described for RXFP2

is the presence of the receptor but not its cognate ligand in

brain. The evidence for the presence of the receptor includes

RT-PCR, in situ hybridization histochemistry, and direct

binding studies with

125

I-INSL3 (239, 460, 469). In con-

trast, there is no convincing evidence for the expression of

INSL3 in the brain of mammals with the exception of bo-

vine hypothalamus, perhaps reﬂecting the lack of a relaxin

system in ruminants. Explanations that have been put for-

ward include that brain RXFP2 remains as part of a now

defunct ligand-receptor pairing (561) or that it is the target

for circulating INSL3 that either is transported across or

acts on receptors outside the blood-brain barrier. Sugges-

tions that relaxin might act as a ligand for RXFP2 in brain

appear unlikely due to lack of colocalization.

2. RXFP3: does it have a role in feeding/weight

control or is it one of many systems controlling

these functions?

The relaxin-3-RXFP3 ligand-receptor pairing is predomi-

nantly a neuropeptide receptor system. The cell bodies are

found mainly in the NI and project to many areas of the

brain. There has been a great deal of interest in this system

because of its reported roles in stress and feeding. Most

neurons that express relaxin-3 in the NI also express CRF-

R1, the receptor that responds to CRF released by stress.

Administration of CRF increases c-fos expression in many

relaxin-3 expressing neurons, and swim stress increases re-

laxin-3 mRNA in the NI (86). Although these interactions

are of interest, more work needs to be done to establish the

relationship between stress and the relaxin-3 system.

The interest in the role of relaxin-3-RXFP3 in feeding was

stimulated by the presence of RXFP3 in the hypothalamic

paraventricular nuclei. ICV injection of relaxin-3 or the

RXFP3 agonist R3/I5 into the brain of rats increased feed-

ing, and the effects are blocked by the antagonist R3(B⌬23–

27)R/I5 (486). Human relaxin-2 given in the same manner

decreased food intake, perhaps reﬂecting its actions on

RXFP1 or alternatively its different spectrum of action at

RXFP3 (545). Exploitation of the contrasting effects of bi-

ased agonists at RXFP3 would establish a novel paradigm

for drug action. However, the actions of relaxin-3 on feed-

ing do not appear to involve changes in expression of other

hypothalamic peptides known to have roles in appetite con-

trol including neuropeptide Y, proopiomelanocortin, or ag-

outi-related peptide (486). It has been suggested that the

actions of relaxin-3 on feeding do not represent a primary

role for this neuropeptide system but rather reﬂect its wider

role as an arousal neuromodulator (486).

3. What is the role of RXFP4?

Studies of the physiological role of RXFP4 are at a much

earlier stage than those of RXFP3. Although human relax-

in-3 was found to activate RXFP4 (GPR142) (313), it was

quickly recognized that this peptide was unlikely to be the

cognate ligand (98) based largely on the tissue expression

proﬁle. Whereas relaxin-3 is largely conﬁned to the brain,

RXFP4 has a wide tissue distribution including colon, kid-

ney, placenta, prostate, thymus, thyroid, and salivary gland

with only small amounts found in brain (313). Subsequent

work provided strong evidence that a related peptide INSL5

was likely the cognate ligand for RXFP4, since it showed a

similar tissue distribution and selectively activated the re-

ceptor (315). Although RXFP4 in the rat is a pseudogene

and in mice shows some sequence variation, in other species

such as human, monkey, cow, and pig, it is highly conserved

and displays similar ligand speciﬁcity (98). At the present

time, the physiological role of INSL5-RXFP4 is unclear,

although a recent report suggests that polymorphisms of

RXFP4 may be associated with obesity (376). This is of

particular interest given the potential role of relaxin-3-

RXFP3 in appetite control (see NOTE ADDED IN PROOF 1).

C. Therapeutic Actions of Relaxin

1. What is the mechanism of action of relaxin in the

treatment of cardiac failure?

Cardiac failure is a complicated disease that reﬂects the

interplay between many different mechanisms. It involves

weakness of the cardiac muscle that can be brought about

by genetic abnormalities, loss of cardiac muscle following

infarction, or secondary to pressure overload due to hyper-

tension. These stressful stimuli cause remodeling of the

heart which is initially compensatory but eventually leads to

ventricular dilatation, reduced cardiac output, and arrhyth-

mias. One of the features of cardiac failure is the presence of

high circulating levels of neurohumoral agents such as cat-

echolamines, angiotensin, endothelin, cytokines, insulin-

like growth factors, and natriuretic peptides (465). Overac-

tivity of the sympathetic nervous system and the renin-an-

giotensin-aldosterone system are particularly important,

and antagonists acting on these systems have proven valu-

able treatments that substantially reduce morbidity and

mortality (296). However, heart failure is characteristically

a chronic disease that is progressive so that even with recent

RELAXIN FAMILY PEPTIDES AND THEIR RECEPTORS

463Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

improvements in treatment novel approaches are required

to produce better outcomes.

Overactivity of the sympathetic nervous system is initially a

compensatory response that improves cardiac output by

increasing the force and rate of the heartbeat. Chronic ac-

tivation, however, leads to desensitization of

␤

-adrenocep-

tors, cardiac hypertrophy, and apoptosis of cardiac myo-

cytes with ﬁbrosis and cardiac arrhythmias. High levels of

angiotensin II and endothelin are associated with cardiac

hypertrophy, ﬁbrosis, and cardiac arrhythmias. Blockade of

␤

-adrenoceptors, ACE inhibitors, or AT

receptor blockade

are highly effective means of treating cardiac failure (296),

whereas the effects of endothelin antagonists are more

equivocal (207). Relaxin is now in phase III clinical trial for

the treatment of cardiac failure, yet the mechanism of action

is not well understood. Although there are a number of

possible mechanisms suggested by mainly animal studies,

clear links between these effects and the response to relaxin

in cardiac failure have yet to be made. The vasodilator

actions of relaxin mediated through NO (31, 33, 154, 343,

346) could have antihypertrophic effects (465), reduce pe-

ripheral resistance and hence cardiac work load, and also

help maintain viability of cardiac muscle. Relaxin is also

angiogenic, since cAMP increases in response to activation

of RXFP1 lead to increased expression of VEGF (541).

Relaxin has long been recognized as a powerful inotropic

and chronotropic agent (271) and has recently been shown

to have similar effects in humans (127). However, in all

species so far studied, these cardiac responses have been

conﬁned to the atria and would be expected to have little

inﬂuence on ventricular ejection fraction. Relaxin also has

anti-inﬂammatory effects (345) and would be expected to

reduce tissue damage in cardiac failure. At present, it is not

clear which if any of these effects is involved in the positive

response to relaxin in cardiac failure. In addition, aspects of

relaxin-RXFP1 signaling have been extensively studied in in

vitro systems, but the situation could be quite different in path-

ological situations such as heart failure, particularly when fur-

ther complicated by additional drug administration. G pro-

tein-coupled receptor activation by catecholamines, angioten-

sin II, and endothelins leads to coupling to a variety of G

proteins including G

␣

and G

␤␥

that in turn can

activate AC, phospholipase C, Akt, ERK1/2, p38MAPK, and

PKA to have hypertrophic effects (465). A compensatory re-

sponse that increases natriuretic peptides and leads to NO

stimulation of guanylyl cyclase causes an antihypertrophic

effect (465). Since relaxin is known to release NO and va-

sodilatation in systemic arteries is endothelium dependent

(170), this may underlie some of the beneﬁcial effects of

relaxin. However, this study also showed that the effects of

relaxin were inﬂuenced by treatment of patients with ACE

inhibitors and responses of blood vessels to relaxin could be

altered by treatment with indomethacin or blockade of NO

synthase. In the phase I trial of relaxin, all of the patients

were treated with an ACE inhibitor or AT

receptor antag-

onist, as well as a diuretic and a

␤

-adrenoceptor antagonist

(134). In the phase II clinical trial, 62–69% of patients were

treated with ACE inhibitors, 51–59% with

␤

-adrenoceptor

antagonists, 24–38% with aldosterone antagonists, and

14–30% with digoxin (526). Clearly, there are many op-

portunities for interactions between relaxin and a variety of

pathways, and examination of potential interactions in in

vitro systems may help clarify the mechanism of action of

relaxin in vivo.

2. Is there a future for relaxin in the treatment

of ﬁbrosis?

Although the early clinical trials of relaxin for the treatment

of sclerosis appeared encouraging (92) and a subsequent

placebo-controlled trial indicated that low-dose relaxin

produced signiﬁcant improvement in skin and lung param-

eters (462), the phase II/III trial that followed was unsuc-

cessful (149, 461). It is possible that the failure of this trial

reﬂects the severity of the scleroderma in the patients chosen

for the trial (54). Nonetheless, the large number of animal

studies in which relaxin was successfully shown to have

antiﬁbrotic properties still provide a strong case for its po-

tential utility in the treatment of pulmonary, renal, cardiac,

and hepatic ﬁbrosis. Certainly work continues in this area

to attempt to demonstrate antiﬁbrotic effects that will be

useful in humans (420, 437, 518).

D. Agonist and Antagonist Development for

RXFP Receptors

There is now a basic understanding of the mode of endog-

enous peptide ligand binding at RXFP1–3 with limited

knowledge of the mode of interaction of INSL5 with

RXFP4 (sect. III). There are currently peptide-based antag-

onists for all of the receptors with the most efﬁcacious com-

pounds targeting RXFP3 (213, 299, 463). These com-

pounds have already been used to assist in determining the

role of relaxin-3 in the brain. Relaxin-3 peptide analogs

have also been developed that are speciﬁc agonists for

RXFP3 over RXFP1 (312, 463). However, these peptide-

based analogs are unlikely to cross the blood-brain barrier

so must be injected directly into the brain to be effective.

Currently there are no small molecule RXFP3 agonists or

antagonists reported, although a recent study has described

an RXFP3-speciﬁc relaxin-3-positive allosteric modulator

that was effective when used with relaxin-3 COOH-termi-

nal amide peptides but was unfortunately not effective with

the native relaxin COOH-terminal acid peptide (7).

The complex mechanism whereby relaxin binds to RXFP1 has

meant that there are currently no high-afﬁnity peptide antag-

onists, and since the relaxin peptide itself rapidly loses activity

when truncated, it is likely that a small peptide agonist acting

at the orthosteric sites utilized by relaxin will be difﬁcult to

develop (232, 233). However, what is clear is that the peptide

BATHGATE ET AL.

464 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

can be modiﬁed at the NH

terminus of both the A- and

B-chains, meaning that potentially it can be modiﬁed to im-

prove its pharmacokinetic properties and make it more drug-

like (232, 233). Small peptide antagonists have been devel-

oped that target RXFP2 that exploit the different binding

mechanism of INSL3 to RXFP2 compared with relaxin bind-

ing to RXFP1 (121, 464). However, the complex activation

mechanism associated with leucine-rich repeat contain-

ing GPCRs has precluded the development of small peptide

agonists. The isolated LDLa module has been demonstrated to

act as a RXFP1 antagonist (456, 457), and it is possible that if

the mechanism of action of the LDLa module in activating

RXFP1 and RXFP2 is determined, then speciﬁc agonists or

antagonists could be designed. Currently, no small molecule

RXFP1 or RXFP2 agonists or antagonists have been reported

(see NOTE ADDED IN PROOF 2). A possible approach that has

currently only been adopted for RXFP3 is the use of allosteric

modulators that typically target binding sites distinct from the

orthosteric sites utilized by the endogenous ligands.

An interesting recent development has been the discovery of

short linear peptides derived from an unidentiﬁed natural pre-

cursor protein that were able to activate RXFP1 and RXFP2,

but not RXFP3 receptors expressed in CHO cells (466, 467).

Further in vitro studies demonstrated that one of these pep-

tides (CGEN-25009) is able to activate ﬁbroblasts and THP1

cells expressing RXFP1. The peptide was able to reduce lung

inﬂammation and injury as well as ameliorate adverse airway

remodeling and peribronchial ﬁbrosis in a mouse model of

pulmonary ﬁbrosis in a manner similar to relaxin (420).

CGEN-25009 and other peptides (CGEN-25010 and -25011)

produced complex concentration-response relationships in

cell systems expressing RXFP1 or RXFP2 with either inhibi-

tion or stimulation of cAMP accumulation being observed

(466). Some of the differences are attributable to assay condi-

tions such as the use of forskolin to amplify responses but also

could be explained by measurement of cAMP directly or by

the use of CRE reporter gene assays that display responses not

only to cAMP but also other signaling pathways and in par-

ticular MAPKs. It has also yet to be deﬁnitely demonstrated

that these peptides mediate their actions at RXFP1 or RXFP2

and even if they do the mechanism of binding is likely to be

very different from that of the endogenous ligands. However,

it is clear that some promising tools for the study of relaxin

family peptides are starting to emerge that will begin to answer

some of the questions raised in this section.

NOTE ADDED IN PROOF

1) A recent study has demonstrated that IN5L5⫺/⫺mice

are glucose intolerant and show elevated blood glucose lev-

els with advancing age. This results from a reduced area of

pancreatic islets and decreased numbers of

␤

cells (Bur-

nicka-Turek et al., Endocrinology 153: 4655–4665, 2012).

2) Evidence was presented at the Relaxin 2012 meeting for

ML290, a small molecule agonist at RXFP1 which acts at a

site distinct from those at which relaxin binds (Chen et al. J

Biomol Screen doi:10.1177/1087057112469406.2012).

ACKNOWLEDGMENTS

We thank Dr. Bronwyn Evans for the analysis illustrated in

FIGURES 1 AND 2, Dr. Daniel Scott for FIGURE 2D, Dr.

Johan Rosengren for FIGURE 1C, Prof. Andrew Gundlach

and Dr. Craig Smith for contributions to FIGURE 8, and Dr.

Chrishan Samuel for helpful discussions. Some of the tissue

illustrations in FIGURE 4 were downloaded from www.med.

ars.it with permission from Dr. Davide Brunelli.

R. A. D. Bathgate and M. L. Halls contributed equally to

the writing of this review.

Present addresses: R. A. D. Bathgate, M. L. Halls, M. Ko-

can, and R. J. Summers, Drug Discovery Biology, Monash

Institute of Pharmaceutical Sciences & Department of Phar-

macology, Monash University, Victoria, Australia; G. E.

Callander, Florey Neurosciences Institutes and Department

of Biochemistry and Molecular Biology, The University of

Melbourne, Victoria, Australia; and E. T. van der Westhui-

zen, Université de Montréal, Institut de Recherche en Im-

munologie et Cancérologie, Pharmacologie Moleculaire,

Montréal, Canada.

Address for reprint requests and other correspondence: R. J.

Summers, Drug Discovery Biology, Monash Institute of

Pharmaceutical Sciences & Department of Pharmacology,

Monash University, 399 Royal Parade, Victoria 3052, Aus-

tralia (e-mail: roger.summers@monash.edu).

GRANTS

This work was funded by the National Health and Medical

Research Council (NHMRC) of Australia Program Grant

519461 (to P. M. Sexton, A. Christopoulos, and R. J. Sum-

mers), NHMRC Career Development Awards 519581 (to

M. L. Halls) and 1013819 (to E. Van Der Westhuizen),

NHMRC Senior Research Fellowship 509011 (to R. A. D.

Bathgate), and NHMRC Project Grant 628427 (to R. A. D.

Bathgate).

DISCLOSURES

No conﬂicts of interest, ﬁnancial or otherwise, are declared

by the authors.

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480 Physiol Rev •VOL 93 •JANUARY 2013 •www.prv.org

Relaxin Modulates the Genomic Actions and Biological Effects of Estrogen in the Myometrium by Reducing Estrogen Receptor Alpha Phosphorylation

Preprint

Full-text available

Apr 2024

A bstract Estradiol (E2) and relaxin (Rln) are steroid and polypeptide hormones, respectively, with important roles in the female reproductive tract, including myometrium. Some actions of Rln, which are mediated by its membrane receptor RXFP1, require or are augmented by E2 signaling through its cognate nuclear steroid receptor, estrogen receptor alpha (ERα). In contrast, other actions of Rln act in opposition to the effects of E2. Here we explore the molecular and genomic mechanisms that underlie the functional interplay between E2 and Rln in the myometrium. We used both ovariectomized female mice and immortalized human myometrial cells expressing wild type or mutant ERα (hTERT-HM-ERα cells). Our results indicate that Rln attenuates the genomic actions and biological effects of estrogen in the myometrium and myometrial cells by reducing phosphorylation ERα on serine 118 (S118). Interestingly, we observed a potent inhibitory effect of Rln on the E2-dependent binding of ERα across the genome. The reduction in ERα binding was associated with changes in the hormone-regulated transcriptome, including a decrease in the E2-dependent expression of neighboring genes. The inhibitory effects of Rln cotreatment on the E2-dependent phosphorylation of ERα required the nuclear dual-specificity phosphatases DUSP1 and DUSP5. Moreover, the inhibitory effects of Rln were reflected in a concomitant inhibition of the E2-dependent contraction of myometrial cells. Collectively, our results identify a pathway that integrates Rln/RXFP1 and E2/ERα signaling, resulting in a convergence of membrane and nuclear signaling pathways to control genomic and biological outcomes.

Single-cell RNA-seq of the chicken hypothalamic-pituitary-ovarian axis offers new insights into the molecular regulatory mechanisms of ovarian development

Article

Jan 2024

Seleno -relaxin analogues: Effect of internal and external diselenide bonds on the foldability and a fibrosis-related factor of endometriotic stromal cells

Article

Full-text available

Jan 2024

Human relaxin-2 (H2 relaxin) is a peptide hormone of about 6 kDa, first identified as a reproductive hormone involved in vasoregulation during pregnancy. It has recently attracted strong interest because...

Cardiomyocyte-specific RXFP1 overexpression protects against pressure overload-induced cardiac dysfunction independently of Relaxin

Article

May 2024
BIOCHEM PHARMACOL

Identification and Quantification of Human Relaxin Proteins by Immunoaffinity-Mass Spectrometry

Article

May 2024

Relaxin as a treatment for musculoskeletal fibrosis: What we know and future directions

Article

May 2024
BIOCHEM PHARMACOL

Targeting the relaxin-3/RXFP3 system: a patent review for the last two decades

Article

Apr 2024
EXPERT OPIN THER PAT

Human recombinant relaxin-2 (serelaxin) regulates the proteome, lipidome, lipid metabolism and inflammatory profile of rat visceral adipose tissue

Article

Mar 2024
BIOCHEM PHARMACOL

Discovery of Clinical Candidate AZD5462, a Selective Oral Allosteric RXFP1 Agonist for Treatment of Heart Failure

Article

Mar 2024
J MED CHEM

Identification of Novel Series of Potent and Selective Relaxin Family Peptide Receptor 1 (RXFP1) Agonists

Article

Mar 2024
J MED CHEM

Water Drinking in Rats Resulting from Intravenous Relaxin and Its Modification by Other Dipsogenic Factors1

Article

Nov 1999
ENDOCRINOLOGY

The purpose of the study was to determine whether iv infusion of relaxin would acutely stimulate water drinking in rats and, if it did, whether such drinking is affected by other dipsogenic stimuli or is blocked by centrally administered losartan. iv infusions of human gene 2 relaxin at doses of 25, 40, 55, or 80 μg/kg·h for 1 h induced dose-dependent water drinking in both male and female rats within 15–30 min of commencement of infusions. iv infusion of a nondipsogenic dose of angiotensin II (0.5 μg/h), combined with relaxin (40 μg/kg·h), almost tripled the relaxin-induced water intake. iv infusion of hypertonic (1 m) NaCl did not potentiate relaxin-induced drinking. Intracerebroventricular injection of the angiotensin AT1 antagonist losartan (10 μg) reduced water drinking induced by iv infusion of relaxin. The water drinking induced by iv infusion of relaxin in the rat suggests that blood-borne relaxin may be a dipsogenic hormone. Potentiation of this relaxin-induced drinking by moderate levels of circulating angiotensin II is additional evidence in support of this view. The results also indicate that a central angiotensinergic neural pathway, utilizing AT1 receptors, subserves relaxin-induced drinking.

Mice without a Functional Relaxin Gene Are Unable to Deliver Milk to Their Pups * * This work was supported by an institute block grant from the National Health and Medical Research Council of Australia (983001) and in part by a WHO Research Training Fellowship and a University of Melbourne Postgraduate Research Scholarship (to L.Z.)

Article

Jan 1999
ENDOCRINOLOGY

We have used gene targeting to generate relaxin (rlx)-deficient mice. The majority (15 of 17) of homozygous (rlx−/−) mice are fertile and produce normal litters. However their mammary development is deficient; pups are unable to suckle and die within 24 h of birth unless cross-fostered to a wild-type (rlx+/+) foster mother. The nipples of rlx−/− animals do not enlarge significantly during pregnancy, and their histology retains the appearance of the virgin state. Breast parenchyma is somewhat underdeveloped at term even though milk is produced. Mammary ducts become grossly dilated in these animals. Heterozygous (rlx+/−) mice lactate normally. The interpubic ligament does not relax during pregnancy in rlx−/− mice. Plasma osmolality during late gestation was significantly higher (P < 0.001) in rlx−/− mice than in wild-type controls.

Hormonal Characteristics in the Early Luteal Phase of Conceptive and Nonconceptive Menstrual Cycles

Article

Jan 2003
J Soc Gynecol Investig

Objective:To determine whether serum hormone profiles are different in nonconceptive and conceptive menstrual cycles after ovulation and before implantation.

Monoclonal antibodies specific for rat relaxin. IX. Evidence that endogenous relaxin promotes growth of the vagina during the second half of pregnancy in rats.

Article

Feb 1996

It is established that endogenous relaxin promotes the growth and development of the cervix, mammary glands, and nipples in pregnant rats. Additionally, the observation that porcine relaxin promotes growth of the vagina in both nonpregnant and pregnant rats provides indirect evidence that endogenous relaxin may effect growth of the vagina during rat pregnancy. The purpose of this study was to determine whether endogenous relaxin promotes growth of the vagina in pregnant rats. To that end, a monoclonal antibody, specific for rat relaxin, designated MCA1, was used to passively neutralize endogenous relaxin throughout the second half of pregnancy in intact rats. Five milligrams of highly purified MCA1 were injected iv to rats daily from days 12-22 of pregnancy. Controls received either a monoclonal antibody for fluorescein or PBS. The vaginal wet weight, dry weight, length, diameter, inner surface area, DNA content, and percent water content were determined. No differences were found between monoclonal antib...

Protein Kinase A Anchoring to the Myometrial Plasma Membrane Is Required for Cyclic Adenosine 3?,5?-Monophosphate Regulation of Phosphatidylinositide Turnover 1

Article

Nov 1999

Demonstration of a Relaxin Receptor and Relaxin-Stimulated Tyrosine Phosphorylation in Human Lower Uterine Segment Fibroblasts 1

Article

Mar 1998

To elucidate the mechanism of relaxin action, we studied the binding characteristics of human relaxin and its effects on intracellular concentrations of cAMP and tyrosine phosphorylation of cellular proteins in a model system of human cervix, human lower uterine segment fibroblasts. Human relaxin labeled with 125I bound specifically to a single class of high-affinity relaxin binding sites, distinct from insulin receptors, with a mean (±sem) dissociation constant (Kd) of 4.36 ± 1.7 × 10−9 m and a mean of 3220 ± 557 binding sites per cell in human lower uterine segment fibroblasts. Relaxin, in quantities that were shown previously to stimulate intracellular levels of cAMP in other cell types, had no effect on intracellular levels of cAMP in human lower uterine segment fibroblasts even in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Incubation of the cells with relaxin caused a significant increase in tyrosine phosphorylation of a protein with an apparent Mr of approxi...

The Dipsogenic Effects of Rat Relaxin: The Effect of Photoperiod and the Potential Role of Relaxin on Drinking in Pregnancy 1

Article

May 1998

Experiments were done to examine whether rat relaxin is dipsogenic and whether such dipsogenic effects of rat relaxin are related to time of injection during the light-dark cycle. Female rats were fitted with a chronic intra-cerebro-ventricular (icv) cannula. Rat relaxin (2.5, 5, 10, 25, 50, or 100 ng/2 μl in 0.9% saline) was injected into the right lateral ventricle at either morning (0800–1000 h), afternoon (1400–1600 h), or night (2200–2400 h), and water consumption was measured. Relaxin caused a dose-dependent dipsogenesis at doses ≥ 5 ng, but the sensitivity and magnitude of the response varied with the photoperiod. Water consumption was smallest (3.5 ± 0.7 ml at 50 ng) and least sensitive (minimal effective dose at 25 ng) in the afternoon and maximal (17.7 ± 2.3 ml at 50 ng) and most sensitive (minimal effective dose 5 ng) at night. The latency from injection to drinking was 55.8 ± 10.4 sec (mean ± sem) and did not vary significantly with either the dose or time of day. A second set of experiments w...

A polyclonal antibody to a synthetic peptide derived from the rat follicle-stimulating hormone receptor reveals the recombinant receptor as a 74-kilodalton protein.

Article

Nov 1993

We have prepared a polyclonal antibody (AntiF) against a synthetic peptide comprising residues 19-29 of the rat FSH receptor. The specificity of this antibody was documented using human embryonic kidney (293) cells and stable transfectants of 293 cells expressing the recombinant LH/CG and FSH receptors. The data presented show that AntiF inhibits the binding of FSH, but not that of LH/CG, to their cognate receptors. AntiF also recognizes a specific protein(s) representing the FSH receptor in Western blots or by immunoprecipitation of cells transfected with the FSH receptor. This protein(s) is not present in untransfected 293 cells or in 293 cells permanently transfected with the LH/CG receptor. Finally, addition of the 19-29 peptide prevents immunoprecipitation of the FSH receptor by AntiF, whereas an unrelated peptide corresponding to residues 637-647 has no effect. In Western blots of 293 cells transfected with the FSH receptor, AntiF reveals the recombinant receptor as a heterogenous glycoprotein with ...

Neutralization of Relaxin within the Brain Affects the Timing of Birth in Rats 1

Article

Feb 1998

Experiments were performed to determine whether neutralization of relaxin in the brain, by injecting monoclonal antibodies to rat relaxin into the ventricular system of the brain, affected either the timing or the processes of birth in rats. Pregnant rats were injected daily through a chronically implanted intracerebroventricular cannula either with a specific monoclonal antibody raised against rat relaxin from days 12–22 of gestation or with an antibody raised against fluorescein as a control. The rats were watched closely from the afternoon of day 20 of pregnancy, and the process of birth was observed. No sign of dystocia was observed in any of the rats in the experiment. Neutralization of endogenous relaxin caused a significant decrease in the length of gestation (505.4 ± 3.1 h) compared with that in rats treated with PBS (524.6 ± 0.5 h) or that in rats treated with a nonspecific antibody (525.9 ± 0.7 h). The time to the onset of delivery was also shorter in the relaxin-neutralized group (507.8 ± 1.1 h) compared with that in either PBS-treated (526.5 ± 0.6 h) or fluorescein antibody-treated (525.3 ± 0.7 h) animals. In contrast, there was no significant effect of the relaxin antibody on length of straining, duration of parturition, delivery interval, live birth rate, or body weight of the neonates. Premature delivery in the relaxin-neutralized group was accompanied by a 24-h advance in the fall in plasma progesterone. These data support the hypothesis that there may be a central relaxin system that is independent of the peripheral relaxin system. Central relaxin may have a significant physiological role on the timing of pregnancy in the rat, but does not affect the course of labor once it has started.

Evidence that cellular proliferation contributes to relaxin-induced growth of both the vagina and the cervix in the pregnant rat.

Article

Nov 1995

Relaxin Family Peptides and Their Receptors

Abstract

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