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 São Paulo University
School of Medicine CEP 01246 São Paulo, Brazil
Department of Human Genetics (H.G.B.) University Hospital
6500 HB Nijmegen, The Netherlands
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ABSTRACT
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Leydig cell hypoplasia (LCH) is characterized by a
decreased response of the Leydig cells to LH. As a result, patients
with this syndrome display aberrant 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
revealed 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 surface 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
efficiency. For LHR(A593P), the absence of a reduced response resulted
from both poor cell surface expression 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.
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INTRODUCTION
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Male sex differentiation begins when the sex-determining factor
(SRY) is expressed from the Y chromosome, which causes the indifferent
gonads to develop into testes (1). In the testis, testicular cords are
formed containing Sertoli cells and germ cells, while mesenchymal cells
migrate into the interstitial space between the cords, giving rise to
fetal Leydig cells (2). The testicular Sertoli cells and Leydig cells
produce, respectively, anti-Müllerian hormone and testosterone,
two hormones that are essential for correct differentiation of both
primary and secondary sex characteristics (for review see Refs. 35).
Anti-Müllerian hormone triggers the regression of the
müllerian duct, the anlagen of the female urogenital tract, while
testosterone, in some target tissues after its reduction to
dihydrotestosterone, 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, proliferation and differentiation of Leydig cells
and their production of androgens are dependent on the placental
hormone, human CG (hCG). Prenatal disturbance of male sex
differentiation leads to a variety of phenotypes ranging from males
with micropenis, individuals with ambiguous genitalia and hypospadias,
to complete male pseudohermaphrodites (5). These phenotypes are either
due to insensitivity of the target cells to androgens (5, 6) or to
impaired testicular androgen production as a result of a steroidogenic
enzyme defect [e.g. 17ß-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, 12, 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 micropenis (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 partially inactivates LH signaling, which explains the
phenotype. In addition, we have compared this mutation to other
missense mutations previously identified in patients with LCH. Our data
show that a clear correlation exists between receptor activity and the
resulting phenotype.
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RESULTS
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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 testosterone and androstenedione levels, however, were low
and responded poorly to hCG (Table 1
;
data not shown). Some variation in their basal and hCG-stimulated
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 seminiferous
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).

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Figure 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 spermatogenesis arrested in
elongated spermatid stage. In the interstitium, mature Leydig cells are
absent. Magnification 160x. B, Higher magnification (400x) of one
tubule with elongated spermatids (see arrow). Sections
were stained with hematoxylin/eosin.
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DNA Analysis of the Patients
Single-strand conformation polymorphism (SSCP) analysis of the LHR
gene in two of the affected brothers 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 fragments from both affected brothers while
normal individuals showed only control bands (Fig. 2A
). Sequencing of the PCR fragment
revealed a T-to-A transversion 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 cytoplasmic tail. Both tested brothers were
homozygous for the DNA change, and the mutation was not identified in
any of the other samples that were analyzed (n = 25). Therefore,
we conclude that the missense mutation segregates with the disease,
although genomic DNA of the other family members could not be
analyzed.

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Figure 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, respectively. 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 identified at position 1874 of the LHR
gene in the patient.
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I625K Mutation Partially Inactivates Signal Transduction
The I625K mutation was introduced in a wild-type human LHR (hLHR)
cDNA expression vector to produce pLHR(I625K). To determine the effect
of the mutation on LH signal transduction, HEK293 cells were
cotransfected with a luciferase reporter construct containing a
cAMP-responsive promoter (CRE6-Lux) (19) and the wild-type
expression vector (pLHR(WT)) or pLHR(I625K). We used a CRE-driven
luciferase reporter 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 EC50 of
this response in the LHR(I625K) shifted more than a 1 order of
magnitude to the right compared with the EC50 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
).

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Figure 3. hCG-Induced CRE Activation by Wild-Type and Mutant
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, pCRE6Lux. 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 ß-galactosidase activity determined in the same
cell lysate was plotted as means ± SEM (n = 4)
against the dose of hCG. The results of one representative experiment
out of three is shown.
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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. Therefore, 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%, respectively (Table 2
). In addition, the
EC50 of CRE induction 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 possible 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
provided with an hemagglutinin (HA) epitope tag at the 3'-end of the
open reading frame and overexpressed in COS-1 cells. The HA tag had no
effect on the number of binding sites or on the hCG-induced CRE
response (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 observed (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 approximately 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 mutant LHR cDNAs, a similar profile was observed but
the intensity of the LHR-specific bands was less, indicating that the
amount of LHR protein is much less. The total number of binding sites
(Bmax) was also determined 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 number of binding sites, but
the Bmax 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 receptor 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 different 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 µg) of expression vector in HEK293 cells. In these cells, basal
and hCG-induced CRE luciferase activity as well as the number of cell
surface hCG-binding sites was determined (Fig. 5A
). Subsequently, 10 µg 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-induced CRE activity increased proportionally with the
number of cell surface binding sites. Cell surface expression of all
mutant receptor was reduced compared with the wild-type receptor when
10 µg expression vector DNA were also used in the transfection. In
addition, LHR(A593P) completely lacked hormone-dependent 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 densities. The
expression of LHR(I625K) was also reduced although to a lesser extent
than LHR(S616Y). In addition, however, this mutant receptor displayed a
reduced signaling capacity.

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Figure 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 transfection, 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.
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Figure 5. Comparison of LHR Signaling Capacity per Cell
Surface Binding Site
HEK 293 cells were cotransfected with the cAMP responsive reporter
gene, pCRE6Lux in combination with either different amounts
(10, 1, 0.1 µg) of wild-type LHR cDNA expression vector (A) or with
equal amounts (10 µg) of expression vectors containing the wild-type
hLHR cDNA or mutant cDNA A593P, S616Y, or I625K (B). Basal and
hCG-induced luciferase activity (n = 4) determined in cell lysate
is plotted as means ± SEM against the number of
binding sites (n = 2). The results of one representative
experiment out of two is shown.
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DISCUSSION
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The characteristics of LCH II that are found in the family
investigated in the present paper are caused by a homozygous
T1874 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 response, which probably explains why spermatogenesis in
these patients progressed up to late stages of spermatid
differentiation. Spermiation, the last complicated step of sperm
release involving major reconstruction of the spermatogenic epithelium,
did not occur, 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
-helical
turns toward the extracellular 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,
homozygous for S616Y, had a phenotype similar to the patients described
in the present paper, micropenis but no other indications of aberrant
male sex differentiation. 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 signaling, is
associated with complete pseudohermaphroditism. Similar phenotypes are
observed in patients having a premature stop codon in both alleles;
severe truncation of the LHR also results in complete disruption of
signal transduction (12, 13). Homozygocity for a S616Y or a I625K
mutation both cause a milder identical phenotype (micropenis). The
overall receptor activity of these two mutant receptors is equally
reduced, albeit this reduction results from different mechanisms.
LHR(S616Y) is poorly expressed but responds normally to hCG while
LHR(I625K) is both poorly expressed and impaired in responding to hCG.
In addition, both mutant receptors require a higher dose of hCG to
respond compared with the wild-type receptor. 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 correlations 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 pseudohermaphrodism to patients that are only
mildly affected is observed depending on the different effects of
mutations on LHR signal transduction and on specific combinations of
abnormal alleles. During the preparation of this manuscript, another
mutation causing partial LCH was described (16). This patient displayed
sexual ambiguity 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 EC50 is severly affected.
Thus, only the biological response of these receptors is impaired.
Similar discrepancies have been observed in the TSH receptor (21),
which underscores the separate position of the glycoprotein hormone
receptors within the G protein-coupled receptor (GPCR) superfamily and
supports the hypothesis that these receptors contain two independent
domains: a ligand-binding domain and a TMD that transduces the signal
into the cell. Only the function 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 receptors, have indicated that the NPXXY motif is
important for ligand-induced receptor activation (23, 24, 25) and receptor
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
-helices in the membrane (Fig. 6
). In this model, which appears to agree
well with both our own preliminary model (F. Fanelli, personal
communication) and a recently published LHR model (28), the hydrophobic
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
-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
ß2-adrenergic receptor (29), indicating that such a turn
can affect signal transduction. A slight rotation of TMD 7 changes the
position of the conserved NPXXY motif in relation to the rest of the
receptor 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 communication). 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 indicates
that the size of this residue and its ability to form hydrogen bonds
may be important. According to molecular modeling (F. Fanelli, personal
communication) a tyrosine residue at this position may fit in the
receptor 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 indicated 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.

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Figure 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.
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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 impairment in hormone-dependent receptor signaling. Detailed
analysis of three missense mutations that result in LCH revealed a
clear inverse relationship between residual receptor activity and
severity of the clinical phenotype.
 |
MATERIALS AND METHODS
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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 patients, all with a 46
XY karyotype, had male external genitalia with adult-sized testis, but
an undervirilized penis (micropenis). 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 µg 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 µg 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 enanthate (250 mg/3
weeks; Organon International). After 2 yr of treatment, both patients
showed sufficient virilization but penis size remained inadequate. In
only one of the patients did treatment result in a significant increase
in sperm count (from azoospermia to 3 x 106/ml) and
fertility. All procedures were carried out in the course of normal
patient care after appropriate 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).
Construction of Mutant hLHR Expression Vectors
Wild-type hLHR cDNA was introduced in the expression vector pSG5
(33), resulting in pLHR(WT) (34). Mutations were introduced into this
construct using standard PCR mutagenesis (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: 5'-GTC GGT GTC AGC AAT TAC-3';
LHR2182REV: 5'-GTT AAA ATT ACT GGT ACA GG-3';
LHR616SYFOR: 5'-CCC ATC AAT TAT TGC
GCA AAT CCA TTT-3';
LHR616SYREV: 5'-AAA TGG ATT TGC GCA ATA ATT GAT
GGG-3';
LHR625IKFOR: 5'-G TAT GCA AAA TTC ACT
AAG-3';
LHR625IKREV: 5'-CTT AGT GAA TTT TGC ATA
C-3'.
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 constructs
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/Hams F12 (1:1 vol/vol) (GIBCO BRL, Gaithersburg, MD), 2 x
105 IU/liter penicillin (Brocades Pharma, Leiderdorp, The
Netherlands) and 0.2 g/liter streptomycin (Radium Farma, Milan, Italy)
and 5% and 10% FCS (SEBAK, Aidenbach, Germany), respectively, and
were incubated in a humidified incubator at 37 C and 5%
CO2. Before transfection the cells were seeded at 15%
confluence in 75-cm2 flasks (Nunc, Roskilde, Denmark) and
transfected the next day with 1 ml precipitate containing 20 µg DNA
(36).
cAMP Reporter Activity Measurements
For measuring the hormonal response of the different mutants,
HEK293 cells were cotransfected with pCRE6Lux (19),
pRSVlacZ (37) and pSG5, pLHR(WT), pLHR(A593P), pLHR(S616Y) or
pLHR(I625K) (10 µg expression construct, 1 µg pRSVlacZ, 2 µg
pCRE6Lux, and 7 µg carrier DNA per ml precipitate). Three
days after transfection the hCG-dependent 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 concentrations of hCG (0.001 to 1000 ng/ml; urinary hCG;
Organon International). Subsequently, the cells were lysed and
luciferase activity was measured (38). ß-Galactosidase activity of
the lysates was determined to correct for transfection efficiency
(37).
Scatchard Analysis
To determine the binding affinity (Kd) and total
receptor number (Bmax), HEK293 cells were transfected with
pSG5, pLHR(WT), pLHR(A593P), pLHR(S616Y), or pLHR(I625K) (10 µg
expression construct and 10 µg carrier DNA per ml precipitate). Three
days after transfection, Scatchard analysis using chloramine T
125I-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
pCRE6Lux, pRSVlacZ and pSG5, pLHR(WT), pLHR(A593P),
pLHR(S616Y), or pLHR(I625K). Three days after transfection a part of
the transfected cells was used to measure the basal and maximal hCG
(1000 ng/ml) CRE response 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
mM Tris-HCl, pH 7.5, 5 mM MgCl2,
0.1% BSA, 5 mM sodium azide, 200 mM sucrose)
containing 125I-labeled hCG (1.5 x 106
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 for 1 h at 37 C, the cells 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
-counter.
SDS-PAGE
To determine LHR expression on Western blots, LHR cDNAs were
extended with an HA immuno tag (YPYDVPDYAS) at the 3'-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 µg
expression plasmid and 10 µg 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 µg) were separated on 10%
SDS/PAGE (43) and subsequently blotted onto nitrocellulose (Schleicher
& Schuell, Dassel, Germany) using the Mini-Protean II gel
electrophoresis and electro-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 antiserum 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 expression 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.
 |
FOOTNOTES
|
---|
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{at}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).
Received for publication October 23, 1997.
Revision received February 17, 1998.
Accepted for publication March 2, 1998.
 |
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