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A Homozygous Mutation in the
Luteinizing Hormone Receptor
Causes Partial Leydig Cell
Hypoplasia: Correlation between
Receptor Activity and Phenotype
John W. M. Martens, Miriam Verhoef-Post, Neusa Abelin,
Marilza Ezabella, Sergio P. A. Toledo, Han G. Brunner, and
Axel P. N. Themmen
Department of Endocrinology and Reproduction (J.W.M.M., M.V.-P.,
A.P.N.T.)
Faculty of Medicine and Health Sciences
Erasmus University Rotterdam
3000 DR Rotterdam, The Netherlands
Endocrine Genetics Unit (N.A., M.E., S.P.A.T.)
Endocrine Division
Department of Medicine
Sa˜ o Paulo University School of Medicine
CEP 01246 Sa˜ o Paulo, Brazil
Department of Human Genetics (H.G.B.)
University Hospital
6500 HB Nijmegen, The Netherlands
Leydig cell hypoplasia (LCH) is characterized by a
decreased response of the Leydig cells to LH. As a
result, patients with this syndrome display aber-
rant male development ranging from complete
pseudohermaphroditism to males with micropenis
but with otherwise normal sex characteristics. We
have evaluated three brothers with a mild form of
LCH. Analysis of their LH receptor (LHR) gene re-
vealed a homozygous missense mutation resulting
in a substitution of a lysine residue for a isoleucine
residue at position 625 of the receptor. In vitro
analysis of this mutant LHR, LHR(I625K), in HEK293
cells indicated that the signaling efficiency was
significantly impaired, which explains the partial
phenotype. We have compared this mutant LHR to
two other mutant LHRs, LHR(A593P) and
LHR(S616Y), identified in a complete and partial
LCH patient, respectively. Although the ligand-
binding affinity for all three mutant receptors was
normal, the hormonal response of LHR(A593P) was
completely absent and that of LHR(S616Y) and
LHR(I625K) was severely impaired. Low cell sur-
face expression explained the reduced response of
LHR(S616Y), while for LHR(I625K) this diminished
response was due to a combination of low cell
surface expression and decreased coupling effi-
ciency. For LHR(A593P), the absence of a reduced
response resulted from both poor cell surface ex-
pression and a complete deficiency in coupling.
Our experiments further show a clear correlation
between the severity of the clinical phenotype of
patients and overall receptor signal capacity,
which is a combination of cell surface expression
and coupling efficiency. (Molecular Endocrinology
12: 775–784, 1998)
INTRODUCTION
Male sex differentiation begins when the sex-deter-
mining factor (SRY) is expressed from the Y chromo-
some, which causes the indifferent gonads to develop
into testes (1). In the testis, testicular cords are formed
containing Sertoli cells and germ cells, while mesen-
chymal cells migrate into the interstitial space between
the cords, giving rise to fetal Leydig cells (2). The
testicular Sertoli cells and Leydig cells produce, re-
spectively, anti-Mu¨ llerian hormone and testosterone,
two hormones that are essential for correct differenti-
ation of both primary and secondary sex characteris-
tics (for review see Refs. 3–5). Anti-Mu¨ llerian hormone
triggers the regression of the mu¨ llerian duct, the anla-
gen of the female urogenital tract, while testosterone,
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Molecular Endocrinology
Copyright © 1998 by The Endocrine Society
775
in some target tissues after its reduction to dihydrotes-
tosterone, stimulates differentiation and growth of the
epididymides, vasa deferentia, the prostate, seminal
vesicles, and other parts of the male urogenital tract
including the formation of the penis. Before birth, pro-
liferation and differentiation of Leydig cells and their
production of androgens are dependent on the pla-
cental hormone, human CG (hCG). Prenatal distur-
bance of male sex differentiation leads to a variety of
phenotypes ranging from males with micropenis, indi-
viduals with ambiguous genitalia and hypospadias, to
complete male pseudohermaphrodites (5). These phe-
notypes are either due to insensitivity of the target
cells to androgens (5, 6) or to impaired testicular an-
drogen production as a result of a steroidogenic en-
zyme defect [e.g.17
b
-hydroxysteroid dehydrogenase
type II (7)] or reduced sensitivity of the Leydig cels to
LH [Leydig cell hypoplasia (LCH) (8, 9)]. Two types of
LCH have been described (10). LCH type I is the
severe form of LCH identified in 46 XY individuals
displaying a predominantly female phenotype, which
is caused by mutations in the LH receptor (LHR) that
completely disrupt LH signaling (11–13). Milder forms
of LCH (type II), initially described in 1985 by Toledo et
al. (14), are also caused by a mutation in the LHR gene,
but this defect disrupts LHR signaling less severely,
and patients are characterized by hypospadias or mi-
cropenis (13, 15, 16).
In the present paper we report the identification and
characterization of a homozygous mutation in the LHR
in three brothers with LCH type II. This mutation par-
tially inactivates LH signaling, which explains the phe-
notype. In addition, we have compared this mutation
to other missense mutations previously identified in
patients with LCH. Our data show that a clear corre-
lation exists between receptor activity and the result-
ing phenotype.
RESULTS
Clinical Details
Three brothers with normal karyotype were referred at
the age of 28, 35, and 51 with micropenis, absence of
pubertal signs, and infertility. LH and FSH levels were
elevated but responded normally to GnRH. Basal tes-
tosterone and androstenedione levels, however, were
low and responded poorly to hCG (Table 1; data not
shown). Some variation in their basal and hCG-stim-
ulated testosterone levels was observed among the
brothers. However, this variation is probably the result
of interindividual variation that is also observed in the
normal population (Table 1) (17). Histological analysis
of a testis biopsy of one of the patients showed sem-
iniferous tubules with clearly thickened basal lamina
and an interstitium that lacked mature Leydig cells
(Fig. 1A). Spermatogenesis was present but did not
extend beyond the elongated spermatid stage (Fig.
1B). Taken together these observations indicate that
these patients have a mild form of LCH (LCH type II).
DNA Analysis of the Patients
Single-strand conformation polymorphism (SSCP)
analysis of the LHR gene in two of the affected broth-
ers was first performed on exon 11 because in the
transmembrane domain (TMD) of the receptor most of
the subtle mutations have been identified. An aberrant
migration pattern was found in one of the PCR frag-
ments from both affected brothers while normal
individuals showed only control bands (Fig. 2A). Se-
quencing of the PCR fragment revealed a T-to-A trans-
version at position 1874 of the cDNA (18), changing
codon 625 from ATA for isoleucine to AAA for lysine
(Fig. 2B). Isoleucine 625 is located in the seventh
transmembrane segment of the receptor near the cy-
toplasmic tail. Both tested brothers were homozygous
for the DNA change, and the mutation was not iden-
tified in any of the other samples that were analyzed
(n 525). Therefore, we conclude that the missense
mutation segregates with the disease, although
genomic DNA of the other family members could not
be analyzed.
I625K Mutation Partially Inactivates Signal
Transduction
The I625K mutation was introduced in a wild-type
human LHR (hLHR) cDNA expression vector to pro-
duce pLHR(I625K). To determine the effect of the mu-
tation on LH signal transduction, HEK293 cells were
cotransfected with a luciferase reporter construct con-
taining a cAMP-responsive promoter (CRE
6
-Lux) (19)
and the wild-type expression vector (pLHR(WT)) or
pLHR(I625K). We used a CRE-driven luciferase re-
porter because this response is more sensitive and
more conveniently measured than the regularly used
cAMP response (not shown). Transiently transfected
cells expressing the wild-type LHR responded to hCG
with a 32-fold increase of luciferase activity whereas
cells transfected with LHR(I625K) showed only a 18-
fold increase (Fig. 3A). Furthermore, the EC
50
of this
response in the LHR(I625K) shifted more than a 1
order of magnitude to the right compared with the
EC
50
obtained with the wild-type LHR (Table 2). The
total number of binding sites and the affinity for hCG of
LHR(I625K) were not different from those of the wild-
type receptor (Table 2).
Table 1. Basal and hCG-Induced Testosterone Levels of
Three Brothers with Mild Leydig Cell Hypoplasia
hCG test (10000 IU im) Case I Case II Case III Normal
Testosterone (basal)
a
2.28 3.26 2.99 10–33
b
Testosterone (after hCG)
a
4.17 20.4 10.9 36–60
b
a
Testosterone in nanomoles per liter.
b
Basal and hCG-induced levels in normal men (17).
MOL ENDO · 1998 Vol 12 No. 6
776
Correlation of Receptor Activity and Phenotype
To date, three different missense mutations in the LHR
gene, A593P, S616Y, and, in the present report, I625K,
have been identified in patients with LCH (11, 13, 15).
The extent of the syndrome probably depends on the
residual level of androgen production by the Leydig
cells, which in turn might be a function of the severity
of the effect of the mutation on LH signaling. There-
fore, we compared the characteristics of the different
mutant LHRs in HEK293 cells (Fig. 3B and Table 2).
The three mutant receptors bound hCG with similar
affinity as the wild-type receptor (Table 2). The hCG-
induced cAMP regulatory element (CRE) activity of all
three mutant receptors was impaired (Fig. 3B). As
previously shown, LHR(A593P) did not respond to
hCG at all, while the maximal response of LHR(S616Y)
and LHR(I625K) was reduced to 50% and 65%, re-
spectively (Table 2). In addition, the EC
50
of CRE in-
duction by hCG of both mutant receptors shifted to the
right by a factor of 20. The results obtained with
LHR(S616Y) confirm those of Laue et al. (15); however,
they contradict the results of Latronico et al. (13), who
were unable to show signaling of LHR(S616Y). A pos-
sible explanation for this discrepancy may be the poor
sensitivity of the cAMP production assay. The CRE
luciferase assay used here is much more sensitive,
and in this case appears to be clearly superior to a
cAMP assay.
A reduced hormonal response of the mutant LHRs
could be caused by a reduced expression of the LHR.
Therefore, the amount of LHR receptor protein on a
Western blot (Fig. 4) and the total number of binding
sites were determined (Table 2). To visualize the LHR
molecules on Western blot, the LHR cDNAs were pro-
vided with an hemagglutinin (HA) epitope tag at the
39-end of the open reading frame and overexpressed
in COS-1 cells. The HA tag had no effect on the num-
ber of binding sites or on the hCG-induced CRE re-
sponse (data not shown). In COS-1 cells transfected
with the wild-type LHR cDNA, two major LHR-specific
bands of approximately 65 and 220 kDa were ob-
served (indicated with arrows). Furthermore a minor
band of approximately 300 kDa was present. The two
largest bands are probably multimers or aggregates
but they could also represent glycosylated forms of
the receptor. The two additional bands of approxi-
mately 55 and 70 kDa are the result of nonspecific
binding of the HA antibody, as they also occur in cells
transfected with the empty expression vector (Fig. 4,
right lane). In cells transfected with the different mu-
tant LHR cDNAs, a similar profile was observed but the
intensity of the LHR-specific bands was less, indicat-
ing that the amount of LHR protein is much less. The
total number of binding sites (B
max
) was also deter-
mined as a measure of the amount of receptor that has
been inserted properly into the plasma membrane.
The wild-type LHR and LHR(I625K) had the same
Fig. 1. Testis Histology of Testis of a Patient with Mild LCH
A, Testis biopsy taken from one of the patients. It shows seminiferous tubules with thickened basal lamina and spermato-
genesis arrested in elongated spermatid stage. In the interstitium, mature Leydig cells are absent. Magnification 1603. B, Higher
magnification (4003) of one tubule with elongated spermatids (see arrow). Sections were stained with hematoxylin/eosin.
Partial LHR Inactivation in LCH Type II 777
number of binding sites, but the B
max
values of
LHR(A593P) and LHR(S616Y) were reduced to 0.5%
and 14%, respectively (Table 2). These results indicate
that the processing and/or the transport of the latter
two mutant receptors to the cell surface was impaired.
In conclusion, the expression studies show mutant
LHR molecules do not behave as wild-type molecules
with respect to stability and/or trafficking to the cell
surface. We, therefore, decided to measure the recep-
tor activity of those receptor molecules that present at
the cell surface because these are responsible for
signal transduction. Thus, basal and hCG-induced
CRE activity per cell surface binding-site of the differ-
ent mutants was determined and was compared with
activity of the wild-type receptor expressed at different
levels. Thus, a reference curve for the wild type LHR
was constructed by transfecting different amounts
(0.1, 1, and 10
m
g) of expression vector in HEK293
cells. In these cells, basal and hCG-induced CRE lu-
ciferase activity as well as the number of cell surface
hCG-binding sites was determined (Fig. 5A). Subse-
quently, 10
m
g of the various mutant LHR expression
vectors were transfected into HEK293 cells, and the
same parameters were determined and compared
with the wild-type LHR reference curve (Fig. 5B). In
wild-type LHR-expressing cells, both basal and hCG-
Fig. 2. Homozygous Missense Mutation in LHR Gene
A, SSCP in the coding sequence in patient with mild LCH
(patient). For comparison, SSCP patterns of three normal
individuals (contol) are shown. Normal and aberrant SSCP
patterns are indicated by arrows and arrowheads, respec-
tively. B, Genomic sequence of the LHR gene (from position
1867 to 1882) of patient with mild LCH (left) and normal
individual (right). A homozygous T-to-A conversion was iden-
tified at position 1874 of the LHR gene in the patient. Fig. 3. hCG-Induced CRE Activation by Wild-Type and Mu-
tant LHR
HEK 293 cells were transfected with the expression vector
pSG5 containing the wild-type or indicated mutant hLHR
cDNA in combination with the cAMP responsive construct,
pCRE
6
Lux. The expression vector pRSV-lacZ was included in
the transfection assay as a control for transfection efficiency.
Basal and hCG-induced luciferase activity divided by
b
-ga-
lactosidase activity determined in the same cell lysate was
plotted as means 6SEM (n 54) against the dose of hCG. The
results of one representative experiment out of three is
shown.
MOL ENDO · 1998 Vol 12 No. 6
778
induced CRE activity increased proportionally with the
number of cell surface binding sites. Cell surface ex-
pression of all mutant receptor was reduced com-
pared with the wild-type receptor when 10
m
g expres-
sion vector DNA were also used in the transfection. In
addition, LHR(A593P) completely lacked hormone-de-
pendent signaling activity. In contrast, LHR(S616Y),
which is also poorly expressed at the cell surface,
displayed hardly reduced signaling activity compared
with the wild-type receptor at similar receptor densi-
ties. The expression of LHR(I625K) was also reduced
although to a lesser extent than LHR(S616Y). In addi-
tion, however, this mutant receptor displayed a re-
duced signaling capacity.
DISCUSSION
The characteristics of LCH II that are found in the
family investigated in the present paper are caused by
a homozygous T
1874
to A mutation in the LHR gene.
This base change results in a substitution of a neutral
hydrophobic isoleucine residue by a positively
charged lysine residue at position 625 of the LHR. This
residue is located at the border between the seventh
transmembrane segment and the cytoplasmic tail of
the receptor. The substitution severely impairs hCG-
induced receptor activation, without interfering with
the total number of binding sites and the affinity for the
ligand. The reduced hCG response, however, does not
completely preclude all Leydig cell responses. An hCG
challenge elicited a slight plasma testosterone re-
sponse, which probably explains why spermatogene-
sis in these patients progressed up to late stages of
spermatid differentiation. Spermiation, the last com-
plicated step of sperm release involving major recon-
Table 2. Comparison of Different Mutant LHRs Identified in Patients with LCH
Mutation Wild Type I625K S616Y A593P
Binding
K
d
(nM) 2.4 2.5 1.4 0.6
B
max
[pmol/mg protein] 2.5 1.7 0.36 0.01
CRE response
EC
50
(ng/ml) 0.5 10 10 ND
b
Basal
1
1.0 60.1 1.0 60.1 1.3 60.2 1.0 60.1
Max.
1
30 61.4 18 61.6 14 60.6 1.5 60.2
a
Relative luciferase units (means 6SEM).
b
ND, not detectable.
Fig. 4. Western Blot of Mutant LHRs Expressed in COS-1
Cells
COS-1 cells were transfected with the empty expression
vector pSG5 or with pSG5 containing the indicated wild-type
or mutant HA-tagged hLHR cDNA. Three days after transfec-
tion, the cells were harvested and subjected to 10% SDS-
PAGE followed by Western blotting. Specific bands were
visualized using immunostaining with a HA tag-specific
monoclonal antiserum. The arrows indicated the two most
predominant bands that are specific for the wild-type LHR.
On the left the migration and the molecular weights of three
standard proteins are indicated.
Fig. 5. Comparison of LHR Signaling Capacity per Cell Sur-
face Binding Site
HEK 293 cells were cotransfected with the cAMP respon-
sive reporter gene, pCRE
6
Lux in combination with either dif-
ferent amounts (10, 1, 0.1
m
g) of wild-type LHR cDNA ex-
pression vector (A) or with equal amounts (10
m
g) of
expression vectors containing the wild-type hLHR cDNA or
mutant cDNA A593P, S616Y, or I625K (B). Basal and hCG-
induced luciferase activity (n 54) determined in cell lysate is
plotted as means 6SEM against the number of binding sites
(n 52). The results of one representative experiment out of
two is shown.
Partial LHR Inactivation in LCH Type II 779
struction of the spermatogenic epithelium, did not oc-
cur, which is in accordance with the dependence of
this step on sufficient androgens (20). In patients with
severe LCH who display very low levels of androgens,
spermatogenesis does not occur at all (11), indicating
that the first stages of spermatogenesis may also be
androgen-dependent, although an additional effect of
cryptorchidism in complete LCH patients cannot be
excluded.
The only other reported point mutation causing LCH
type II, S616Y, is located in the same receptor domain,
approximately two
a
-helical turns toward the extracel-
lular side in TMD 7 (Fig. 5) (13, 15). Two patients with
this mutation have been identified independently. One
compound heterozygote patient carrying the S616Y
mutation in combination with a completely inactive
LHR gene (deletion of exon 8) had a small penis and
severe hypospadias, while the other patient, homozy-
gous for S616Y, had a phenotype similar to the pa-
tients described in the present paper, micropenis but
no other indications of aberrant male sex differentia-
tion. Both patients with the S616Y mutations were too
young to be informative about the effect of their LHR
mutation on spermatogenesis.
The extent of the phenotype of patients correlates
well with the effect of the mutation on both receptor
expression and responsiveness to hCG. Homozygous
presence of two LHR(A593P) allelles, giving rise to a
receptor that is poorly expressed and deficient in sig-
naling, is associated with complete pseudohermaph-
roditism. Similar phenotypes are observed in patients
having a premature stop codon in both alleles; severe
truncation of the LHR also results in complete disrup-
tion of signal transduction (12, 13). Homozygocity for a
S616Y or a I625K mutation both cause a milder iden-
tical phenotype (micropenis). The overall receptor ac-
tivity of these two mutant receptors is equally reduced,
albeit this reduction results from different mecha-
nisms. LHR(S616Y) is poorly expressed but responds
normally to hCG while LHR(I625K) is both poorly ex-
pressed and impaired in responding to hCG. In addi-
tion, both mutant receptors require a higher dose of
hCG to respond compared with the wild-type recep-
tor. When the LHR(S616Y) allele is combined with a
completely inactive LHR allele (15), the phenotype of
the compound heterozygous patient is intermediate
between the mild and the complete form of LCH.
(micropenis with severe hypospadia). These correla-
tions of patient phenotype with receptor behavior in
vitro suggest that there may be no clear distinction
between complete and partial feminization of external
genitalia due to LH insensitivity (LCH type I and type II)
as proposed by Toledo et al. (14). Rather, a continuous
range of phenotypes from complete pseudohermaph-
rodism to patients that are only mildly affected is ob-
served depending on the different effects of mutations
on LHR signal transduction and on specific combina-
tions of abnormal alleles. During the preparation of this
manuscript, another mutation causing partial LCH was
described (16). This patient displayed sexual ambigu-
ity at birth due to a homozygous mutation (C131R) in
the extracellular domain of the receptor. The signaling
of the receptor was detectable but severely impaired,
which is in line with the observed phenotype.
Remarkably, LHR(A593P), LHR(S616Y) and
LHR(I625K) display normal binding affinity while their
EC
50
is severly affected. Thus, only the biological re-
sponse of these receptors is impaired. Similar discrep-
ancies have been observed in the TSH receptor (21),
which underscores the separate position of the glyco-
protein hormone receptors within the G protein-cou-
pled receptor (GPCR) superfamily and supports the
hypothesis that these receptors contain two indepen-
dent domains: a ligand-binding domain and a TMD
that transduces the signal into the cell. Only the func-
tion of this latter domain is affected in the three mutant
receptors.
The different characteristics of the tested mutants
also provide clues as to the importance of subdomains
of the receptor molecule. Both the S616Y and I625K
mutations are located on the cytoplasmic side of TMD
7, close to a conserved region in the superfamily of
GPCRs, the NPXXY motif (22). In vitro studies of other
GPCRs, including the glycoprotein hormone recep-
tors, have indicated that the NPXXY motif is important
for ligand-induced receptor activation (23–25) and re-
ceptor sequestration (26). Indeed, I625K and S616Y
may affect the function of the NPXXY motif and in this
way decrease hCG-induced receptor activation.
Based on homology studies of a number of GPCRs,
Baldwin (22, 27) has suggested a model of the most
probable orientation of the seven-transmembrane
a
-helices in the membrane (Fig. 6). In this model,
which appears to agree well with both our own pre-
liminary model (F. Fanelli, personal communication)
and a recently published LHR model (28), the hydro-
phobic isoleucine 625 points toward the hydrophobic
phospholipid membrane. Introduction of a positive
charge by the exchange of a lysine residue at this
position may cause the
a
-helix of TMD 7 to rotate and
move residue 625 toward the hydrophilic core of the
TMD (see arrow in Fig. 6). Alternatively, TMD 7 may be
shifted toward the cytoplasm. Movement of parts of
TMD 7 could be facilitated by the presence of two
helix-breaking proline residues. Furthermore, a turn of
TMD 6 has been observed in a constitutively active
mutant
b
2
-adrenergic receptor (29), indicating that
such a turn can affect signal transduction. A slight
rotation of TMD 7 changes the position of the con-
served NPXXY motif in relation to the rest of the re-
ceptor and in that manner may reduce the response to
hCG. However, the change must be small because the
mutation does not severely affect proper folding. In the
same model, serine 616 points toward TMD 1 and 2 in
the hydrophilic pocket between the transmembrane
helices of the receptor (F. Fanelli, personal communi-
cation). This serine is located exactly one helical turn
N-terminal of the asparagine in the NPXXY motif and is
very well conserved in the GPCR family, which indi-
cates that the size of this residue and its ability to form
MOL ENDO · 1998 Vol 12 No. 6
780
hydrogen bonds may be important. According to mo-
lecular modeling (F. Fanelli, personal communication)
a tyrosine residue at this position may fit in the recep-
tor pocket, although its side chain points toward TMD
3 instead of TMD 1 and 2. However, this fit must be
poor because receptor folding is disturbed as indi-
cated by the reduced cell surface expression. The
intrinsic receptor activity is, however, unaffected,
which indicates that the bulky tyrosine side chain does
not interfere with receptor activation, once it is in
place.
In conclusion, partial LCH in the present family is
due to a homozygous missense mutation (I625K) in
TMD 7 of the LHR. This mutation causes severe im-
pairment in hormone-dependent receptor signaling.
Detailed analysis of three missense mutations that
result in LCH revealed a clear inverse relationship be-
tween residual receptor activity and severity of the
clinical phenotype.
MATERIALS AND METHODS
Patients
The patients studied here have a mild form of LCH that was
designated LCH type II (14). In short, three brothers, born to
consanguineous parents were referred at the ages of 28, 31,
and 51, because of infertility due to azoospermia. The pa-
tients, all with a 46 XY karyotype, had male external genitalia
with adult-sized testis, but an undervirilized penis (micrope-
nis). Baseline levels of testosterone were low and a single
hCG injection (10,000 IU Pregnyl; Organon International, Oss,
The Netherlands) elicited a slight but significant increase of
serum testosterone levels (Table 1). Levels of intermediates
of the testosterone biosynthetic route were not elevated,
indicating absence of enzyme defects (not shown). An acute
adrenal cortex stimulation using 250
m
g Cortrosyn (Organon
International) induced a normal elevation of corticosteroids,
showing that adrenal steroid production was normal (data not
shown). LH and FSH levels were elevated, but the pituitary
responded normally to GnRH (100
m
g iv; Ayerst Laboratories,
Rouse Point, IL) (data not shown). LH bioactivity of one of
these patients was tested and was found to be normal. The
two younger brothers were treated with testosterone enan-
thate (250 mg/3 weeks; Organon International). After 2 yr of
treatment, both patients showed sufficient virilization but pe-
nis size remained inadequate. In only one of the patients did
treatment result in a significant increase in sperm count (from
azoospermia to 3 310
6
/ml) and fertility. All procedures were
carried out in the course of normal patient care after appro-
priate informed consent had been obtained.
SSCP and Sequence Analysis
Genomic DNA was extracted from peripheral blood (30) of
two of the affected brothers. Six overlapping fragments of
exon 11 of the LHR gene were amplified by PCR and
analyzed by SSCP as described previously (Ref. 31 and H.
Kremer et al., submitted). For sequencing, PCR fragments
were treated with alkaline phosphatase and exonuclease I
and sequenced using the USB sequencing kit for PCR
fragments (US Biochemical Corporation, Cleveland, OH).
Fig. 6. Model of the TMD of the hLHR
Projection of the cytoplasmic part of the TMD of the LHR according to the model proposed by Baldwin (22, 27). The view is
from the intracellular side toward the outside. The size of the dots indicate the distance of the amino acid to the cytoplasm (a large
dot means that the amino acid is close to the cytoplasm). The amino acids of the NPXXY motif are indicated in squares; the two
amino acids mutated in patients with partial LCH are indicated in circles. The amino acid mutated in the patient with complete
LCH (A593P) is located at the border between TMD 6 and extracellular loop 3 and is therefore absent in the projection.
Partial LHR Inactivation in LCH Type II 781
Construction of Mutant hLHR Expression Vectors
Wild-type hLHR cDNA was introduced in the expression vec-
tor pSG5 (33), resulting in pLHR(WT) (34). Mutations were
introduced into this construct using standard PCR mutagen-
esis (34, 35) with the primers (Pharmacia, Uppsala, Sweden)
described below. The nucleotides that differ from the wild-
type hLHR cDNA are indicated in bold.
LHR1512FOR: 59-GTC GGT GTC AGC AAT TAC-39;
LHR2182REV: 59-GTT AAA ATT ACT GGT ACA GG-39;
LHR616SYFOR: 59-CCC ATC AAT TATTGCGCAAAT
CCA TTT-39;
LHR616SYREV: 59-AAA TGG ATT TGC GCA ATA ATT
GAT GGG-39;
LHR625IKFOR: 59-G TAT GCA AAA TTC ACT AAG-39;
LHR625IKREV: 59-CTT AGT GAA TTT TGC ATA C-39.
For constructing pLHR(I625K), primer sets LHR1512FOR/
LHR625IKREV and LHR625IKFOR/LHR2181REV were used
separately to perform the first PCR amplification. After mixing
of the fragments, the final mutant fragment was obtained by
PCR using the primer set LHR1512FOR and LHR2181REV.
To construct the mutant hLHR expression vector, a BstXI-
HpaI fragment of the reamplified fragment (669 bp) was used
to replace the wild-type sequence in pLHR(WT), resulting in
pLHR(I625K). For the construction of pLHR(S616Y), a similar
strategy was used using the LHR616 primer set. Both con-
structs were checked by DNA sequencing. The construct
pLHR(A593P) was described previously (11).
Transfection of COS-1 and HEK293 Cells
COS-1 and HEK293 cells were maintained in culture medium
(DMEM/Ham’s F12 (1:1 vol/vol) (GIBCO BRL, Gaithersburg,
MD), 2 310
5
IU/liter penicillin (Brocades Pharma, Leiderdorp,
The Netherlands) and 0.2 g/liter streptomycin (Radium
Farma, Milan, Italy) and 5% and 10% FCS (SEBAK, Aiden-
bach, Germany), respectively, and were incubated in a hu-
midified incubator at 37 C and 5% CO
2
. Before transfection
the cells were seeded at 15% confluence in 75-cm
2
flasks
(Nunc, Roskilde, Denmark) and transfected the next day with
1 ml precipitate containing 20
m
g DNA (36).
cAMP Reporter Activity Measurements
For measuring the hormonal response of the different mu-
tants, HEK293 cells were cotransfected with pCRE
6
Lux
(19), pRSVlacZ (37) and pSG5, pLHR(WT), pLHR(A593P),
pLHR(S616Y) or pLHR(I625K) (10
m
g expression construct, 1
m
g pRSVlacZ, 2
m
g pCRE
6
Lux, and 7
m
g carrier DNA per ml
precipitate). Three days after transfection the hCG-depen-
dent CRE response was determined in 24-well tissue culture
plates (Costar, Cambridge, MA) by incubating the cells for 4 h
in culture medium containing 0.1% BSA with increasing con-
centrations of hCG (0.001 to 1000 ng/ml; urinary hCG; Or-
ganon International). Subsequently, the cells were lysed and
luciferase activity was measured (38).
b
-Galactosidase activ-
ity of the lysates was determined to correct for transfection
efficiency (37).
Scatchard Analysis
To determine the binding affinity (K
d
) and total receptor num-
ber (B
max
), HEK293 cells were transfected with pSG5,
pLHR(WT), pLHR(A593P), pLHR(S616Y), or pLHR(I625K) (10
m
g expression construct and 10
m
g carrier DNA per ml pre-
cipitate). Three days after transfection, Scatchard analysis
using chloramine T
125
I-labeled hCG (39) was performed on
purified membranes according to Ketelslegers and Catt (40).
cAMP Reporter Activity per Cell Surface Receptor
Number
For determining the receptor activity per receptor number
expressed at the cell surface, HEK293 cells were trans-
fected with pCRE
6
Lux, pRSVlacZ and pSG5, pLHR(WT),
pLHR(A593P), pLHR(S616Y), or pLHR(I625K). Three days af-
ter transfection a part of the transfected cells was used to
measure the basal and maximal hCG (1000 ng/ml) CRE re-
sponse as described above while the rest of the cells were
used to measure LHR cell surface expression. Cell surface
expression was determined as described previously (41, 42).
Briefly, transfected cells were harvested and resuspended in
binding buffer (10 mMTris-HCl, pH 7.5, 5 mMMgCl
2
, 0.1%
BSA, 5 mMsodium azide, 200 mMsucrose) containing
125
I-
labeled hCG (1.5 310
6
cpm) in the presence or absence of
a 1,000 fold excess of unlabeled hCG in a volume of 0.2 ml.
The sodium azide was added to prevent internalization. After
incubation for1hat37C,thecells were washed twice with
excess of binding buffer, after which the binding as measured
by the amount of radioactive hCG bound to the cells was
counted in a
g
-counter.
SDS-PAGE
To determine LHR expression on Western blots, LHR cDNAs
were extended with an HA immuno tag (YPYDVPDYAS) at the
39-end. This HA tag did not affect the number of binding sites
or hormone-dependent signaling (not shown). COS-1 cells
were transfected with pSG5, pLHR(WT)HA, pLHR(A593P)HA,
pLHR(S616Y)HA, or pLHR(I625K)HA (10
m
g expression plas-
mid and 10
m
g carrier DNA per ml precipitate). Three days
after transfection, the cells were washed twice with PBS and
harvested in 1 ml PBS. After the protein concentration was
determined (Bradford), equal amounts of protein (3.5
m
g)
were separated on 10% SDS/PAGE (43) and subsequently
blotted onto nitrocellulose (Schleicher & Schuell, Dassel, Ger-
many) using the Mini-Protean II gel electrophoresis and elec-
tro-blotting apparatus (Bio-Rad, Hercules, CA). The tagged
LHR proteins were visualized using the Renaissance Western
blot chemiluminescence detection kit (DuPont/NEN, Du Pont
de Nemours GmbH, Dreieich, Germany) using as primary
antibody a 1:500 dilution of the HA-specific monoclonal an-
tiserum 12C5.
Acknowledgments
We would like to thank Dr. H. Kremer for her support during
the initial stages of this project, Marianna Timmerman for
technical assistence, Dr. A. Himmler for supplying the CRE
luciferase construct, Dr. E. Milgrom for supplying the expres-
sion vector containing the wild-type hLHR cDNA, Dr. F.
Fanelli for performing molecular modeling on the LHR, and
Dr. Th. van der Kwast for evaluating the testis pathology.
Received October 23, 1997. Revision received February
17, 1998. Accepted March 2, 1998.
Address requests for reprints to: J. W. M. Martens,
Department of Endocrinology and Reproduction, Erasmus
University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam,
The Netherlands. E-mail: martens@endov.fgg.eur.nl.
The research of J. Martens is financed by the Netherlands
Organisation of Scientific Research and that of S. Toledo and
N. Abelin by the National Research Council of Brazil (CNPq),
the State Research Foundation (FAPESP), and the Medical
Research Laboratory (LIM).
MOL ENDO · 1998 Vol 12 No. 6
782
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