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Negative cooperativity in H2 relaxin binding to a dimeric relaxin family peptide receptor 1
Abstract
H2 relaxin, a member of the insulin superfamily, binds to the G-protein-coupled receptor RXFP1 (relaxin family peptide 1), a receptor that belongs to the leucine-rich repeat (LRR)-containing subgroup (LGRs) of class A GPCRs. We recently demonstrated negative cooperativity in INSL3 binding to RXFP2 and showed that this subgroup of GPCRs functions as constitutive dimers. In this work, we investigated whether the binding of H2 relaxin to RXFP1 also shows negative cooperativity, and whether this receptor functions as a dimer using BRET(2). Both binding and dissociation were temperature dependent, and the pH optimum for binding was pH 7.0. Our results showed that RXFP1 is a constitutive dimer with negative cooperativity in ligand binding, that dimerization occurs through the 7TM domain, and that the ectodomain has a stabilizing effect on this interaction. Dimerization and negative cooperativity appear to be general properties of LGRs involved in reproduction as well as other GPCRs.
... CHEN Svendsen, Zalesko, et al., 2008). Cellular pH levels control ligand/receptor complex stability and play an important role in modulating the downstream signaling pathway (Svendsen, Zalesko, et al., 2008). ...
... CHEN Svendsen, Zalesko, et al., 2008). Cellular pH levels control ligand/receptor complex stability and play an important role in modulating the downstream signaling pathway (Svendsen, Zalesko, et al., 2008). Heterodimerization of WT RXFP1 with its splicing variants or other receptors exhibit negative cooperativity in relaxin/RXFP1 binding (Kern et al., 2008;Svendsen, Zalesko, et al., 2008). ...
... Cellular pH levels control ligand/receptor complex stability and play an important role in modulating the downstream signaling pathway (Svendsen, Zalesko, et al., 2008). Heterodimerization of WT RXFP1 with its splicing variants or other receptors exhibit negative cooperativity in relaxin/RXFP1 binding (Kern et al., 2008;Svendsen, Zalesko, et al., 2008). Furthermore, overexpression of RXFP1 in HEK-293T cells resulted in its intracellular accumulation and inhibition of relaxin/RXFP1 signaling (Hoare et al., 2019). ...
Background:
Relaxin/relaxin family peptide receptor 1 (RXFP1) signaling is important for both normal physiology and disease. Strong preclinical evidence supports relaxin as a potent antifibrotic molecule. However, relaxin-based therapy failed in clinical trial in patients with systemic sclerosis. We and others have discovered that aberrant expression of RXFP1 may contribute to the abnormal relaxin/RXFP1 signaling in different diseases. Reduced RXFP1 expression and alternative splicing transcripts with potential functional consequences have been observed in fibrotic tissues. A relative decrease in RXFP1 expression in fibrotic tissues-specifically lung and skin-may explain a potential insensitivity to relaxin. In addition, receptor dimerization also plays important roles in relaxin/RXFP1 signaling.
Methods:
This review describes the tissue specific expression, characteristics of the splicing variants, and homo/heterodimerization of RXFP1 in both normal physiological function and human diseases. We discuss the potential implications of these molecular features for developing therapeutics to restore relaxin/RXFP1 signaling and to harness relaxin's potential antifibrotic effects.
Results:
Relaxin/RXFP1 signaling is important in both normal physiology and in human diseases. Reduced expression of RXFP1 in fibrotic lung and skin tissues surrenders both relaxin/RXFP1 signaling and their responsiveness to exogenous relaxin treatments. Alternative splicing and receptor dimerization are also important in regulating relaxin/RXFP1 signaling.
Conclusions:
Understanding the molecular mechanisms that drive aberrant expression of RXFP1 in disease and the functional roles of alternative splicing and receptor dimerization will provide insight into therapeutic targets that may restore the relaxin responsiveness of fibrotic tissues.
... 7 There is evidence that the LDLa module is a tethered agonist that interacts with and activates the transmembrane domain of RXFP1, 8,9 and there is also evidence that RXFP1 forms dimers/oligomers in the cell membrane. 10,11 It has therefore been previously postulated that RXFP1 may be activated as a homodimer, with the LDLa module of one receptor subunit activating the transmembrane domain of the other receptor subunit via a trans-activation mechanism ( Figure 1B). 12 However, there is weak evidence that RXFP1 forms stable homodimers at the cell surface, so a mechanism involving relaxin activating a homodimer of RXFP1 requires further investigation. ...
... 13,16,[18][19][20] This type of experiment has been applied to RXFP1, indicating "constitutive" homodimerization of the receptor which is not affected by relaxin stimulation. 10,11 Currently, however, this is the main evidence that RXFP1 forms homodimers. ...
... Data representative of a single experiment performed 3 times in duplicate, presented as mean ± SD. (E) and (F) Saturation BRET curve using HiBiT-RXFP1 and mCitrine-RXFP1 (E) or mCitrine-GABA B2 (F). Data points represent single wells in which filtered luminescence was measured, followed by measurement of mCitrine fluorescence, and is pooled from three independent experiments cell surface and what role, if any, homodimerization of RXFP1 plays on its mechanism of activation.A common method for determining GPCR proximity is the use of saturation BRET experiments, and these have been published for RXFP1 showing "constitutive" homodimerization, which appear to be unaffected by relaxin binding.11 Our primary aim was to further investigate RXFP1 homodimerization in order to assess whether it is indeed a necessary requirement for relaxin-mediated activation of RXFP1, thus we sought appropriate tools to investigate RXFP1 proximity in live cells. ...
Relaxin family peptide 1 (RXFP1) is the receptor for relaxin a peptide hormone with important therapeutic potential. Like many G protein‐coupled receptors (GPCRs), RXFP1 has been reported to form homodimers. Given the complex activation mechanism of RXFP1 by relaxin, we wondered whether homodimerization may be explicitly required for receptor activation, and therefore sought to determine if there is any relaxin‐dependent change in RXFP1 proximity at the cell surface. Bioluminescence resonance energy transfer (BRET) between recombinantly tagged receptors is often used in GPCR proximity studies. RXFP1 targets poorly to the cell surface when overexpressed in cell lines, with the majority of the receptor proteins sequestered within the cell. Thus, any relaxin‐induced changes in RXFP1 proximity at the cell surface may be obscured by BRET signal originating from intracellular compartments. We therefore, utilized the newly developed split luciferase system called HiBiT to specifically label the extracellular terminus of cell surface RXFP1 receptors in combination with mCitrine‐tagged receptors, using the GABAB heterodimer as a positive control. This demonstrated that the BRET signal detected from RXFP1‐RXFP1 proximity at the cell surface does not appear to be due to stable physical interactions. The fact that there is also no relaxin‐mediated change in RXFP1‐RXFP1 proximity at the cell surface further supports these conclusions. This work provides a basis by which cell surface GPCR proximity and expression levels can be specifically studied using a facile and homogeneous labeling technique such as HiBiT.
... Serelaxin treatment also prevented downregulation of Notch-1 expression by TGF-b 1 , as well as the inhibition of NICD and Jagged-1 release [42] (Figure 2). Like many other class A GPCRs, there is evidence from bioluminescence resonance energy transfer (BRET) studies that RXFP1 receptors form homo-and heterodimers [83][84][85] in the absence of, and independent of, ligand occupation of the receptor [84,85]. It has been suggested that dimer formation is necessary for signal transduction with ligand binding occurring at the leucine-rich repeat (LRR) region of one dimer partner, followed by interaction of the bound ligand with the extracellular loop (ECL)2 of the second partner [86]. ...
... Serelaxin treatment also prevented downregulation of Notch-1 expression by TGF-b 1 , as well as the inhibition of NICD and Jagged-1 release [42] (Figure 2). Like many other class A GPCRs, there is evidence from bioluminescence resonance energy transfer (BRET) studies that RXFP1 receptors form homo-and heterodimers [83][84][85] in the absence of, and independent of, ligand occupation of the receptor [84,85]. It has been suggested that dimer formation is necessary for signal transduction with ligand binding occurring at the leucine-rich repeat (LRR) region of one dimer partner, followed by interaction of the bound ligand with the extracellular loop (ECL)2 of the second partner [86]. ...
... It has been suggested that dimer formation is necessary for signal transduction with ligand binding occurring at the leucine-rich repeat (LRR) region of one dimer partner, followed by interaction of the bound ligand with the extracellular loop (ECL)2 of the second partner [86]. One functional consequence of dimerization can be negative cooperativity [84,85] where the two binding sites do not have a fixed affinity and the affinity of each remaining unoccupied receptor binding site decreases as occupancy increases. There are two intriguing, functionally relevant consequences of negative cooperativity: an increased functional range of the ligand and a decrease in ligand residence time at the receptor as the free ligand concentration increases, potentially allowing selective activation of different signaling pathways [87]. ...
Fibrosis represents a failed wound healing response to tissue injury. It is characterized by the accumulation of excess connective tissue and is a significant cause of organ failure, morbidity, and mortality. Fibrotic disorders accompany a wide spectrum of conditions including both systemic and organ-specific diseases, for which there is currently no effective cure. Serelaxin, the recombinant form of the major stored and circulating form of human relaxin, has emerged as a pleiotropic drug with rapidly occurring antifibrotic actions. This review discusses the effectiveness of serelaxin as an antifibrotic, and how it augments the actions of several other therapeutics leading to its potential use not only as a monotherapy but also as an adjunct therapy with other antifibrotic agents.
... Figure 3A shows the accelerated dissociation of labeled insulin in the presence of unlabeled insulin (167 nM). We recently showed a similar phenomenon in the dissociation of labeled INSL3 from RXFP2 (Fig. 3B), 17 as well as H2 relaxin to RXFP1, 18 suggesting the existence of negative cooperativity in these hormone-receptor interactions as well. The development of the accelerating effect of cold INSL3 is much slower than that seen for insulin, IGF-I, or TSH. ...
... The dose response for negative cooperativity for INSL3 showed a bellshaped curve, as also seen for insulin ( Fig. 3C and D). The negative cooperativity of H2 relaxin binding to RXFP1 in contrast shows a sigmoid curve like that seen for IGF-I binding to the IGF-I receptor 18 (see also the chapter by De Meyts et al. in this issue). We have also shown, using the infinite dilution procedure, that negative cooperativity occurs in the H3 relaxin/RXFP3 (GPCR135) system and that therefore this receptor most likely also functions as a dimer. ...
... (Modified from Ref. 17 and used with permission.) The curves for both insulin and INSL3 have a reverse bell shape, in contrast to what is seen for IGF-I (see the chapter by De Meyts et al. in this volume)and H2 relaxin.18 ...
Peptides of the relaxin family bind to the relaxin family peptide receptors or RXFPs, members of the G-protein-coupled receptor (GPCR) superfamily. For many years, ligand binding to GPCRs was thought to take place as monomeric complexes, ignoring early evidence of negative cooperativity. However, recent research has shown that most GPCRs form constitutive dimers or larger oligomers. The connection between dimerization and negative cooperativity has now been shown for several GPCRs, including the thyroid-stimulating hormone, luteinizing hormone, and follicle-stimulating hormone receptors, which like RXFP1 and -2 belong to the leucine-rich repeat-containing subgroup of class A GPCRs. We recently demonstrated homodimerization and negative cooperativity for RXFP1 and RXFP2 as well as their heterodimerization. Another study showed that RXFP1 has to homodimerize in order to be transported from the endoplasmic reticulum to the cell membrane.
... As with other GPCRs, RXFP1 is subject to homodimerization [51,52], but also heterodimerizes with angiotensin type-2 receptor (AT2R) [53]. ...
... Frequently, bell-shaped dose-response curves to hRlx-2 are observed both in vitro and in vivo [64,68]. This phenomenon is usually explained by the interaction of different mechanisms: the negative cooperativity of the RXFP1 homodimer [52] gradually alters the residence time of hRlx-2 at its receptor, which then may differentially affect the utilization of the various G proteins and signaling paths [21]. In contrast, RXFP1 desensitization and internalization do not play a major role herein [69] which, on the other hand, represents the molecular basis for the usually prolonged action of hRlx-2. ...
Acute heart failure (AHF) syndrome, characterized by pulmonary and/or venous congestion owing to increased cardiac filling pressures with or without diminished cardiac output, is still associated with high post-discharge mortality and hospitalization rates. Many novel and promising therapeutic approaches, among them endothelin-1, vasopressin and adenosine antagonists, calcium sensitization, and recombinant B-type natriuretic hormone, have failed in large studies. Likewise, the classic drugs, vasodilators, diuretics, and inotropes, have never been shown to lower mortality.
The phase III trial RELAX-AHF tested recombinant human relaxin-2 (rhRlx) and found it to improve clinical symptoms moderately, to be neutral regarding the combination of death and hospitalization at day 60, to be safe, and to lower mortality at day 180.
This review focuses on basic research and pre-clinical findings that may account for the benefit of rhRlx in AHF. The drug combines short-term hemodynamic advantages, such as moderate blood pressure decline and functional endothelin-1 antagonism, with a wealth of protective effects harboring long-term benefits, such as anti-inflammatory, anti-fibrotic, and anti-oxidative actions. These pleiotropic effects are exerted through a complex and intricate signaling cascade involving the relaxin-family peptide receptor-1, the glucocorticoid receptor, nitric oxide, and a cell type-dependent variety of kinases and transcription factors.
... The LRRs are the primary binding site for the cognate ligands, relaxin (RXFP1) and insulin-like peptide 3 (INSL3) (RXFP2), while the LDLa module is essential for signaling (4). As the receptors have been shown to form constitutive homodimers (5, 6) it is hypothesized that the LDLa module acts as a secondary ligand, possibly interacting with the extracellular loops of the TM region of a homodimer partner receptor to induce the conformational change necessary for signaling (7). RXFP1 and RXFP2 are the only known mammalian GPCRs to contain an LDLa module and thus the potential role of this module in signal activation is unique. ...
... It should also be pointed out that it is highly unlikely that changes in potency or efficacy in the receptor chimeras is related to decreased efficiency of homodimerization of the receptors. Our previous studies on dimerization of RXFP1 (6) and RXFP2 (5) have demonstrated that homodimerization is primarily driven by TM interactions with some potential stabilizing influence of the ectodomains. As the TM and LRR regions would be matched in homodimers of the chimeric receptors and there was no evidence of profound changes in cell surface expression or efficacy changes on our chimeras or with RXFP1/2 and RXFP2/1 previously (11), it is therefore likely that the chimeric receptors are constitutive homodimers like the WT receptors. ...
Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism.
... Hoare et al. [101] exploited the cell-impermeability of LgBiT in a novel assay to investigate homomerization of relaxin family peptide 1 receptor (RXFP1) on the cell surface. Previous reports suggested RXFP1 forms homomers at the cell surface and that this may be important to the receptor's activation [102,103]. However, this study observed transiently overexpressed RXFP1 expressed poorly on the plasma membrane, likely sequestering within internal cellular compartments [101]. ...
G protein‐coupled receptors (GPCRs) represent a major target for the development of pharmaceuticals. As such, they are subject to extensive research to understand their function, as well as how their signaling is influenced by therapeutics. Bioluminescence Resonance Energy Transfer (BRET) is a biophysical technique that enables real‐time monitoring of protein and small molecule proximity in live cells. BRET has seen widespread use as an important research tool to elucidate many aspects of GPCR pharmacology. Recently, multiple technological advances have furthered the scope and power of BRET techniques. New assay platforms such as NanoBRET have enabled the development of novel applications of the BRET technique, while the combination of BRET with newly developed biosensors has been shown to be a powerful research strategy. Here, we review recent BRET assay developments and their uses, demonstrating the rapidly expanding facets of GPCR pharmacology that can be studied utilizing BRET technologies. These tools will likely prove to be fundamental in furthering our understanding of GPCR function and pharmaceutical development.
... The reasons for this bell-shaped dose-response curve are not completely clear. Several mechanisms are discussed, e.g., that ligand dissociation is accelerated at higher RLX concentrations, thus shortening the time of RLX binding to its receptor [62]. As another possible explanation, different dose-response curves for venous and arterial dilator properties of RLX are discussed [27]. ...
Acute hypoxia impairs left ventricular (LV) inotropic function and induces development of pulmonary edema (PE). Enhanced and uneven hypoxic pulmonary vasoconstriction is an important pathogenic factor of hypoxic PE. We hypothesized that the potent vasodilator relaxin might reduce hypoxic pulmonary vasoconstriction and prevent PE formation. Furthermore, as relaxin has shown beneficial effects in acute heart failure, we expected that relaxin might also improve LV inotropic function in hypoxia. Forty-two rats were exposed over 24 h to normoxia or hypoxia (10% N 2 in O 2 ). They were infused with either 0.9% NaCl solution (normoxic/hypoxic controls) or relaxin at two doses (15 and 75 μg kg ⁻¹ day ⁻¹ ). After 24 h, hemodynamic measurements and bronchoalveolar lavage were performed. Lung tissue was obtained for histological and immunohistochemical analyses. Hypoxic control rats presented significant depression of LV systolic pressure by 19% and of left and right ventricular contractility by about 40%. Relaxin did not prevent the hypoxic decrease in LV inotropic function, but re-increased right ventricular contractility. Moreover, hypoxia induced moderate interstitial PE and inflammation in the lung. Contrasting to our hypothesis, relaxin did not prevent hypoxia-induced pulmonary edema and inflammation. In hypoxic control rats, PE was similarly distributed in the apical and basal lung lobes. In relaxin-treated rats, PE index was 35–40% higher in the apical than in the basal lobe, which is probably due to gravity effects. We suggest that relaxin induced exaggerated vasodilation, and hence pulmonary overperfusion. In conclusion, the results show that relaxin does not prevent but rather may aggravate PE formation.
... Instead, it could be due to alterations in RXFP1 signaling under persistent pain conditions, including possible RXFP1 oligomerization. 61,62 Another possibility is that specific neuronal systems must be physiologically challenged to become sensitive to RXFP1 activation, as observed for other neuropeptides that play a particular role in pathophysiological conditions. 31 ...
La douleur chronique souvent accompagnée d’anxiété et de dépression est un fléau mondial. La modulation de la douleur par les neuropeptides (NP) est bien connu au niveau des afférences primaires et de la moelle épinière. Peu de données sont toutefois disponibles sur leur rôle dans la douleur dans le cerveau. La famille relaxine comprend la relaxine, présente dans le système nerveux central (SNC) et qui possède des propriétés antifibrotiques, et la relaxine-3, strictement exprimée dans le SNC et qui présente des effets anxiolytiques et antidépressifs. Notre objectif est d’étudier la modulation de la douleur par les neuropeptides relaxine et relaxine-3 dans un modèle de douleur inflammatoire persistante chez la souris.Nos résultats démontrent que non seulement le système relaxine-3 / RXFP3, mais aussi le système relaxine / RXFP1 encore très peu exploré dans le SNC, ont des effets analgésiques en conditions de douleur inflammatoire. Les sites d'action de ces systèmes peptidergiques comprennent des régions corticales (cortex cingulaire, claustrum) et sous-corticales (amygdale) qui régulent les voies descendantes et l'intégration sensorielle dans la moelle épinière. Nos données mettent en évidence le potentiel thérapeutique de cette famille peptidergique dont les rôles dans la douleur n'avaient jamais été testés.
... [23][24][25] Negative cooperativity of binding and its association with receptor dimerization/oligomerization are frequently reported for GPCRs from different classes. 23,26,27 These findings were also validated for the full-length GLP-1R by ligand accelerated dissociation, 11,17 although a concave Scatchard curve in the ligand binding assay was not observed. 28 Both the ligand accelerated dissociation and the concave-up Scatchard curve are typical indices of the negative cooperativity originally described for insulin receptor binding. ...
The glucagon‐like peptide‐1 receptor (GLP‐1R), a family B G‐protein coupled receptor (GPCR), regulates the insulin secretion following stimulation by ligands. The transmembrane domain (TM) mediates GLP‐1R homodimerization, which modulates its ligand binding and signaling. We investigated the possible involvement of the N‐terminal extracellular domain (NTD) in dimerization/oligomerization and dimer‐associated ligand binding by NanoLuc Binary Technology (NanoBiT). With improved NanoBiT detection using a decreasing substrate concentration, the negative cooperativity of ligand binding to the NTD was confirmed by accelerated dissociation and Scatchard analysis. The dimerization/oligomerization of the isolated NTD was observed by NanoBiT and validated by analytical ultracentrifugation, deriving the comparable dimerization affinity (~10⁵ M⁻¹). The NTD was also involved in the dimerization/oligomerization of the full‐length GLP‐1R with mutated TM4 at the cellular level. In an analysis of the parameters of the NTD binding, the Kd for the probe GLP‐1 (7‐36, A8G) was similar (6‐8 μM) in both the 1:1 binding model and the receptor dimerization model. Compared with GLP‐1 and dulaglutide, exenatide showed two‐site binding with Ki values of 1.4 pM and 8.7 nM. Our study indicates the involvement of NTD in the GLP‐1R dimerization/oligomerization and suggests that further investigations on the role in other family B GPCRs are needed.
... In addition, saturation binding experiments in HEK293 cells lacking G as , the primary G protein to which RXFP1 is coupled in HEK cells, did not show any significant difference in relaxin binding affinity, and previous studies have found no change in relaxin binding affinity in membrane preparations in the presence of the non-hydrolysable GTP analogs, Gpp(NH)p and GTPgS (Scott et al., 2006). Another alternative explanation for a two-phase TamRLX dissociation relates to possible homodimerization of RXFP1 (Svendsen et al., 2008), or differing localization of the receptor within the cell (for example, intracellular pools of receptor, receptor clustering within different membrane microdomains, or receptor internalization), providing a framework by which two relaxin-binding sites might exist with two slightly different affinities. These explanations also fall short for three reasons. ...
The peptide hormone H2 relaxin has demonstrated promise as a therapeutic, but mimetic development has been hindered by the poorly understood relaxin receptor RXFP1 activation mechanism. H2 relaxin is hypothesized to bind to two distinct ECD sites, which reorientates the N-terminal LDLa module to activate the transmembrane domain. Here we provide evidence for this model in live cells by measuring bioluminescence resonance energy transfer (BRET) between nanoluciferase-tagged RXFP1 constructs and fluorescently labeled H2 relaxin (NanoBRET). Additionally, we validate these results using the related RXFP2 receptor and chimeras with an inserted RXFP1-binding domain utilizing NanoBRET and nuclear magnetic resonance studies on recombinant proteins. We therefore provide evidence for the multi-component molecular mechanism of H2 relaxin binding to RXFP1 on the full-length receptor in cells. Also, we show the utility of NanoBRET real-time binding kinetics to reveal subtle binding complexities, which may be overlooked in traditional equilibrium binding assays.
... Nevertheless, there are two intriguing, functionally relevant consequences of negative cooperativity: an increased functional range of the ligand over a wider concentration range and a decrease in ligand residence time at the receptor as the free ligand concentration increases, potentially allowing selective activation of different signaling pathways (Shymko et al., 1997). The negative cooperativity concentration-response curve for relaxin binding to RXFP1 is linear, although the absence of a protein structure precludes conclusions regarding the functional consequences of this observation (Svendsen et al., 2008b). ...
Relaxin, insulin-like peptide 3 (INSL3), relaxin-3, and INSL5 are the cognate ligands for the relaxin family peptide (RXFP) receptors 1-4, respectively. RXFP1 activates pleiotropic signaling pathways including the signalosome protein complex that facilitates high-sensitivity signaling; coupling to Gαs, Gαi, and Gαo proteins; interaction with glucocorticoid receptors; and the formation of hetero-oligomers with distinctive pharmacological properties. In addition to relaxin-related ligands, RXFP1 is activated by Clq-tumor necrosis factor-related protein 8 and by small-molecular-weight agonists, such as ML290 [2-isopropoxy-N-(2-(3-(trifluoromethylsulfonyl)phenylcarbamoyl)phenyl)benzamide], that act allosterically. RXFP2 activates only the Gαs- and Gαo-coupled pathways. Relaxin-3 is primarily a neuropeptide, and its cognate receptor RXFP3 is a target for the treatment of depression, anxiety, and autism. A variety of peptide agonists, antagonists, biased agonists, and an allosteric modulator target RXFP3. Both RXFP3 and the related RXFP4 couple to Gαi/Gαo proteins. INSL5 has the properties of an incretin; it is secreted from the gut and is orexigenic. The expression of RXFP4 in gut, adipose tissue, and β-islets together with compromised glucose tolerance in INSL5 or RXFP4 knockout mice suggests a metabolic role. This review focuses on the many advances in our understanding of RXFP receptors in the last 5 years, their signal transduction mechanisms, the development of novel compounds that target RXFP1-4, the challenges facing the field, and current prospects for new therapeutics.
Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.
... Insl3 functions through Rxfp2. Insl3/Rxfp2 signaling mediates male and female reproduction [26][27][28] and the pathogenesis of male osteoporosis associated with hypogonadism [29][30][31]. The effects of Insl3 on skeletal metabolism include induction of macrophage colony-stimulating factor and alkaline phosphatase (ALP) [29]. ...
... At the in vitro level, H2 relaxin (1Á 100 ng/ml) administration to TGF-b1-stimulated human lung fibroblasts dose-dependently decreased the overexpression of collagen and fibronectin that was induced by TGF-b1, while increasing MMP-1 expression in a biphasic, dose-dependent manner. 34 The biphasic actions of relaxin are potentially explained from recent studies, which have shown that RXFP1 undergoes dimerization and ''negative cooperativity'' 38,39 to accelerate the dissociation rate of relaxin peptides with ascending concentrations, leading to lower physiological responses of these peptides at higher concentrations. A similar H2 relaxin (100 ng/ml)-mediated reduction in collagen and fibronectin production was demonstrated in TGF-b1-treated rat pulmonary artery fibroblasts. ...
INTRODUCTION
Fibrosis is a pathophysiological response to injury characterized by excess, abnormal extracellular matrix (ECM) deposition disrupting the normal tissue architecture and function of affected organs. Lung fibrosis may occur as a component of systemic or local diseases, but the most pernicious clinical form is idiopathic pulmonary fibrosis (IPF), which represents a major challenge for pneumologists, being refractory to any conventional therapy. This has prompted the development of suitable animal models of lung fibrosis to test novel therapies.
OBJECTIVES
This mini-review summarizes the current knowledge on the antifibrotic properties of relaxin, a natural hormone capable of modulating ECM turnover in multiple target organs, which can be viewed as a promising antifibrotic drug. In fact, human recombinant relaxin has been and is currently being investigated in clinical trials due to its favorable bioactivity profile.
RESULTS AND CONCLUSION
Based on several in vitro and in vivo studies, relaxin has emerged as a key endogenous factor involved in the regulation of ECM synthesis and remodeling by fibroblasts. Knockout mice for relaxin or its receptor are prone to heart, kidney, and lung fibrosis: in the former animal model, the profibrotic trend can be reverted by exogenous administration of the hormone, implicating its therapeutic potential. To overcome the intrinsic limitations of the peptide properties of the hormone, low molecular weight relaxin receptor agonists have been synthesized and are currently being evaluated as alternative treatments. As shown using a mouse model of bleomycin-induced lung fibrosis, these
compounds are capable of blunting the pulmonary inflammatory reaction to bleomycin and the subsequent enhanced collagen deposition.
Taken together, these existing notions and clues suggest that relaxin and related molecules deserve to be further investigated as possible antifibrotic drugs.
... At the in vitro level, H2 relaxin (1Á 100 ng/ml) administration to TGF-b1-stimulated human lung fibroblasts dose-dependently decreased the overexpression of collagen and fibronectin that was induced by TGF-b1, while increasing MMP-1 expression in a biphasic, dose-dependent manner. 34 The biphasic actions of relaxin are potentially explained from recent studies, which have shown that RXFP1 undergoes dimerization and ''negative cooperativity'' 38,39 to accelerate the dissociation rate of relaxin peptides with ascending concentrations, leading to lower physiological responses of these peptides at higher concentrations. A similar H2 relaxin (100 ng/ml)-mediated reduction in collagen and fibronectin production was demonstrated in TGF-b1-treated rat pulmonary artery fibroblasts. ...
INTRODUCTION
Fibrosis is a pathophysiological response to injury characterized by excess, abnormal extracellular matrix (ECM) deposition disrupting the normal tissue architecture and function of affected organs. Lung fibrosis may occur as a component of systemic or local diseases, but the
most pernicious clinical form is idiopathic pulmonary fibrosis (IPF), which represents a major challenge for pneumologists, being refractory to any conventional therapy. This has prompted the development of suitable animal models of lung fibrosis to test novel therapies.
OBJECTIVES
This mini-review summarizes the current knowledge on the antifibrotic properties of relaxin, a natural hormone capable of modulating ECM turnover in multiple target organs, which can be viewed as a promising antifibrotic drug. In fact, human recombinant relaxin has been and is
currently being investigated in clinical trials due to its favorable bioactivity profile.
RESULTS AND CONCLUSION
Based on several in vitro and in vivo studies, relaxin has emerged as a key endogenous factor involved in the regulation of ECM synthesis and remodeling by fibroblasts. Knockout mice for relaxin or its receptor are prone to heart, kidney, and lung fibrosis: in the former animal model, the profibrotic trend can be reverted by exogenous administration of the hormone, implicating its therapeutic potential. To overcome the intrinsic limitations of the peptide properties of the hormone, low molecular weight relaxin receptor agonists have been synthesized and are currently being evaluated as alternative treatments. As shown using a mouse model of bleomycin-induced lung fibrosis, these compounds are capable of blunting the pulmonary inflammatory reaction to bleomycin and the subsequent enhanced collagen deposition. Taken together, these existing notions and clues suggest that relaxin and related molecules deserve to be further investigated as possible antifibrotic drugs.
Keywords: relaxin, lung fibrosis, RXFP1 agonist, animal models of fibrosis
... These findings resemble other heteromerization paradigms among G-protein-coupled receptors. 35,36 In addition, we now demonstrate that H2 relaxin appeared to signal through these constitutive RXFP1-AT 2 R heterodimers to induce downstream functional effects, at the level of pERK1/2 (which had previously been shown to be activated by relaxin-RXFP1 12,13 or Ang II-AT 2 R 28 to mediate organ protection), to inhibit the TGF-b1/Smad2 axis. Our added finding that pERK1/2 responses were almost completely blocked by PD123319 under conditions where RXFP1-AT 2 R dimers are present also suggested that these RXFP1-AT 2 R heteromers may be regulated by AT 2 R blockade, as a novel means by which the actions of H2 relaxin may be inhibited. ...
Fibrosis is a hallmark of chronic kidney disease, for which there is currently no effective cure. The hormone relaxin is emerging as an effective antifibrotic therapy; however, its mechanism of action is poorly understood. Recent studies have shown that relaxin disrupts the profibrotic actions of transforming growth factor-β1 (TGF-β1) by its cognate receptor, relaxin family peptide receptor 1 (RXFP1), extracellular signal-regulated kinase phosphorylation, and a neuronal nitric oxide synthase-dependent pathway to abrogate Smad2 phosphorylation. Since angiotensin II also inhibits TGF-β1 activity through its AT2 receptor (AT2R), we investigated the extent to which relaxin interacts with the AT2R. The effects of the AT2R antagonist, PD123319, on relaxin activity were examined in primary rat kidney myofibroblasts, and in kidney tissue from relaxin-treated male wild-type and AT2R-knockout mice subjected to unilateral ureteric obstruction. Relaxin's antifibrotic actions were significantly blocked by PD123319 in vitro and in vivo, or when relaxin was administered to AT2R-knockout mice. While heterodimer complexes were formed between RXFP1 and AT2Rs independent of ligand binding, relaxin did not directly bind to AT2Rs but signaled through RXFP1-AT2R heterodimers to induce its antifibrotic actions. These findings highlight a hitherto unrecognized interaction that may be targeted to control fibrosis progression.Kidney International advance online publication, 15 January 2014; doi:10.1038/ki.2013.518.
... The mechanism of activation whereby ligand binding alone is unable to induce an active receptor conformation but requires distinct side chain-driven interactions involving the N-terminal LDLa module presents a novel paradigm in GPCR signal activation. Based on the discovery that RXFP1 and RXFP2 form constitutive homodimers (42,43), we postulate that ligand binding at one monomer of a dimer may position the LDLa module for interaction with the transmembrane domain of its dimerization partner to form the active receptor complex capable of signaling (5). Although hypothetical, this model highlights the complex signaling and binding mechanism of the RXFP1 receptor. ...
The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true “ligand” of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation.
Background: Activation of the relaxin receptor RXFP1 is driven by the LDLa module at the RXFP1 N terminus.
Results: LDLa residues Leu-7, Tyr-9, and Lys-17 all contribute to receptor activation via interactions involving their hydrophobic side chains.
Conclusion: These interactions induce the active receptor conformation, suggesting a novel mode of GPCR activation.
Significance: This novel mechanism of GPCR activation may lead to new drug development.
... In 2005 and 2006, negative cooperativity was found in the binding of chemokines to chemokine receptor (CCR) homo- and heteromers (El-Asmar et al., 2005; Springael et al., 2006). Furthermore, in 2008, a possible link between negative cooperativity and GPCR oligomerization was indicated for the relaxin family peptide receptor 1 (RXFP1) and RXFP2 (Svendsen et al., 2008a,b). Existence of positive cooperativity has also been reported in the binding of ligands to GPCRs. ...
The superfamily of the seven transmembrane G-protein-coupled receptors (7TM/GPCRs) is the largest family of membrane-associated receptors. GPCRs are involved in the pathophysiology of numerous human diseases, and they constitute an estimated 30–40% of all drug targets. During the last two decades, GPCR oligomerization has been extensively studied using methods like bioluminescence resonance energy transfer (BRET) and today, receptor–receptor interactions within the GPCR superfamily is a well-established phenomenon. Evidence of the impact of GPCR oligomerization on, e.g., ligand binding, receptor expression, and signal transduction indicates the physiological and pharmacological importance of these receptor interactions. In contrast to the larger and more thoroughly studied GPCR subfamilies A and C, the B1 subfamily is small and comprises only 15 members, including, e.g., the secretin receptor, the glucagon receptor, and the receptors for parathyroid hormone (PTHR1 and PTHR2). The dysregulation of several family B1 receptors is involved in diseases, such as diabetes, chronic inflammation, and osteoporosis which underlines the pathophysiological importance of this GPCR subfamily. In spite of this, investigation of family B1 receptor oligomerization and especially its pharmacological importance is still at an early stage. Even though GPCR oligomerization is a well-established phenomenon, there is a need for more investigations providing a direct link between these interactions and receptor functionality in family B1 GPCRs. One example of the functional effects of GPCR oligomerization is the facilitation of allosterism including cooperativity in ligand binding to GPCRs. Here, we review the currently available data on family B1 GPCR homo- and heteromerization, mainly based on BRET investigations. Furthermore, we cover the functional influence of oligomerization on ligand binding as well as the link between oligomerization and binding cooperativity.
... However, its receptor RXFP1 was only discovered a decade ago (8) which has limited the ability to characterize the structural basis of the mechanism of action of the hormone. The activation of the rather unusual RXFP1 and RXFP2 receptors is complex, requiring several interactions involving the LRR domain (15), the extracellular loops (43), and the N-terminal LDLa module (28), eventually leading to the activation of the receptor (44,45). Although one key feature, the RXXXRXXI relaxin cassette in the mid-region of the H2 relaxin B-chain and its interaction with the leucine-rich repeats of the ectodomain (14 -16), this interaction alone is not sufficient to explain all of the activation events involved. ...
H2 relaxin is a peptide hormone associated with a number of therapeutically relevant physiological effects, including regulation of collagen metabolism and multiple vascular control pathways. It is currently in phase III clinical trials for the treatment of acute heart failure due to its ability to induce vasodilation and influence renal function. It comprises 53 amino acids and is characterized by two separate polypeptide chains (A-B) that are cross-linked by three disulfide bonds. This size and complex structure represents a considerable challenge for the chemical synthesis of H2 relaxin, a major limiting factor for the exploration of modifications and derivatizations of this peptide, to optimize effect and drug-like characteristics. To address this issue, we describe the solid phase peptide synthesis and structural and functional evaluation of 24 analogues of H2 relaxin with truncations at the termini of its peptide chains. We show that it is possible to significantly truncate both the N and C termini of the B-chain while still retaining potent biological activity. This suggests that these regions are not critical for interactions with the H2 relaxin receptor, RXFP1. In contrast, truncations do reduce the activity of H2 relaxin for the related receptor RXFP2 by improving RXFP1 selectivity. In addition to new mechanistic insights into the function of H2 relaxin, this study identifies a critical active core with 38 amino acids. This minimized core shows similar antifibrotic activity as native H2 relaxin when tested in human BJ3 cells and thus represents an attractive receptor-selective lead for the development of novel relaxin therapeutics.
... The inverse "bell-shaped" dose-response effects of H3 relaxin on TGF-β1-stimulated collagen deposition, which resulted in the highest concentration of H3 relaxin evaluated (500 ng/mL) not having the same inhibitory effects as lower doses (100-250 ng/mL), are consistent with previous findings for H2 relaxin (35,(41)(42)(43) and other related peptides (44,45), which also produce biphasic dose-response curves, where higher concentrations of these peptides likewise do not produce the same maximum physiological responses. This is potentially explained from recent studies which have shown that RXFP1 undergoes dimerization and "negative cooperativity" (45,46) to accelerate the dissociation rate of relaxin peptides with ascending concentrations, leading to lower physiological responses of these peptides at higher concentrations. Additional studies though are required to fully understand this complex process and more importantly in 2) and TGF-β1 alone (lanes 3 and 4), TGF-β1 þ H3 relaxin (lanes 5 and 6), TGF-β1 þ H2 relaxin (lanes 7 and 8), or H3 relaxin alone (lanes 9 and 10) treated cells after 72 h in culture. ...
Human gene 3 (H3) relaxin is the most recently discovered member of the relaxin peptide family and can potentially bind all of the defined relaxin family peptide receptors (RXFP1-4). While its effects as a neuromodulator are being increasingly studied through its primary receptor, RXFP3, its actions via other RXFPs are poorly understood. Hence, we specifically determined the antifibrotic effects and mechanisms of action of H3 relaxin via the RXFP1 receptor using primary rat ventricular fibroblasts in vitro, which naturally express RXFP1, but not RXFP3, and a mouse model of fibrotic cardiomyopathy in vivo. Transforming growth factor β1 (TGF-β1) administration to ventricular fibroblasts significantly increased Smad2 phosphorylation, myofibroblast differentiation, and collagen deposition (all p < 0.05 vs untreated controls), while having no marked effect on matrix metalloproteinase (MMP) 9, MMP-13, tissue inhibitor of metalloproteinase (TIMP) 1, or TIMP-2 expression over 72 h. H3 relaxin (at 100 and 250 ng/mL) almost completely abrogated the TGF-β1-stimulated collagen deposition over 72 h, and its effects at 100 ng/mL were equivalent to that of the same dose of H2 relaxin. Furthermore, H3 relaxin (100 ng/mL) significantly inhibited TGF-β1-stimulated cardiac myofibroblast differentiation and TIMP-1 and TIMP-2 expression to an equivalent extent as H2 relaxin (100 ng/mL), while also inhibiting Smad2 phosphorylation to approximately half the extent of H2 relaxin (all p < 0.05 vs TGF-β1). Lower doses of H3 (50 ng/mL) and H2 (50 ng/mL) relaxin additively inhibited TGF-β1-stimulated collagen deposition in vitro, while H3 relaxin was also found to reverse left ventricular collagen overexpression in the model of fibrotic cardiomyopathy in vivo. These combined findings demonstrate that H3 relaxin exerts antifibrotic actions via RXFP1 and may enhance the collagen-inhibitory effects of H2 relaxin.
... Radioligand binding experiments are one of the fundamental methods for establishing conventional "ontarget" allosterism at GPCRs and have themselves provided early evidence of cooperative homomeric GPCR interactions well before the first GPCRs were even cloned (Limbird et al., 1975;Davis et al., 1977;Powell-Jones et al., 1979). More recently, negative cooperativity has been demonstrated by radioligand binding at a number of GPCR homomers, such as H2 relaxin at relaxin RXFP1 homomers (Svendsen et al., 2008b) and insulinlike peptide 3 at RXFP2 receptors (Svendsen et al., 2008a), neurotensin at neurotensin 1 receptors (White et al., 2007), secretin at secretin receptors (Gao et al., 2009), vasopressin and oxytocin at their respective receptors (Albizu et al., 2006), atypical antipsychotics clozapine and risperidone at the serotonin 5-HT 2A (Brea et al., 2009), quinuclidinylbenzilate and N-methylscopolamine at muscarinic acetylcholine M 2 receptors (Wreggett and Wells, 1995;Chidiac et al., 1997;Sum et al., 2001;Park et al., 2002), raclopride, spiperone, and nemonapride at dopamine D 2 homomers (Armstrong and Strange, 2001;Vivo et al., 2006), and thyroid-stimulating hormone (TSH) and follicle-stimulating hormone at their respective GPCRs (Urizar et al., 2005). ...
For many years seven transmembrane domain G protein-coupled receptors (GPCRs) were thought to exist and function exclusively as monomeric units. However, evidence both from native cells and heterologous expression systems has demonstrated that GPCRs can both traffic and signal within higher-order complexes. As for other protein-protein interactions, conformational changes in one polypeptide, including those resulting from binding of pharmacological ligands, have the capacity to alter the conformation and therefore the response of the interacting protein(s), a process known as allosterism. For GPCRs, allosterism across homo- or heteromers, whether dimers or higher-order oligomers, represents an additional topographical landscape that must now be considered pharmacologically. Such effects may offer the opportunity for novel therapeutic approaches. Allosterism at GPCR heteromers is particularly exciting in that it offers additional scope to provide receptor subtype selectivity and tissue specificity as well as fine-tuning of receptor signal strength. Herein, we introduce the concept of allosterism at both GPCR homomers and heteromers and discuss the various questions that must be addressed before significant advances can be made in drug discovery at these GPCR complexes.
... Constitutive homodimerization of RXFP1 was initially shown to occur in the endoplasmic reticulum via bioluminescence resonance energy transfer (BRET) (Kern et al., 2008). Another study revealed that dimerization occurs via the seven transmembrane region with the extracellular domain stabilizing the interaction (Svendsen et al., 2008b). RXFP2 also homodimerizes in a manner similar to RXFP1; dimerization occurs via the transmembrane region with the ectodomain stabilizing the dimer (Svendsen et al., 2008a). ...
The receptors for members of the relaxin peptide family have only recently been discovered and are G-protein-coupled receptors (GPCRs). Relaxin and insulin-like peptide 3 (INSL3) interact with the leucine-rich-repeat-containing GPCRs (LGRs) LGR7 and LGR8, respectively. These receptors show closest similarity to the glycoprotein hormone receptors and contain large ectodomains with 10 leucine-rich repeats (LRRs) but are unique members of the LGR family (class C) as they have an LDL class A (LDLa) module at their N-terminus. In contrast, relaxin-3 and INSL5 interact with another class of type I GPCRs which lack a large ectodomain, the peptide receptors GPCR135 and GPCR142, respectively. These receptors are now classified as relaxin family peptide (RXFP) receptors, RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 (GPCR135) and RXFP4 (GPCR142). This review outlines the identification of the peptides and receptors, their expression profiles and physiological roles and the functional interactions of the peptides with their unique receptors.
... 110 Evidence that RXFP1 receptors form oligomers is emerging and this oligomerization may be responsible for the negative cooperativity observed in receptor binding studies. [111][112][113] The accelerated dissociation of ligand that occurs at higher concentrations shortens the time the ligand remains bound to the receptor and may influence the pattern and degree of activation of signaling pathways. ...
Although substantial advances have been achieved in recent decades in the clinical management of heart diseases, new therapies that provide better or additional efficacy with minimal adverse effects are urgently required. Evidence that has accumulated since the 1990s indicates that the peptide hormone relaxin has multiple beneficial actions in the cardiovascular system under pathological conditions and, therefore, holds promise as a novel therapeutic intervention. Clinical trials for heart failure therapy using relaxin revealed several beneficial actions. Here we review findings from mechanistic and applied research in this field, comment on the outcomes of recent phase I/II clinical trails on patients with heart failure, and highlight settings of cardiovascular diseases where relaxin might be effective.
Class A, rhodopsin‐like, G‐protein‐coupled receptors (GPCRs) are by far the largest class of GPCRs and are integral membrane proteins used by various cells to convert extracellular signals into intracellular responses. Initially, class A GPCRs were believed to function as monomers, but a growing body of evidence has emerged to suggest that these receptors can function as homodimers and heterodimers and can undergo functional crosstalk to influence the actions of agonists or antagonists acting at each receptor. This review will focus on the angiotensin type 1 (AT1) and type 2 (AT2) receptors, as well as the relaxin family peptide receptor 1 (RXFP1), each of which have their unique characteristics but have been demonstrated to undergo some level of interaction when appropriately co‐expressed, which influences the function of each receptor. In particular, this receptor functional crosstalk will be discussed in the context of fibrosis, the tissue scarring that results from a failed wound‐healing response to injury, and which is a hallmark of chronic disease and related organ dysfunction.
LINKED ARTICLES
This article is part of a themed issue Therapeutic Targeting of G Protein‐Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc
Introduction:
The relaxin peptide signaling system is involved in diverse physiological processes, but its possible roles in the brain, including nociception, are largely unexplored.
Objective:
In light of abundant expression of relaxin receptor (RXFP1) mRNA/protein in brain regions involved in pain processing, we investigated the effects of central RXFP1 activation on nociceptive behavior in a mouse model of inflammatory pain and examined the neurochemical phenotype and connectivity of relaxin and RXFP1 mRNA-positive neurons.
Methods:
Mice were injected with Complete Freund Adjuvant (CFA) into a hind paw. After 4 days, the RXFP1 agonist peptides, H2-relaxin or B7-33, ± the RXFP1 antagonist, B-R13/17K-H2, were injected into the lateral cerebral ventricle, and mechanical and thermal sensitivity were assessed at 30 to 120 minutes. Relaxin and RXFP1 mRNA in excitatory and inhibitory neurons were examined using multiplex, fluorescent in situ hybridization. Relaxin-containing neurons were detected using immunohistochemistry and their projections assessed using fluorogold retrograde tract-tracing.
Results:
Both H2-relaxin and B7-33 produced a strong, but transient, reduction in mechanical and thermal sensitivity of the CFA-injected hind paw alone, at 30 minutes postinjection. Notably, coinjection of B-R13/17K-H2 blocked mechanical, but not thermal, analgesia. In the claustrum, cingulate cortex, and subiculum, RXFP1 mRNA was expressed in excitatory neurons. Relaxin immunoreactivity was detected in neurons in forebrain and midbrain areas involved in pain processing and sending projections to the RXFP1-rich, claustrum and cingulate cortex. No changes were detected in CFA mice.
Conclusion:
Our study identified a previously unexplored peptidergic system that can control pain processing in the brain and produce analgesia.
“Healthy” aging drives structural and functional changes in the heart including maladaptive electrical remodeling, fibrosis and inflammation, which lower the threshold for cardiovascular diseases such as heart failure (HF) and atrial fibrillation (AF). Despite mixed results in clinical trials, Relaxin-therapy for 2-days reduced mortality by 37% at 180-days post-treatment, in patients with acute decompensated HF.
Relaxin's short lifespan (2 − 3 h) but long-lasting protective actions suggested that relaxin acts at a genomic level to reverse maladaptive remodeling in AF, HF and aging. Our recent studies showed that a 2-week treatment with Relaxin (0.4 mg/kg/day) of aged (24 months old F-344 rats) increases the expression of voltage-gated Na⁺ channels (mRNA, Nav1.5 and INa), connexin-43, abrogates inflammatory and immune responses and reverses myocardial fibrosis and cellular hypertrophy of the aged hearts. Relaxin acts directly at a wide range of cell types in the cardiovascular system that express its cognate GPCR receptor, RXFP1. RNA-seq analysis of young and aged hearts with and without Relaxin treatment revealed that “normal” aging altered the expression of ~ 10% of genes expressed in the ventricles, including: ion channels, components of fibrosis, hemodynamic biomarkers, immune and inflammatory responses which were reversed by Relaxin. The extensive cardiovascular remodeling caused by Relaxin was mediated through the activation of the Wnt/β-catenin signaling pathway which was otherwise suppressed by in adult cardiomyocytes intracellular by cytosolic Dickkopf1 (Dkk1). Wnt/β-catenin signaling is a mechanism that can explain the pleiotropic actions of Relaxin and the marked reversal of genomic changes that occur in aged hearts.
The pancreas has both an exocrine (digestive) function exerted by acinar cells and endocrine functions exerted by five different cell types secreting at least eight peptide hormones: insulin, glucagon, somatostatin, neuronostatin, pancreatic polypeptide, amylin, ghrelin, urocortin III, and stanniocalcin 2. In this chapter, we discuss the development, general organization, and cellular composition of the endocrine islets of Langerhans, the paracrine relationships between the different cell types, and the structure and function and mechanism of action of the various peptide hormones, and their role in metabolic pathophysiology.
This study aimed to functionally characterize β2-adrenergic (β2AR) and insulin receptor (IR) heteromers in regard to β-arrestin 2 (βarr2) recruitment and cAMP signaling and to examine the involvement of the cytoplasmic portion of the IR β chain in heteromerization with β2AR. Evidence for β2AR:IR:βarr2 complex formation and the specificity of the IR:βarr2 interaction was first provided by bioinfomatics analysis. Receptor-heteromer investigation technology (HIT) then provided functional evidence of β2AR:IR heterodimerization by showing isoproterenol-induced but not insulin-induced GFP²-βarr2 recruitment to the β2AR:IR complex; the IR:βarr2 interaction was found to only be constitutive. The constitutive IR:βarr2 BRET signal (BRETconst) was significantly smaller in cells coexpressing IR-RLuc8 and a GFP²-βarr2 1–185 mutant lacking the proposed IR binding domain. β2AR:IR heteromerization also influenced the pharmacological phenotype of β2AR, i.e., its efficacy in recruiting βarr2 and activating cAMP signaling. Evidence suggesting involvement of the cytoplasmic portion of the IR β chain in the interaction with β2AR was provided by BRET² saturation and HIT assays using an IR 1–1271 stop mutant lacking the IR C-terminal tail region. For the complex consisting of IR 1–1271–RLuc8:β2AR-GFP², saturation was not reached, most likely reflecting random collisions between IR 1–1271 and β2AR. Furthermore, in the HIT assay, no substantial agonist-induced increase in the BRET² signal was detected that would be indicative of βarr2 recruitment to the IR 1–1271:β2AR heteromer. Complementary 3D visualization of β2AR:IR provided supporting evidence for stability of the heterotetramer complex and identified amino acid residues involved in β2AR:IR heteromerization.
This article is part of the Special Issue entitled ‘Receptor heteromers and their allosteric receptor-receptor interactions’.
Human gene-2 relaxin (H2 relaxin) is a pleiotropic hormone with powerful vasodilatory and anti-fibrotic properties which has led to its clinical evaluation and provisional FDA approval as a treatment for acute heart failure. The diverse effects of H2 relaxin are mediated via its cognate G protein coupled-receptor (GPCR), Relaxin Family Peptide Receptor (RXFP1), leading to stimulation of a combination of cell signalling pathways that includes cyclic adenosine monophosphate (cAMP) and extracellular-signal-regulated kinases (ERK)1/2. However, its complex two-chain (A and B), disulfide-rich insulin-like structure is a limitation to its facile preparation, availability and affordability. Furthermore, its strong activation of cAMP signaling is likely responsible for reported detrimental tumor-promoting actions that may preclude long-term use of this drug for treating human disease. Here we report the design and synthesis of a H2 relaxin B-chain-only analogue, B7-33, which was shown to bind to RXFP1 and preferentially activate the pERK pathway over cAMP in cells that endogenously expressed RXFP1. Thus, B7-33 represents the first functionally selective agonist of the complex GPCR, RXFP1. Importantly, this small peptide agonist prevented or reversed organ fibrosis and dysfunction in three pre-clinical rodent models of heart or lung disease with similar potency to H2 relaxin. The molecular mechanism behind the strong anti-fibrotic actions of B7-33 involved its activation of RXFP1-angiotensin II type 2 receptor heterodimers that induced selective downstream signaling of pERK1/2 and the collagen-degrading enzyme, matrix metalloproteinase (MMP)-2. Furthermore, in contrast to H2 relaxin, B7-33 did not promote prostate tumor growth in vivo. Our results represent the first known example of the minimisation of a two-chain cyclic insulin-like peptide to a single-chain linear peptide that retains potent beneficial agonistic effects.
The BRET technology is a relatively new, robust and versatile platform suitable for studying protein-protein interactions and protein conformational changes in living cells. BRET occurs when energy is transferred from the bioluminescent donor to the complementary fluorescent acceptor by the resonance energy transfer (RET). For the specific labeling of proteins, Renilla luciferase (Rluc) is used as the donor and the fluorescent label, usually the mutant form of the green fluorescent protein (GFP) variant GFP2, is used as the acceptor molecule. Upon the addition of the substrate for Rluc, BRET can occur. Bioluminescence is caused by catalytic degradation of the substrate by the Rluc energy donor, which enables the excitation of GFP, and, consequently, fluorescence is re-emitted by GFP. Several versions of BRET have been developed that use different substrates and/or energy donor/acceptor couples to improve stability and specificity of the BRET signal. The review presents development and utilization of the BRET technology in the field of seven-transmembrane receptors (7TMRs), which are targets for more than 40% of clinically marketed drugs and represent the majority of new drug discovery targets. This study summarizes mainly our own results originating from 7TMRs dimerization studies, 7TMR/-arrestins interaction and ligand-induced conformational changes in -arrestins as well as different approaches (e.g. mutations inarrestins, 7TMR C-terminal tail swapping) to optimize compound medium/high-throughput BRET-based screening assays.
Objective: To highly express human relaxin-2 analogue (HR2) in Pichia pastoris, and determine the bioactivity of expressed product. Methods: HR2 gene was synthesized by modification of codons, based on which recombinant plasmid pPICZaA-HR2 was constructed and transformed to P. pastoris PGS115 for expression under induction of methanol. The expressed product was identified by Tricine-SDS-PAGE and Western blot, then purified by ultrafiltration and column chromatography, and determined for bioactivity after removal of synthetic C peptide. Results: Restriction analysis and sequencing proved that recombinant plasmid pPICZαA-HR2 was constructed correctly. Tricine-SDS-PAGE showed that the expressed recombinant protein in a secretory form, with a relative molecular mass of about 6 500, contained 47.6% (451 μg/ml) of total somatic protein. Western blot showed good reactogenicity of the recombinant protein. The purified recombinant protein reached a purity of 96% and, after removal of C peptide, showed stimulating activity to THP-1 cells. Conclusion: Recombinant HR2 was highly expressed in P. pastoris GS115, which showed a certain bioactivity.
We demonstrate two novel mutations of the LHCGR, each homozygous, in a 46,XY patient with severe Leydig cell hypoplasia. One is a mutation in the signal peptide (p.Gln18_Leu19ins9; referred to herein as SP) that results in an alteration of the coding sequence of the N-terminus of the mature mutant receptor. The other mutation (p.G71R) is also within the ectodomain. Similar to many other inactivating mutations, the cell surface expression of recombinant hLHR(SP,G71R) is greatly reduced due to intracellular retention. However, we made the unusual discovery that the intrinsic efficacy for agonist-stimulated cAMP in the reduced numbers of receptors on the cell surface was greatly increased relative to the same low number of cell surface wild-type receptor. Remarkably, this appears to be a general attribute of misfolding mutations in the ectodomains, but not serpentine domains, of the gonadotropin receptors. These findings suggest that there must be a common, shared mechanism by which disparate mutations in the ectodomain that cause misfolding and therefore reduced cell surface expression concomitantly confer increased agonist efficacy to those receptor mutants on the cell surface. Our data further suggest that, due to their increased agonist efficacy, extremely small changes in cell surface expression of misfolded ectodomain mutants cause larger than expected alterations in the cellular response to agonist. Therefore, for inactivating LHCGR mutations causing ectodomain misfolding, the numbers of cell surface mutant receptors on fetal Leydig cells of 46,XY individuals exert a more exquisite effect on the relative severity of the clinical phenotypes than already appreciated.
Relaxin-like factor (RLF), generally known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development. However, its role in adult males is not fully understood. We investigate the function of INSL3 in male Saanen goats by identifying cell types expressing its receptor, relaxin/insulin-like family peptide receptor (RXFP)2 and by characterizing the developmental expression pattern of INSL3 and RXFP2 and the binding of INSL3 to target cells in the male reproductive system. A highly specific RXFP2 antibody that co-localizes with an anti-FLAG antibody in HEK-293 cells recognizes RXFP2-transcript-expressing cells in the testis. INSL3 and RXFP2 mRNA expression is upregulated in the testis, starting from puberty. INSL3 mRNA and protein expression has been detected in Leydig cells, whereas RXFP2 mRNA and protein localize to Leydig cells, to meiotic and post-meiotic germ cells and to the epithelium and smooth muscle of the cauda epididymis and vas deferens. INSL3 binds to all of these tissues and cell types, with the exception of Leydig cells, in a hormone-specific and saturable manner. These results provide evidence for a functional intra- and extra-testicular INSL3 ligand-receptor system in adult male goats.
Relaxin is a member of the relaxin/insulin peptide hormone superfamily and is characterized by a two-chain structure constrained by three disulfide bonds. Relaxin is a pleiotropic hormone and involved in a number of physiological and pathogenic processes, including collagen and cardiovascular regulation, tissue remodelling during pregnancy and cancer. Crystallographic and ultracentrifugation experiments have revealed that the human form of relaxin, H2 relaxin, self-associates into dimers, but the significance of this is poorly understood. Here we present the NMR structure of a monomeric, amidated form of H2 relaxin, and compare its features and behaviour in solution to those of native H2 relaxin. The overall structure of H2 relaxin is retained in the monomeric form. H2 relaxin amide is fully active at the relaxin receptor RXFP1 and thus dimerisation is not required for biological activity. Analysis of NMR chemical shifts and relaxation parameters identified internal motion in H2 relaxin at the pico-nanosecond and milli-microsecond time scales, which is commonly seen in other relaxin and insulin peptides, and might be related to function.
The Relaxin receptor, RXFP1, is a complex G-protein coupled receptor (GPCR). It has a rhodopsin-like 7 transmembrane helix region and a large ecto-domain containing Leucine-rich repeats and a Low Desnsity Lipoprotein Class-A module at the N-terminus. RXFP1 and the closely related receptor for INSL3, RXFP2 are the only mammalian GPCRs to contain an LDL-A module. The LDL-A module has been shown to be essential for receptor signal activation. RXFP1, like other GPCRs, has been shown to form dimers however the interface upon association is currently unknown. As LDL-A modules are commonly found as repeats we hypothesized that the LDL-A module may associate at the dimer interface and play a role in receptor activation. To this end we analyzed the ability for the LDL-A module to oligomerise via Analytical Ultracentrifugation (AUC).
The Encyclopedia of Signaling Molecules functions as a dictionary and compendium of over 350 biologically important signaling molecules. In this chapter we summarize: 1) historical background and function of Rab7, 2) Rab7a activation and localization, 3) Rab7a effectors in the control of endocytic trafficking, and 4) Rab7a in endosomal signaling. Rab7a GTPase regulators, effectors and their functions are given in tabular form.
The mechanisms whereby insulin analogues may cause enhanced mitogenicity through activation of either the insulin receptor (IR) or the IGF-I receptor (IGF-IR) are incompletely understood. We demonstrate that in L6 myoblasts expressing only IGF-IRs as well as in the same cells overexpressing the IR, IGF-I, insulin, and AspB10 insulin (X10) downregulate the mRNA expression level of the cell cycle inhibitor cyclin G2, as measured by qRT-PCR, and induce cell growth measured by [6-3H] thymidine incorporation into DNA. Western blotting showed a marked downregulation of cyclin G2 at the protein level in both cell lines. Overexpression of cyclin G2 in the two cell lines diminished the mitogenic effect of all three ligands. The use of specific inhibitors indicated that both the MAPK and the PI3K pathways mediate the downregulation of Ccng2. The downregulation of CCNG2 by the three ligands was also observed in other cell lines: MCF-7, HMEC, Saos-2, R-/IR, and INS-1. These results indicate that regulation of cyclin G2 is a key mechanism whereby insulin, insulin analogues and IGF-I stimulate cell proliferation.
Seven transmembrane (7TM) receptors constitute the largest and the most diverse superfamily of proteins encoded in the human genome comprising at least 850 putative members. These receptors are widely expressed in the body and play a fundamental role in physiology and pathophysiology. Not only do they regulate many physiological processes, but drugs that target these receptors and either activate or inactivate them, account for the most prosperous drugs sold worldwide. Of approximately 500 currently marketed drugs, more then 30 % are modulators of 7TM receptor function. In the last two decades, impressive progress in the understanding of 7TM receptor function has been achieved, though dimerization or oligomerization of 7TM receptors is still a novel and controversial concept. Although a large quantity of data, obtained by different biophysical, biochemical, structural and functional approaches e.g. coimunoprecipitation, Western blot, bioluminescence and fluorescence resonance energy transfer (BRET and FRET, respectively) argue for dimerization or oligomerization of these receptors, several publications criticized the applied methods and challenged the concept. In this paper the main and the most important techniques are presented and complemented with our ideas.
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.
Relaxin is a naturally occurring peptide hormone that plays a central role in the hemodynamic and renovascular adaptive changes
that occur during pregnancy. Triggering similar changes could potentially be beneficial in the treatment of patients with
heart failure. The effects of relaxin include the production of nitric oxide, inhibition of endothelin, inhibition of angiotensin
II, production of VEGF, and production of matrix metalloproteinases. These effects lead to systemic and renal vasodilation,
increased arterial compliance, and other vascular changes. The recognition of this has led to the study of relaxin for the
treatment of heart failure. An initial pilot study has shown favorable hemodynamic effects in patients with heart failure,
including reduction in ventricular filling pressures and increased cardiac output. The ongoing RELAX-AHF clinical program
is designed to evaluate the effects of relaxin on the symptoms and outcomes in a large group of patients admitted to hospital
for acute heart failure. This review will summarize both the biology of relaxin and the data supporting its potential efficacy
in human heart failure.
The classical second messenger cAMP is important in diverse physiological processes, where its spatial and temporal compartmentalization allows precise control over multiple cellular events. Within this context, G‐protein‐coupled receptors (GPCRs) govern specialized pools of cAMP, which are functionally specific for the unique cellular effects attributed to a particular system. The relaxin receptor, RXFP1, is a GPCR that exerts pleiotropic physiological effects including a potent anti‐fibrotic response, increased cancer metastases, and has efficacy as a vasodilator in heart failure. On a cellular level, relaxin stimulation of RXFP1 results in the activation of multiple G‐protein pathways affecting cAMP accumulation. Specificity and diversity in the cAMP signal generated by RXFP1 is controlled by differential G‐protein coupling dependent upon the background of cellular expression, and cAMP compartmentalization. Further complexity in cAMP signalling results from the constitutive assembly of an RXFP1–signalosome, which specifically responds to low concentrations of relaxin, and activates a distinct cAMP pathway. The RXFP1–signalosome is a higher‐order protein complex that facilitates receptor sensitivity to attomolar concentration of peptide, exhibits constitutive activity and dual coupling to G‐proteins and β‐arrestins and reveals a concentration‐biased agonism mediated by relaxin. The specific and directed formation of GPCR‐centered signalosomes allows an even greater spatial and temporal control of cAMP, thus rationalizing the considerable physiological scope of this ubiquitous second messenger.
LINKED ARTICLES This article is part of a themed section on the Molecular Pharmacology of G Protein‐Coupled Receptors (GPCRs). To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue‐6 . To view the 2010 themed section on the same topic visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2010.159.issue‐5/issuetoc
The receptors for relaxin and insulin-like peptide 3 (INSL3) are now well-characterized as the relaxin family peptide (RXFP) receptors RXFP1 and RXFP2, respectively. They are G-protein-coupled receptors (GPCRs) with closest similarity to the glycoprotein hormone receptors, with both containing large ectodomains with 10 leucine-rich repeats (LRRs). Additionally, RXFP1 and RXFP2 are unique in the LGR family in that they contain a low-density lipoprotein class A (LDL-A) module at their N-terminus. Ligand-mediated activation of RXFP1 and RXFP2 is a complex process involving various domains of the receptors. Primary ligand binding occurs via interactions between B-chain residues of the peptides with specific residues in the LRRs of the ectodomain. There is a secondary binding site in the transmembrane exoloops which may interact with the A chain of the peptides. Receptor signaling through cAMP then requires the unique LDL-A module, as receptors without this domain bind ligand normally but do not signal. This is an unconventional mode of activation for a GPCR, and the precise mode of action of the LDL-A module is currently unknown. The specific understanding of the mechanisms underlying ligand-mediated activation of RXFP1 and RXFP2 is crucial in terms of targeting these receptors for future drug development.
The insulin/relaxin superfamily of peptide hormones comprises 10 members in humans. The three members of the insulin-related subgroup bind to receptor tyrosine kinases (RTKs), while four of the seven members of the relaxin-like subgroup are now known to bind to G-protein-coupled receptors (GPCRs), the so-called relaxin family peptide receptors (RXFPs). Both systems have a long evolutionary history and play a critical role in fundamental biological processes, such as metabolism, growth, survival and longevity, and reproduction. The structural biology and ligand-binding kinetics of the insulin and insulin-like growth factor I receptors have been studied in great detail, culminating in the recent crystal structure of the insulin receptor extracellular domain. Some of the fundamental properties of these receptors, including constitutive dimerization and negative cooperativity, have recently been shown to extend to other RTKs and GPCRs, including RXFPs, confirming kinetic observations made over 30 years ago.
Most patients admitted for acute heart failure have normal or increase blood pressure. Relaxin is a natural human peptide that affects multiple vascular control pathways, suggesting potential mechanisms of benefit for such patients. We assessed the dose response of relaxin's effect on symptom relief, other clinical outcomes, and safety.
In a placebo-controlled, parallel-group, dose-ranging study, 234 patients with acute heart failure, dyspnoea, congestion on chest radiograph, and increased brain natriuretic peptide (BNP) or N-terminal prohormone of BNP, mild-to-moderate renal insufficiency, and systolic blood pressure greater than 125 mm Hg were recruited from 54 sites in eight countries and enrolled within 16 h of presentation. Patients were randomly assigned, in a double-blind manner via a telephone-based interactive voice response system, to standard care plus 48-h intravenous infusion of placebo (n=62) or relaxin 10 microg/kg (n=40), 30 microg/kg (n=43), 100 microg/kg (n=39), or 250 microg/kg (n=50) per day. Several clinical endpoints were explored to assess whether intravenous relaxin should be pursued in larger studies of acute heart failure, to identify an optimum dose, and to help to assess endpoint selection and power calculations. Analysis was by modified intention to treat. This study is registered with ClinicalTrials.gov, number NCT00520806.
In the modified intention-to-treat population, 61 patients were assessed in the placebo group, 40 in the relaxin 10 microg/kg per day group, 42 in the relaxin 30 microg/kg per day group, 37 in the relaxin 100 microg/kg per day group, and 49 in the relaxin 250 microg/kg per day group. Dyspnoea improved with relaxin 30 microg/kg compared with placebo, as assessed by Likert scale (17 of 42 patients [40%] moderately or markedly improved at 6 h, 12 h, and 24 h vs 14 of 61 [23%]; p=0.044) and visual analogue scale through day 14 (8214 mm x h [SD 8712] vs 4622 mm x h [9003]; p=0.053). Length of stay was 10.2 days (SD 6.1) for relaxin-treated patients versus 12.0 days (7.3) for those given placebo, and days alive out of hospital were 47.9 (10.1) versus 44.2 (14.2). Cardiovascular death or readmission due to heart or renal failure at day 60 was reduced with relaxin (2.6% [95% CI 0.4-16.8] vs 17.2% [9.6-29.6]; p=0.053). The number of serious adverse events was similar between groups.
When given to patients with acute heart failure and normal-to-increased blood pressure, relaxin was associated with favourable relief of dyspnoea and other clinical outcomes, with acceptable safety.
We report here the desensitization and internalization of the relaxin receptor (RXFP1) after agonist activation in both primary human decidual cells and HEK293 cells stably transfected with RXFP1. The importance of beta-arrestin 2 in these processes has also been demonstrated. Thus, in HEK-RXFP1 cells the desensitization of RXFP1 was significantly increased when beta-arrestin 2 was overexpressed. After relaxin activation, beta-arrestin 2 was translocated to the cell membrane and RXFP1 underwent rapid internalization. We have previously shown that RXFP1 forms dimers/oligomers during its biosynthesis and trafficking to the plasma membrane, we now show that internalization of RXFP1 occurs through this dimerization/oligomerization. In nonagonist stimulated cells, it is known that the majority of the RXFP1 is located intracellularly and was confirmed in the cells used here. Constitutive internalization of RXFP1 could account for this and indeed, slow but robust constitutive internalization, which was increased after agonist stimulation was demonstrated. A carboxyl-terminal deleted RXFP1 variant had a similar level of constitutive agonist-independent internalization as the wild-type RXFP1 but lost sensitivity to agonist stimulation. This demonstrated the importance of the carboxyl terminus in agonist-stimulated receptor internalization. These data suggest that the autocrine/paracrine actions of relaxin in the decidua are under additional controls at the level of expression of its receptor on the surface of its target cells.
Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [125I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.
The phenomenon of receptor-receptor interactions was hypothesized by Agnati and Fuxe in the 1980s, and several indirect proofs were provided in the following years by means of in vitro binding experiments and in vivo experiments in physiological and pathological animal models. This paper aims to outline some of the most important features and consequences of this phenomenon in the frame of the structural and functional aspects of molecular networks. In particular, the concepts of receptor mosaic (RM), and of horizontal and vertical molecular networks (HMNs, VMNs, respectively) are illustrated. To discuss some aspects of the functional organization of molecular networks, not only new data on protein-protein interactions but also the biochemical mechanism of cooperativity will be used. On this basis, some theoretical deductions can be drawn that allow a tentative classification of the RMs and the proposal of the extension of the concept of branching point introduced for enzymes to the possible switching role of some RMs in directing signals to various VMNs. Finally, the cooperativity phenomenon and the so-called symmetry rule will be used to introduce a proper mathematical approach that characterizes RMs as to their receptor composition, receptor topography, and order of receptor activation inside the RM. These new data on G protein-coupled receptors and molecular network organization indicate possible new approaches for drug development.
The monomeric model of rhodopsin-like G protein-coupled receptors (GPCRs) has progressively yielded the floor to the concept of GPCRs being oligo(di)mers, but the functional correlates of dimerization remain unclear. In this report, dimers of glycoprotein hormone receptors were demonstrated in living cells, with a combination of biophysical (bioluminescence resonance energy transfer and homogenous time resolved fluorescence/fluorescence resonance energy transfer), functional and biochemical approaches. Thyrotropin (TSHr) and lutropin (LH/CGr) receptors form homo- and heterodimers, via interactions involving primarily their heptahelical domains. The large hormone-binding ectodomains were dispensable for dimerization but modulated protomer interaction. Dimerization was not affected by agonist binding. Observed functional complementation indicates that TSHr dimers may function as a single functional unit. Finally, heterologous binding-competition studies, performed with heterodimers between TSHr and LH/CG–TSHr chimeras, demonstrated the unsuspected existence of strong negative cooperativity of hormone binding. Tracer desorption experiments indicated an allosteric behavior in TSHr and, to a lesser extent, in LH/CGr and FSHr homodimers. This study is the first report of homodimerization associated with negative cooperativity in rhodopsin-like GPCRs. As such, it may warrant revisitation of allosterism in the whole GPCR family.
Disclosure statement: The authors have nothing to disclose.
By studying the dissociation of 125I-instulin from its receptors in the absence and phe negatively cooperative type for the insulin receptors. In the present study we extend oy purified mouse and rat liver membranes as well as in human circulating monocytes and human cultured lymphocytes demonstrated negative cooperativity that was extraordinarily simn membranes more slowly than it does from its receptors on whole cells. The dissociaty a small percentage of the receptor sites (1 to 5%), are sufficient to accelerate dissociation of hormone from receptor. At these insulin concentrations insulin is entirely monomeric, and in fact at higher concentrations of insulin (greater than 10(-7) M) where insulin dimers predominate, the cooperativity effect is progressively lost. The dissociation rate of 125I-insulin alone (that is at very low fractional saturation of receptors) was markedly accelerated by dripping the pH from 8.0 to 5.0, whereas the dissociation of 125I-insulin at high receptor occupancy was only slightly accelerated by the fall in pH. The dissociation rate was directly related to temperature, but the dissociation rate of 125I-insulin at low receptor occupancy was much more affected by reduction in temperature and showed a sharp transition at 21 degrees. Urea at concentrations as low as 1 M produced a marked acceleration of 125I-insulin dissociation. Divalent cations (calcium and magnesium) appear to stabilize the insulin-receptor interaction, since higher degrees of receptor occupancy were required to achieve a given rate of dissociation of 125I-insulin. These data make it likely that the insulin receptors exist as oligomeric structures or clusters in the plasma membrane. Insulin receptor sites appear to switch from a "slow dissociating" state to a "fast dissociating" state when their occupancy increases; the proportion of sites in each state is a function of occupancy of the receptor sites by the insulin monomer as well as of the physiochemical environment. Other models which could explain apparent negative cooperativity besides site-site interactions, i.e. polymerization of the hormone, steric or electrostatic hindrance due to ligand-ligand interactions, or unstirred (Noyes-Whitney) layers are considered unlikely in the case of insulin receptors on both experimental and theoretical grounds.
Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [125I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.
Insulin interactions with human lymphocytes in established cultures and with isolated peripheral lymphocytes have been studied using ¹²⁵I-insulin. Receptors in circulating lymphocytes are indistinguishable from those found in cultured cells, and the lymphocyte receptors show striking similarities to those structures described in fat cell and liver preparations. In lymphocytes, the ability of insulin or insulin analogues to inhibit binding of ¹²⁵I-insulin is directly proportional to the ability of that preparation to inhibit binding of labeled hormone to purified liver membranes or to stimulate glucose oxidation in the fat cell.
Binding of ¹²⁵I-insulin to lymphocytes is a rapid and reversible process. Binding is maximal at 15°. Dissociation rate studies revealed a biphasic curve and the constants obtained were 8.3 x 10⁻⁵s⁻¹ and 8.3 x 10⁻⁴s⁻¹. The apparent affinity constants of the two orders of binding sites in the cultured lymphocytes were 1.2 x 10¹⁰m⁻¹ and 1.1 x 10⁹m⁻¹, whereas in the normal circulating lymphocytes the values were 2.0 x 10⁹m⁻¹ and 1.4 x 10⁸m⁻¹. At 30° there is very little degradation of labeled insulin by lymphocytes, and at 15° there is no detectable degradation with cultured or circulating cells.
Binding of ¹²⁵I-insulin to cultured lymphocytes is unaffected by Ca⁺⁺, Mg⁺⁺, or EDTA. The optimum pH for binding occurs at about 7.8. Insulin binding to cells is unaffected by digestion with DNase, RNase, or neuraminidase. However, tryptic digestion destroys the capacity of the cells to bind insulin.
A model is presented to study quantitative the effect of pH on a ligand-receptor interaction. Assuming that binding is only possible if all the "active groups" are in the correct ionic state, and that the ionic state of the other residues does not affect the association constant, it is possible to measure the number and pK values of the active groups. This model is applied to insulin and insulin analogs binding to its cellular receptor. Two active groups are responsible for the marked pH dependence of the reaction: a deprotonated residue of pK 7.6 at 25 degrees C (ionization heat: 1.5 kcal . mol-1) and a protonated residue of pK 8.0 at 25 degrees C (ionization heat: 12 kcal . mol-1). The first active group might be a carboxyl residue, in a hydrophobic environment, probably belonging to the receptor molecule. The second active group was further identified as the A1 alpha-amino residue of insulin by the study of insulin analogs.
We present a method for logical analysis of signal-transduction networks, focusing on metabolic and mitogenic signalling by the insulin receptor, with specific emphasis on dependence of the signalling properties on the timing of binding events. We discuss a basic model which demonstrates this dependence (hormone binding leads to activation of the receptor which can lead to a commitment to mitogenic signalling), and show how residence time of the hormone on the receptor can determine the specificity of signalling between the alternative metabolic or mitogenic pathways. The method gives conditions for the selection of specific branches in the signalling pathway expressed in terms of inequalities among the characteristic activation or deactivation times of components of that pathway. In this way, the conditions for mitogenic signalling can be given in terms of a required range of values of the hormone residence time on the receptor, which is directly related to the kinetic dissociation rate.
Impaired testicular descent (cryptorchidism) is one of the most frequent congenital abnormalities in humans, involving 2% of male births. Cryptorchidism can result in infertility and increases risk for development of germ-cell tumours. Testicular descent from abdomen to scrotum occurs in two distinct phases: the trans-abdominal phase and the inguino-scrotal phase. Currently, little is known about the factors that regulate the trans-abdominal phase of testicular descent. Leydig insulin-like hormone (Insl3) is a member of the insulin hormone superfamily expressed in the developing testis. We show here that mice mutant for Insl3 are viable, but exhibit bilateral cryptorchidism due to developmental abnormalities of the gubernaculum, resulting in abnormal spermatogenesis and infertility. Female homozygotes have impaired fertility associated with deregulation of the oestrus cycle. These findings reveal roles for Insl3 in the development of the urogenital tract and in female fertility. Insl3 may act as a hormone to regulate the growth and differentiation of the gubernaculum, thereby mediating intra-abdominal testicular descent.
Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein-protein interaction assay in whole cells, we unambiguously demonstrate that the human beta(2)-adrenergic receptor (beta(2)AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between beta(2)AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3, a cell-impermeable cross-linking agent, increased BRET between beta(2)AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the beta(2)AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the beta(2)AR and the regulatory protein beta-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein-protein interactions.
To examine thyrotropin (TSH) receptor homophilic interactions we fused the human TSH receptor (hTSHR) carboxyl terminus to green fluorescent protein (GFP) and the corresponding chimeric cDNA was expressed in Chinese hamster ovary cells. Fluorescent TSH receptors on the plasma membrane were functional as assessed by TSH-induced cAMP synthesis. The binding of TSH, as well as TSHR autoantibodies, induced time- and dose-dependent receptor capping. Fluorescence resonance energy transfer between receptors differentially tagged with GFP variants (RFP and YFP) provided evidence for the close proximity of individual receptor molecules. This was consistent with previous studies demonstrating the presence of TSHR dimers and oligomers in thyroid tissue. Co-immunoprecipitation of GFP-tagged and Myc-tagged receptor complexes was performed using doubly transfected cells with Myc antibody. Western blotting of the immunoprecipitated complex revealed the absence of noncleaved TSH holoreceptors. This further suggested that cleavage of the holoreceptor into its two-subunit structure, comprising disulfide-linked TSHR-alpha and TSHR-beta subunits, was required for the formation of TSHR dimers and higher order complexes.
Relaxin is a hormone important for the growth and remodeling of reproductive and other tissues during pregnancy. Although binding sites for relaxin are widely distributed, the nature of its receptor has been elusive. Here, we demonstrate that two orphan heterotrimeric guanine nucleotide binding protein (G protein)-coupled receptors, LGR7 and LGR8, are capable of mediating the action of relaxin through an adenosine 3',5'-monophosphate (cAMP)-dependent pathway distinct from that of the structurally related insulin and insulin-like growth factor family ligand. Treatment of antepartum mice with the soluble ligand-binding region of LGR7 caused parturition delay. The wide and divergent distribution of the two relaxin receptors implicates their roles in reproductive, brain, renal, cardiovascular, and other functions.
Luteinizing hormone receptor is a G protein-coupled receptor and consists of two halves: the N-terminal extracellular half (exodomain) and C-terminal membrane-associated half (endodomain). Hormone binds to the exodomain, and the resulting hormone-exodomain complex modulates the endodomain to generate signals. There are mutations that impair either hormone binding or signal generation. We report that the coexpression of a binding defective mutant and a signal-defective mutant rescues signal generation to produce cAMP. This rescue requires both types of mutant receptors and is dependent on the human chorionic gonadotropin dose, the surface concentration of mutant receptors, and the amino acid position of mutations. Furthermore, random collisions among mutant receptors are not involved in the rescue. Our observations provide new insights into the mechanisms of the functional and structural relationship of the exo- and endodomain, signal transduction, and receptor genetics, in particular for defective heterozygotes.
Recently, several studies have reported oligomerization of G protein-coupled receptors, although the functional implications of this phenomenon are still unclear. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation (COIP), we previously reported that the human thyrotropin (TSH) receptor tagged with green fluorescent protein (TSHR(GFP)) and expressed in a heterologous system was present as oligomeric complexes on the cell surface. Here, we have extended this biophysical and biochemical approach to study the regulation of such oligomeric complexes. Co-expression of TSHR(GFP) and TSHR(Myc) constructs in Chinese hamster ovary cells resulted in FRET-positive cells. The specificity of the FRET signal was verified by the absence of energy transfer in individually transfected TSHR(GFP) and TSHR(Myc):Cy3 cells cultured together and also by acceptor photobleaching. Occupation of the receptor molecule by the ligand (TSH) resulted in a dose-dependent decrease in the FRET index from 20% in the absence of TSH to <1% with 10(3) microunits/ml of TSH. Such reduction in oligomeric forms was also confirmed by coimmunoprecipitation. Exposure of TSHR(GFP/Myc) cells to forskolin or cytochalasin D caused no change in the FRET index, confirming that the decrease in the oligomeric complexes was a receptor-dependent phenomenon and free of energy or microtuble requirements. The TSH-induced decrease in TSHR oligomers was found to be secondary to dissociation of the TSHR complexes as evidenced by an increase in fluorescent intensity of photobleached spots of GFP fluorescence with 10(3) microunits/ml of TSH. These data indicated that the less active conformation of the TSHR was comprised of receptor complexes and that such complexes were dissociated on the binding of ligand. Such observations support the concept of a constitutively active TSHR dimer or monomer that is naturally inhibited by the formation of higher order complexes. Inhibition of these oligomeric forms by ligand binding returns the TSHR to an activated state.
Type 2 diabetes mellitus -- in which the body produces insufficient amounts of insulin or the insulin that is produced does not function properly to control blood glucose -- is an increasingly common disorder. Prospective clinical studies have proven the benefits of tighter glucose control in reducing the frequency and severity of complications of the disease, leading to the advocation of earlier and more aggressive use of insulin therapy. Given the reluctance of patients with type 2 diabetes to inject themselves with insulin, orally active insulin mimetics would be a major therapeutic advance. Here, we discuss recent progress in understanding the structure-function relationships of the insulin and insulin-like growth factor 1 (IGF1) receptors, their mechanism of activation and their implications for the design of insulin-receptor agonists for diabetes therapy and IGF1-receptor antagonists for cancer therapy.
G protein-coupled receptors (GPCRs), which constitute the largest and structurally best conserved family of signaling molecules,
are involved in virtually all physiological processes. Crystal structures are available only for the detergent-solubilized
light receptor rhodopsin. In addition, this receptor is the only GPCR for which the presumed higher order oligomeric state
in native membranes has been demonstrated (Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A., and Palczewski,
K. (2003) Nature 421, 127–128). Here, we have determined by atomic force microscopy the organization of rhodopsin in native membranes obtained
from wild-type mouse photoreceptors and opsin isolated from photoreceptors of Rpe65–/– mutant mice, which do not produce the chromophore 11-cis-retinal. The higher order organization of rhodopsin was present irrespective of the support on which the membranes were
adsorbed for imaging. Rhodopsin and opsin form structural dimers that are organized in paracrystalline arrays. The intradimeric
contact is likely to involve helices IV and V, whereas contacts mainly between helices I and II and the cytoplasmic loop
connecting helices V and VI facilitate the formation of rhodopsin dimer rows. Contacts between rows are on the extracellular
side and involve helix I. This is the first semi-empirical model of a higher order structure of a GPCR in native membranes,
and it has profound implications for the understanding of how this receptor interacts with partner proteins.
To study the fate of TSH receptor (TSHR) on TSH binding, we constructed a chimeric cDNA that encodes a yellow fluorescent protein (YFP) fused to the carboxyl terminus of human TSHR. The protein expression in transfected cells was confirmed using flow cytometry. The functionality of the chimeric protein was determined by its ability to transduce signal leading to activation of cAMP in a TSH dose-dependent manner. The levels of cAMP produced by these cells were comparable with the levels seen in cells transfected with unfused TSHR without the YFP. Using deconvolution microscopy, we observed that the receptor is largely expressed on the cell surface, but on addition of TSH, some of the receptors were rapidly internalized. This conclusion was supported by several independent observations involving different cells expressing either native or recombinant TSHR. On TSH treatment, we observed internalization of human TSHR-YFP and human TSHR, expressed on 293 and CHO cells, respectively. This was further substantiated when we observed colocalization of rhodamine-labeled TSH with TSHR-YFP within the cell and by the uptake of radiolabeled TSH. Furthermore, shortly after ligand binding, there was a profound change in the morphology of the cells and some of the receptors accumulated in the perinuclear region of the cell. The TSHR-YFP was colocalized with RhoB-cyan fluorescent protein, indicating that it accumulated within the endosomes. These results indicate that the receptor internalization might in part be responsible for TSHR desensitization on TSH binding.
The classical idea that G-protein-coupled receptors (GPCRs) function as monomeric entities has been unsettled by the emerging concept of GPCR dimerization. Recent findings have indicated not only that many GPCRs exist as homodimers and heterodimers, but also that their oligomeric assembly could have important functional roles. Several studies have shown that dimerization occurs early after biosynthesis, suggesting that it has a primary role in receptor maturation. G-protein coupling, downstream signalling and regulatory processes such as internalization have also been shown to be influenced by the dimeric nature of the receptors. In addition to raising fundamental questions about GPCR function, the concept of dimerization could be important in the development and screening of drugs that act through this receptor class. In particular, the changes in ligand-binding and signalling properties that accompany heterodimerization could give rise to an unexpected pharmacological diversity that would need to be considered.
This study has focused on enhancing the signal generated from the interaction between a G-protein-coupled receptor (GPCR) and beta-arrestin 2 (beta-arr2), measured by the bioluminescence resonance energy transfer (BRET(2)) technology. Both class A (beta(2)-adrenergic receptor [beta(2)-AR]) and class B (neurokinin-type 1 receptor [NK1-R]) GPCRs, classified based on their internalization characteristics, have been analyzed. It was evaluated whether the BRET(2) signal can be enhanced by using (1) beta-arr2 phosphorylation-independent mutant (beta-arr2 R169E) and (2) beta-arr2 mutants deficient in their ability to interact with the components of the clathrin-coated vesicles (beta-arr2 R393E, R395E and beta-arr2 373 stop). For the class B receptor, there was no major difference in the agonist-promoted BRET(2) signal when comparing results obtained with wild-type (wt) and mutant beta-arr2. However, with the class A receptor, a more than 2-fold increase in the BRET(2) signal was observed with beta-arr2 mutants lacking the AP-2 or both AP-2 and clathrin binding sites. This set of data suggests that the inability of these beta-arr2 mutants to interact with the components of the clathrin-coated vesicle probably prevents their rapid dissociation from the receptor, thus yielding an increased and more stable BRET(2) signal. The beta-arr2 R393E, R395E mutant also enhanced the signal window with other members of the GPCR family (neuropeptide Y type 2 receptor [NPY2-R] and TG1019 receptor) and was successfully applied in full-plate BRET(2)-based agonist and antagonist screening assays.
Relaxin family peptide 1 (RXFP1) receptor (LGR7) and RXFP2 receptor (LGR8) were recently identified as the receptor targets for H2 relaxin and insulin-like peptide 3 (INSL3), respectively. In this study, we define the pharmacology of these two receptors by using a number of receptor chimeras and relaxin family peptides. We have identified two binding sites on these receptors: one primary, high-affinity site within the ectodomain and a secondary, lower affinity site within the transmembrane region. The primary site was found to dictate receptor binding characteristics, although the lower affinity site also exerts some influence and modulates ligand affinity for the primary site in a manner dependent upon the peptide in question. Not all relaxin peptides were able to bind to the RXFP2 receptor, indicating that the relaxin-RXFP2 receptor interaction is species-specific. INSL3 was found to exhibit characteristics of a partial agonist at the RXFP2 and chimeric RXFP1/2 receptors, with low maximal cAMP responses but high potency in coupling to this pathway. cAMP accumulation studies also revealed that the binding sites couple to cAMP signaling pathways with differing efficiency: the high-affinity site signals with high efficiency, whereas the lower affinity site signals with little to no efficiency. Comparisons between RXFP1, RXFP2, the chimeric receptors, and the truncated receptors revealed that the interaction between receptor sites is critical for optimal ligand binding and signal transduction and that the ectodomain is essential for signaling. Evidence obtained in this study supports a two-stage binding model of receptor activation: binding to the primary site allows a conformational change and interaction with the low-affinity transmembrane site.
The relaxin-like peptide family belongs in the insulin superfamily and consists of 7 peptides of high structural but low sequence similarity; relaxin-1, 2 and 3, and the insulin-like (INSL) peptides, INSL3, INSL4, INSL5 and INSL6. The functions of relaxin-3, INSL4, INSL5, INSL6 remain uncharacterised. The evolution of this family has been contentious; high sequence variability is seen between closely related species, while distantly related species show high similarity; an invertebrate relaxin sequence has been reported, while a relaxin gene has not been found in the avian and ruminant lineages.
Sequence similarity searches of genomic and EST data identified homologs of relaxin-like peptides in mammals, and non-mammalian vertebrates such as fish. Phylogenetic analysis was used to resolve the evolution of the family. Searches were unable to identify an invertebrate relaxin-like peptide. The published relaxin cDNA sequence in the tunicate, Ciona intestinalis was not present in the completed C. intestinalis genome. The newly discovered relaxin-3 is likely to be the ancestral relaxin. Multiple relaxin-3-like sequences are present in fugu fish (Takifugu rubripes) and zebrafish (Danio rerio), but these appear to be specific to the fish lineage. Possible relaxin-1 and INSL5 homologs were also identified in fish and frog species, placing their emergence prior to mammalia, earlier than previously believed. Furthermore, estimates of synonymous and nonsynonymous substitution rates (dN/dS) suggest that the emergence of relaxin-1, INSL4 and INSL6 during mammalia was driven by positive Darwinian selection, hence these peptides are likely to have novel and in the case of relaxin-1, which is still under positive selection in humans and the great apes, possibly still evolving functions. In contrast, relaxin-3 is constrained by strong purifying selection, demonstrating it must have a highly conserved function, supporting its hypothesized important neuropeptide role.
We present a phylogeny describing the evolutionary history of the relaxin-like peptide family and show that positive selection has driven the evolution of the most recent members of the family.
Relaxin-3 (INSL-7) is a recently discovered member of the insulin superfamily. Relaxin-3 mRNA is expressed in the nucleus incertus of the brainstem, which has projections to the hypothalamus. Relaxin-3 binds with high affinity to the LGR7 receptor and to the previously orphan G protein-coupled receptor GPCR135. GPCR135 mRNA is expressed predominantly in the central nervous system, particularly in the paraventricular nucleus (PVN). The presence of relaxin-3 and these receptors in the PVN led us to investigate the effect of central administration of relaxin-3 on food intake in male Wistar rats. The receptor involved in mediating these effects was also investigated. Intracerebroventricular injections of human relaxin-3 (H3) to satiated rats significantly increased food intake 1 h post administration in the early light phase [0.96 +/- 0.16 g (vehicle) vs. 1.81 +/- 0.21 g (180 pmol H3), P < 0.05] and the early dark phase [2.95 +/- 0.45 g (vehicle) vs. 4.39 +/- 0.39 g (180 pmol H3), P < 0.05]. Intra-PVN H3 administration significantly increased 1-h food intake in satiated rats in the early light phase [0.34 +/- 0.16 g (vehicle) vs. 1.23 +/- 0.30 g (18 pmol H3), P < 0.05] and the early dark phase [4.43 +/- 0.32 g (vehicle) vs. 6.57 +/- 0.42 g (18 pmol H3), P < 0.05]. Feeding behavior increased after intra-PVN H3. Equimolar doses of human relaxin-2, which binds the LGR7 receptor but not GPCR135, did not increase feeding. Hypothalamic neuropeptide Y, proopiomelanocortin, or agouti-related peptide mRNA expression did not change after acute intracerebroventricular H3. These results suggest a novel role for relaxin-3 in appetite regulation.
Relaxin-3 is a member of the human relaxin peptide family, the gene for which, RLN3, is predominantly expressed in the brain. Mapping studies in the rodent indicate a highly developed network of RLN3, RLN1, and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides have important functional roles in the central nervous system. A regioselective disulfide-bond synthesis protocol was developed and used for the chemical synthesis of human (H3) relaxin-3. The selectively S-protected A and B chains were combined by stepwise formation of each of the three insulin-like disulfides via aeration, thioloysis, and iodolysis. Judicious positioning of the three sets of S-protecting groups was crucial for acquisition of synthetic H3 relaxin in a good overall yield. The activity of the peptide was tested against relaxin family peptide receptors. Although the highest activity was demonstrated on the human relaxin-3 receptor (GPCR135), the peptide also showed high activity on relaxin receptors (LGR7) from various species and variable activity on the INSL3 receptor (LGR8). Recombinant mouse prorelaxin-3 demonstrated similar activity to H3 relaxin, suggesting that the presence of the C peptide did not influence the conformation of the active site. H3 relaxin was also able to activate native LGR7 receptors. It stimulated increased MMP-2 expression in LGR7-expressing rat ventricular fibroblasts in a dose-dependent manner and, following infusion into the lateral ventricle of the brain, stimulated water drinking in rats, activating LGR7 receptors located in the subfornical organ. Thus, H3 relaxin is able to interact with the relaxin receptor LGR7 both in vitro and in vivo.
Insulin-like peptide 3 (INSL3) is a member of the insulin superfamily that plays an important role in mediating testes descent during fetal development. More recently, it has also been demonstrated to initiate oocyte maturation and suppress male germ cell apoptosis. These actions are mediated via a specific G-protein-coupled receptor, LGR8. Little is known regarding the structure and function relationship of INSL3, although it is believed that the principal receptor binding site resides within its B-chain. We subsequently observed that the linear B-chain alone (INSL3B-(1-31)) bound to LGR8 and was able to antagonise INSL3 stimulated cAMP accumulation in HEK-293T cells expressing LGR8. Sequentially N- and C-terminally shortened linear analogs were prepared by solid phase synthesis and subsequent assay showed that the minimum length required for binding was residues 11-27. It was also observed that increased binding affinity correlated with a corresponding increase in alpha-helical content as measured by circular dichroism spectroscopy. Molecular modeling studies suggested that judicious placement of a conformational constraint within this peptide would increase its alpha-helix content and result in increased structural similarity to the B-chain within native INSL3. Consequently, intramolecularly disulfide-linked analogs of the B-chain showed a potentiation of INSL3 antagonistic activity, as well as exhibiting increased proteolytic stability, as assessed in rat serum in vitro. Administration of one of these peptides into the testes of rats resulted in a substantial decrease in testis weight probably due to the inhibition of germ cell survival, suggesting that INSL3 antagonists may have potential as novel contraceptive agents.
Two orphan leucine-rich repeat-containing G protein-coupled receptors were recently identified as targets for the relaxin family peptides relaxin and insulin-like peptide (INSL) 3. Human gene 2 relaxin is the cognate ligand for relaxin family peptide receptor (RXFP) 1, whereas INSL3 is the ligand for RXFP2. Constitutively active mutants of both receptors when expressed in human embryonic kidney (HEK) 293T cells signal through Galphas to increase cAMP. However, recent studies using cells that endogenously express the receptors revealed greater complexity: cAMP accumulation after activation of RXFP1 involves a time-dependent biphasic pathway with a delayed phase involving phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) zeta, whereas the RXFP2 response involves inhibition of adenylate cyclase via pertussis toxin-sensitive G proteins. The aim of this study was to compare and contrast the cAMP signaling pathways used by these two related receptors. In HEK293T cells stably transfected with RXFP1, preliminary studies confirmed the biphasic cAMP response, with an initial Galphas component and a delayed response involving PI3K and PKCzeta. This delayed pathway was dependent upon G-betagamma subunits derived from Galphai3. An additional inhibitory pathway involving GalphaoB affecting cAMP accumulation was also identified. In HEK293T cells stably transfected with RXFP2, the cAMP response involved Galphas and was modulated by inhibition mediated by GalphaoB and release of inhibitory G-betagamma subunits. Thus, initially both RXFP1 and RXFP2 couple to Galphas and an inhibitory GalphaoB pathway. Differences in cAMP accumulation stem from the ability of RXFP1 to recruit coupling to Galphai3, release G-betagamma subunits and thus activate a delayed PI3K-PKCzeta pathway to further increase cAMP accumulation.
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...
This chapter discusses physiology and molecular biology of the relaxin peptide family. Relaxin-a member of a family of peptide hormones-includes seven members in humans-three relaxin peptides (H1, H2, and H3) and insulin-like peptides (INSL) 3, 4, 5, and 6. All these genes evolved from an ancestral relaxin-3 gene in (lower) vertebrates. The reproductive hormone relaxin is the product of one gene in most species, RLN1. Relaxin-3 is likely an important neuropeptide but has no known roles in reproduction. In contrast, relaxin has vital physiological roles during pregnancy, although its actions vary between species. Hence, relaxin has essential actions on the cervix, pubic symphysis, vagina, uterus, and mammary apparatus during pregnancy. It is also a mediator of the important cardiovascular changes that occur during pregnancy in many species. Relaxin plays a crucial role in implantation, especially in primates. In addition, relaxin has nonreproductive actions in wound healing, cardiac protection, and as an antifibrotic agent. INSL3 is essential for testis descent by promoting gubernacular development in the fetus. It also plays an important role in female and male germ cell maturation and survival, respectively.
Relaxin in human pregnancy is both a systemic hormone from the corpus luteum and an autocrine/paracrine hormone at the maternal-fetal interface formed by the decidua/placenta and fetal membranes. We have focused our studies on the autocrine/paracrine roles of relaxin, especially in the preterm premature rupture of the fetal membranes, which causes 30-40% of preterm births. By using different techniques and different tissue collections, our laboratory has shown that expression of the relaxin genes and proteins in the decidua and placenta is increased in patients with preterm premature rupture of the fetal membranes. Relaxin binding and the expression of LGR7 are primarily in the chorion and decidua and are downregulated after spontaneous labor and delivery both at term and preterm. However, expression of LGR7 in the fetal membranes is significantly greater in all clinical situations at preterm than term, suggesting an important role for relaxin in these tissues at that time. The roles of the relaxin system in three potential causes of preterm birth are discussed: in the growth and proliferation of the membranes important for fetal membrane accommodation to fetal and placental growth, in acute infection, and in the inflammatory response leading to the initiation of labor.
The specific binding of [3H] (−)alprenolol to sites in frog erythrocyte membranes provides a tool for directly assessing ligand binding to adenylatecyclase coupled β-adrenergic receptors. Hill Plots of such binding data yield slopes (nH=“Hill Coefficients”) less than 1.0, suggesting that negatively cooperative interactions among the β-adrenergic receptors may occur. The existence of such negative cooperativity was confirmed by a direct kinetic method. The dissociation of receptor bound [3H] (−)alprenolol was studied under two conditions: 1) with dilution of the ligand-receptor complex sufficient to prevent rebinding of the dissociated tracer and 2) with this same dilution in the presence of excess unlabeled (−)alprenolol. If the sites are independent, the dissociation rates must be the same in both cases. However, the presence of (−)alprenolol increases the rate of [3H] (−)alprenolol dissociation, indicating that negatively cooperative interactions among the β-adrenergic receptor binding sites do occur.
The recent crystallographic structure of the insulin receptor (IR) extracellular domain has brought us closer to ending several decades of speculation regarding the stoichiometry and mechanism of insulin-receptor binding and negative cooperativity. It supports a bivalent crosslinking model whereby two sites on the insulin molecule alternately crosslink two partial-binding sites on each insulin-receptor half. Ligand-induced or -stabilized receptor dimerization or oligomerization is a general feature of receptor tyrosine kinases (RTKs), in addition to cytokine receptors, but the kinetic consequences of this mechanism have been less well studied in other RTKs than in the IR. Surprisingly, recent studies indicate that constitutive dimerization and negative cooperativity are also ubiquitous properties of G-protein-coupled receptors (GPCRs), which show allosteric mechanisms similar to those described for the IR.
The binding of many polypeptide hormones to cell surface receptors does not appear to follow the law of mass action. While steady-state binding data are consistent in many cases with either heterogeneous populations of binding sites or interactions of the type known as negative cooperativity, study of the kinetics of dissociation of the type known as negative cooperativity, study of the kinetics of dissociation of the hormone receptor complex allows an unambiguous demonstration of cooperative interactions. Negative cooperativity, which seems to be wide-spread among hormone receptors, provides exquisite sensitivity of the cell at low hormone concentrations while buffering against acutely elevated hormone levels. The molecular mechanisms underlying the cooperativity are still largely unknown. Cooperativity may stem from a conformational transition in individual receptors or involve receptor aggregation in the fluid membrane (clustering) or more extensive membrane phenomena. Thus, new models of hormone action must be considered which integrate the progress in our knowledge of both the complex mechanisms regulating hormone binding to their surface receptors, and the dynamic properties of the cell membrane.
A simple method is reported to detect cooperative interactions in the binding of polypeptide hormones to their membrane receptors. The dissociation of radioiodinated hormone from the receptor is studied under two conditions: first, by diluting the hormone-receptor complex sufficiently to prevent rebinding of the dissociated tracer; second, by dilution to the same extent in a medium containing an excess of unlabeled hormone. If the sites are independent, the dissociation rates must be the same in both cases. If the presence of unlabeled hormone increases the dissociation rate of the tracer, negatively cooperative interactions must occur. Insulin receptors on cultured lymphocytes and liver plasma membranes show negative cooperative interactions. Growth hormone receptor sites lack these interactions.
Intrauterine pressure was recorded from nonpregnant guinea pigs linked to pregnant guinea pigs by cross circulation. A frequency-modulating effect was observed which could not be demonstrated when the pregnant partner was replaced by either another nonpregnant or a male animal. The effect was rapid in onset and was short lasting when cross circulation was discontinued. It was postulated that a myometrial inhibitory factor was present in the blood of the pregnant animal which for reasons discussed could not be progesterone. Infusions of porcine–relaxin (1 mg/h) simulated the changes observed in the cross-circulation experiments and the suggestion is made that relaxin or a relaxin-like substance or substances is a major myometrial regulating factor in the guinea pig.
Relaxin is a peptide hormone synthesized in the corpora lutea of ovaries during pregnancy and is released into the blood stream prior to parturition. Its major biological effect is to remodel the mammalian reproductive tract to facilitate the birth process. Determination of the structure of human relaxin is thus a first step in opening up the possibility of clinical intervention in cases of difficult labour. However, the limited availability of human ovaries during pregnancy has prevented both direct amino acid sequence determination and isolation of cDNA clones obtained from relaxin producing tissue. Our approach has therefore been to screen directly for a human relaxin gene using an homologous porcine relaxin cDNA probe. We report here the successful identification of a genomic clone from which the structure of the entire coding region of a human preprorelaxin gene has been determined. Synthesis of biologically active relaxin has shown that the novel gene structure described herein codes for an authentic human relaxin. We believe this is the first successful synthesis of a biologically active hormone whose structure was predicted solely from the structure of a genomic clone.
The TSH receptor (TSHR) has a large glycosylated ectodomain comprising the amino-terminal half of the molecule (394 of 743 residues) implicated in TSH binding, as well as autoantibody recognition in Graves' disease. In this study we employed antibodies specific for the amino-terminus (Ab1), midportion (Ab2), and carboxyl-terminus (Ab3) of the TSHR-ectodomain, previously mapped using recombinant receptor proteins, to detect the natural receptor present in detergent-solubilized porcine thyroid cell membranes via immunoblotting. Several forms of the receptor were detected. In reduced samples Ab1 detected full-length holoreceptors present in both nonglycosylated and glycoslylated forms of apparent molecular masses 80 and 90 kDa, respectively, as well as apparent dimeric nonglycosylated and dimeric glycosylated holoreceptor forms resistant to reduction. Also detected by Ab1 were a glycosylated amino-terminal 47- to 52-kDa fragment of the holoreceptor (gly alpha-subunit), reduced to 42 kDa (alpha-subunit) by Endo F deglycosylation. Ab2 detected all of the same forms. Ab3 detected primarily a carboxy-terminal, nonglycosylated fragment of 35 kDa (beta-subunit). In unreduced samples, the recognition pattern was unchanged with Ab1. Ab2 detected monomeric and dimeric beta-subunits, as well as higher order complexes. The different TSHR forms present in unreduced preparations were resolved by ammonium sulfate precipitation, confirming their autonomy. The data demonstrate the presence of multiple forms of the natural TSHR. Their roles in TSH action and TSHR autoimmunity require further exploration.
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 x 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 approximately 220 kDa in these cells. In concert with results of recent studies that demonstrated that the Mr of the relaxin receptor is approximately 220 kDa, our data suggest that the phosphorylated protein is likely to be the relaxin receptor.
G protein-coupled receptors (GPCRs) are seven transmembrane proteins that form the largest single family of integral membrane receptors. GPCRs transduce information provided by extracellular stimuli into intracellular second messengers via their coupling to heterotrimeric G proteins and the subsequent regulation of a diverse variety of effector systems. Agonist activation of GPCRs also initiates processes that are involved in the feedback desensitization of GPCR responsiveness, the internalization of GPCRs, and the coupling of GPCRs to heterotrimeric G protein-independent signal transduction pathways. GPCR desensitization occurs as a consequence of G protein uncoupling in response to phosphorylation by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes the binding of beta-arrestins, which not only uncouple receptors from heterotrimeric G proteins but also target many GPCRs for internalization in clathrin-coated vesicles. beta-Arrestin-dependent endocytosis of GPCRs involves the direct interaction of the carboxyl-terminal tail domain of beta-arrestins with both beta-adaptin and clathrin. The focus of this review is the current and evolving understanding of the contribution of GRKs, beta-arrestins, and endocytosis to GPCR-specific patterns of desensitization and resensitization. In addition to their role as GPCR-specific endocytic adaptor proteins, beta-arrestins also serve as molecular scaffolds that foster the formation of alternative, heterotrimeric G protein-independent signal transduction complexes. Similar to what is observed for GPCR desensitization and resensitization, beta-arrestin-dependent GPCR internalization is involved in the intracellular compartmentalization of these protein complexes.
The hormone relaxin is known to be important in pregnancy but its receptor has long remained elusive. In a Perspective, [Ivell][1] discusses the identification of receptors for relaxin and its close relative relaxin-like factor ([ Hsu et al .][2]), and the finding that relaxin is far more than just a reproductive hormone.
[1]: http://www.sciencemag.org/cgi/content/full/295/5555/637
[2]: http://www.sciencemag.org/cgi/content/short/295/5555/671
The relaxin-like factor (RLF), which is the product of the insulin-like factor 3 (INSL3) gene, is a new circulating peptide hormone of the relaxin-insulin family. In male mammals, it is a major secretory product of the testicular Leydig cells, where it appears to be expressed constitutively but in a differentiation-dependent manner. In the adult testis, RLF expression is a good marker for fully differentiated adult-type Leydig cells, but it is only weakly expressed in prepubertal immature Leydig cells or in Leydig cells that have become hypertrophic or transformed. It is also an important product of the fetal Leydig cell population, where it has been demonstrated using knockout mice to be responsible for the second phase of testicular descent acting on the gubernaculum. INSL3 knockout mice are cryptorchid, and in estrogen-induced cryptorchidism, RLF levels in the testis are significantly reduced. RLF is also made in female tissues, particularly in the follicular theca cells of small antral follicles and in the corpus luteum of the cycle and pregnancy. The ruminant ovary has a very high level of RLF expression, and analysis of primary cultures of ovarian theca-lutein cells indicated that, as in the testis, expression is probably constitutive but differentiation dependent. Female INSL3 knockout mice have altered estrous cycles, where RLF may be involved in follicle selection, an idea strongly supported by observations on bovine secondary follicles. Recently, a novel 7-transmembrane domain receptor (LGR8 or Great) has been tentatively identified as the RLF receptor, and its deletion in mice leads also to cryptorchidism.
Relaxin has vital physiological roles in pregnant rats, mice, and pigs. Relaxin promotes growth and softening of the cervix, thus facilitating rapid delivery of live young. Relaxin also promotes development of the mammary apparatus, thus enabling normal lactational performance. The actions of relaxin on the mammary apparatus vary among species. Whereas relaxin is required for development of the mammary nipples in rats and mice, it is essential for prepartum development of glandular parenchyma in pregnant pigs. During pregnancy relaxin also inhibits uterine contractility and promotes the osmoregulatory changes of pregnancy in rats. Recent studies with male and nonpregnant female rodents revealed diverse therapeutic actions of relaxin on nonreproductive tissues that have clinical implications. Relaxin has been reported to reduce fibrosis in the kidney, heart, lung, and liver and to promote wound healing. Also, probably through its vasodilatory actions, relaxin protects the heart from ischemia-induced injury. Finally, relaxin counteracts allergic reactions. Knowledge of the diverse physiological and therapeutic actions of relaxin, coupled with the recent identification of relaxin receptors, opens numerous avenues of investigation that will likely sustain a high level of research interest in relaxin for the foreseeable future.
To perform functional cell-based screening assays on seven-transmembrane (7TM) receptors, also known as G-protein coupled receptors, at least three distinct assays are currently needed to screen for G(alphas), G(alphai/0) or G(alphaq/11) signaling receptors. Therefore, there has long been a desire for a universal screening assay that could be used to screen all 7TM receptors independent of their signaling pathway. The receptor/beta-arrestin interaction is common to virtually all 7TM receptors. Therefore, an assay based on this interaction should achieve just that. Bioluminescence resonance energy transfer technology can be used to measure the receptor/beta-arrestin interaction in living cells but due to various technical and biological reasons, the use of the technology for compound screening has been limited. The recent development of beta-arrestin mutants that significantly improve the assay signal, in combination with new improved instrumentation, has transformed bioluminescence resonance energy transfer technology from being a highly specialized research tool in molecular pharmacology to a more drug screening-friendly technique that is useful in an industrial setting.
Follicle-stimulating hormone (FSH) is central to reproduction in mammals. It acts through a G-protein-coupled receptor on the surface of target cells to stimulate testicular and ovarian functions. We present here the 2.9-A-resolution structure of a partially deglycosylated complex of human FSH bound to the extracellular hormone-binding domain of its receptor (FSHR(HB)). The hormone is bound in a hand-clasp fashion to an elongated, curved receptor. The buried interface of the complex is large (2,600 A2) and has a high charge density. Our analysis suggests that all glycoprotein hormones bind to their receptors in this mode and that binding specificity is mediated by key interaction sites involving both the common alpha- and hormone-specific beta-subunits. On binding, FSH undergoes a concerted conformational change that affects protruding loops implicated in receptor activation. The FSH-FSHR(HB) complexes form dimers in the crystal and at high concentrations in solution. Such dimers may participate in transmembrane signal transduction.
Insulin-like factor 3 (INSL3), previously known as the relaxin-like factor (RLF), is a major peptide hormone secreted from the testicular Leydig cells of adult men and circulating in the blood at a concentration of approximately 1 ng/mL. Women also produce INSL3 in the theca interna cells of ovarian follicles, but circulating levels remain below 100 pg/mL. INSL3 is structurally related to relaxin and insulin, but unlike the latter, signals through a novel G-protein-coupled receptor, LGR8. Ablation of the gene for INSL3 leads primarily to cryptorchidism because of a defect in the first, transabdominal phase of testicular descent. In the adult knockout mouse, a mild phenotype is evident in the testis and ovary. We have developed a panel of antibodies specific for INSL3 from various species, which are suitable for immunohistochemistry and, more recently, for immunoassays. INSL3 is an important marker for the mature Leydig cell phenotype, where it appears to be expressed constitutively, once the mature differentiation state is achieved. It is also an indicator of differentiation status not only for Leydig cells but also for the theca interna cells of the ovary.
Recent studies have identified four receptors that are the physiological targets for relaxin family peptides. All are class I (rhodopsin like) G-protein-coupled receptors with LGR7 (RXFP1) and LGR8 (RXFP2) being type C leucine-rich repeat-containing receptors, whereas GPCR135 (RXFP3) and GPCR142 (RXFP4) resemble receptors that respond to small peptides such as somatostatin and angiotensin II. The cognate ligands for the receptors have been identified: relaxin for RXFP1; INSL3 for RXFP2; relaxin 3 for RXFP3 and INSL5 for RXFP4. RXFP1 and RXFP2 receptors produce increases in intracellular cAMP levels upon stimulation, although the response is complex and contains a component sensitive to PI-3-kinase inhibitors. There is also evidence that RXFP1 can activate Erk1/2 and nitric oxide synthase, and relaxin has been reported to enter cells and activate glucocorticoid receptors. In contrast, RXFP3 and RXFP4 couple to Gi by a pertussis toxin-sensitive mechanism to cause inhibition of cAMP production. Now that the receptors for relaxin family peptides and their cognate ligands have been identified, we suggest a nomenclature for both the peptides and the receptors that we hope will be helpful to researchers in this rapidly advancing field.
This study defines the pharmacologic characteristics of LGR7 and LGR8, the receptors for H2 relaxin and INSL3 respectively, and determines the relative activity of relaxin-related peptides. We show, for the first time, the availability of two binding sites at LGR8 and confirm the presence of two sites at LGR7. Relaxin-related peptides had differing rank orders of affinity and potency at LGR7 and LGR8, but chimeric receptors were highly similar to their ectodomain-origin native receptors. The high-affinity site on the ectodomain coupled efficiently to cAMP production, whereas the low-affinity site in the transmembrane region coupled with decreased efficiency.
It became clear over the recent years that most, if not all, G protein-coupled receptors (GPCR) are able to form dimers or higher order oligomers. Chemokine receptors make no exception to this new rule and both homo- and heterodimerization were demonstrated for CC and CXC receptors. Functional analyses demonstrated negative binding cooperativity between the two subunits of a dimer. The consequence is that only one chemokine can bind with high affinity onto a receptor dimer. In the context of receptor activation, this implies that the motions of helical domains triggered by the binding of agonists induce correlated changes in the other protomer. The impact of the chemokine dimerization process in terms of co-receptor function and drug development is discussed.
The glycoprotein hormones regulate reproduction and development through their interactions with receptors in ovarian, testicular, and thyroid tissues. Efforts to design hormone agonists and antagonists useful for treat-ing infertility and hyperthyroidism would benefit from a molecular understanding of hormone-receptor interaction. The structure of a complex containing FSH bound to a fragment of its receptor has been determined at 2.9 Angstroms resolution, but this does not explain several observations made with cell-surface G protein receptors and may reflect the manner in which FSH binds a short alternate spliced receptor form. We discuss observations that must be explained by any model of the cell-surface G protein-coupled glycoprotein hormone receptors and suggest structures for these receptors that satisfy these requirements. Glycoprotein hormones appear to contact two distinct sites in the extracellular domains of their receptors, not just the leucine-rich repeat domain. These dual contacts contribute to ligand binding specificity and appear to be essential for signal transduction. As outlined in this minireview, differences in the manners in which these ligands contact their receptors explain why some ligands and ligand analogs interact with more than one class of receptor and why some receptors and receptor analogs bind more than one ligand. The unique manner in which these ligands appear to interact with their receptors may have facilitated hormone and receptor co-evolution during early vertebrate speciation.