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Hospital das Clínicas (A.C.L., I.J.P.A., B.B.M.)
Unidade de Endocrinologia do Desenvolvimento Universidade de
São Paulo, Brazil 3671
Department of Physiology and
Biophysics (Y.C., X.L., D.L.S.) The University of Iowa College of
Medicine Iowa City, Iowa 52242
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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L608,V609) revealed that
very little of the mutant receptor was expressed at the cell surface.
This was due to both a decrease in the total amount of receptor
expressed as well as to an increased intracellular retention of the
mutant receptor. In spite of the decreased cell surface expression of
the mutant, sufficient amounts were present to allow for assessment of
its functions. Equilibrium binding assays showed that the cell
surface hLHR(L608,V609) binds hCG with an affinity comparable
to that of the wild-type receptor. However, the cells expressing the
hLHR(L608,V609) exhibit only a 1.5- to 2.4-fold stimulation of cAMP
production in response to hCG. In contrast, cells expressing comparably
low levels of hLHR(wt) responded to hCG with 11- to 30-fold increases
of cAMP levels. Therefore, the testicular and ovarian unresponsiveness
to LH in these patients appears to be due to a mutation of the hLHR
gene in which Leu-608 and Val-609 are deleted. As a consequence, the
majority of the mutant receptor is retained intracellularly. The small
percentage of mutant receptor that is expressed at the cell surface
binds hormone normally but is unable to activate Gs. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We recently described the first occurrence of an inactivating mutation in the hLHR gene in a fully developed, infertile 46,XX woman with secondary amenorrhea (5). This 46,XX woman had a homozygous Arg554Stop mutation, which truncated the hLHR within the third transmembrane domain (5). Similar clinical features were later described in another 46,XX female with a homozygous missense inactivating Ala593Pro mutation (9). Both women were sisters of male pseudohermaphrodites with LCH (5, 9).
In this report, we examined the genomic DNA of a 46,XY patient with
female external genitalia (IV:8 in Fig. 1
) and a 46,XX sister with
menstrual irregularities and infertility (IV:5 in Fig. 1). Six
consecutive nucleotides within the region encoding the seventh
transmembrane helix of the hLHR, and hence two consecutive amino acids,
were found to be missing. This microdeletion caused impaired expression
and reduced signal transduction activity of the hLHR.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). This microdeletion results in the
absence of two consecutive amino acids, Leu-608 and Val-609, within the
seventh transmembrane helix of the hLHR. Automatic sequencing also
revealed a silent homozygous substitution of C for T (TTT/TTC) at
nucleotide 297 of exon 2 of the LHR gene in both patients. This
mutation, however, did not change the amino acid sequence of the
receptor.
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L608,V609)L608,V609), or the plasmid
encoding the wild-type hLHR, hLHR(wt), were transiently transfected
into 293 cells and [125I]hCG binding assays were
performed. Equilibrium binding assays to intact cells revealed that the
binding affinities of the mutant and wild-type cell surface receptors
for hCG were fairly comparable (Table 1).
If anything, mutant receptor bound hCG with a slightly higher (but not
statistically significant) affinity than the wild-type receptor.
However, as shown in Table 2, the maximal
binding of [125I]hCG to intact cells expressing
hLHR(L608,V609) was only 11% of that observed in intact cells
expressing the wild-type hLHR. This decreased cell surface expression
of hLHR(L608,V609) appears to be due, in part, to decreased total
expression of the mutant receptor, since the binding activity of
detergent-solubilized extracts of cells expressing hLHR(L608,V609)
was 32% of that observed in extracts from cells expressing the
wild-type receptor. These data suggest that the mutant receptor may be
synthesized more slowly and/or degraded more rapidly than the wild-type
receptor, resulting in lower steady state levels of mutant
receptor.
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L608,V609) is also due to an increased retention of the mutant
receptor intracellularly. Since the binding of [125I]hCG
to intact cells detects cell surface hLHR only, whereas the binding of
[125I]hCG to detergent-solubilized extracts of cells
detects both the cell surface as well as intracellular forms of the
hLHR, a comparison of the two binding assays indicates the percentage
of hLHR present at the cell surface. As shown in Table 2
, whereas 60%
of the total wild-type hLHR is present at the cell surface, only 19%
of the mutant receptor is located in the plasma membrane.
Responsiveness of Cells Transfected with hLHR(L608,V609)
Although the cell surface expression of the mutant receptor
containing the deletion of Leu-608 and Val-609 was greatly decreased as
compared with the cell surface expression of the wild-type receptor, it
was important to ascertain whether the small amount of mutant receptor
present at the cell surface was responsive to hCG. To address this
question, cells were transfected with decreased amounts of plasmid
encoding hLHR(wt) to deliberately decrease the level of cell surface
expression of the wild-type receptor to levels comparable to that of
the mutant. In the same experiment, cell surface levels of receptor
were measured by [125I]hCG binding to intact cells, and
basal and hCG-stimulated cAMP levels were also assayed. Table 3 summarizes the results from three
independent experiments. It can be seen that in all three experiments
the basal levels of cAMP in cells expressing the mutant were similar to
those expressing the wild-type receptor. However, whereas cells
expressing hLHR(wt) displayed an 11- to 30-fold stimulation in cAMP
production in response to hCG, cells expressing hLHR(L608,V609)
showed only a 1.5- to 2.4-fold increase. Of the three experiments, the
levels of cell surface receptor were most closely matched in Exp 3. In
that case, cells expressing hLHR(wt) exhibited a 30-fold increase in
cAMP production in response to hCG, whereas cells expressing
hLHR(L608,V609) exhibited only a 2.4-fold increase. It is also
worthwhile to examine the results of Exp 1, in which the binding to
cells expressing hLHR(wt) is only 0.21 ng hCG bound/106
cells. This is less than the lowest level of hCG binding to intact
cells expressing hLHR(L608,V609) observed in any of the three
experiments. Yet, even at this very low level of cell surface
expression of the wild-type receptor as seen in Exp 1, the cells were
capable of an 11-fold stimulation of cAMP, far more than that observed
for cells expressing hLHR(L608,V609) in any of the three
experiments. Therefore, the small amount of hLHR(L608, V609)
receptor present on the cell surface appears to bind hCG with normal
affinity, but it is severely impaired in its ability to transduce the
binding of hCG into an increase in cAMP accumulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Amino acids Leu-608 and Val-609 are conserved among the human, porcine,
rat, and mouse LHR as well as among the human TSH and FSH receptors (1, 2). They are predicted to be situated within the seventh transmembrane
helix relatively close to the extracellular surface. We have determined
that there are several consequences of the deletion of Leu-608 and
Val-609. Two of these are decreased total level of expression of the
mutant (possibly due to decreased receptor synthesis and/or increased
receptor degradation) and increased retention of the mutant within the
cell. Both of these phenomena result in a marked decrease in the levels
of cell surface receptors for hLHR(L608,V609). Although this alone
would be predicted to result in an impaired sensitivity of the target
cells to LH or hCG, the deletion of Leu-608 and Val-609 also results in
the inability of the mutant receptors present on the cell surface to
respond to hormone with increased cAMP production. Since the binding
affinity of the mutant is comparable to that of the wild-type receptor,
it can be concluded that the deletion of Leu-608 and Val-609 prevents
the hormone-occupied receptor from activating its effector system.
It is not entirely unexpected that one major consequence of the deletions of Leu-608 and Val-609 from the seventh transmembrane helix results in intracellular retention of the mutant receptor, since it has been previously shown that many different mutations of the LHR result in retention of the mutants in the endoplasmic reticulum due to their altered conformations (2, 10, 11, 12, 13, 14, 15, 16). Interestingly, there does not appear to be a particular region of the receptor that is particularly sensitive to conformational perturbations. Mutations causing intracellular retention have been observed in the extracellular domain, extracellular loops, transmembrane helices, intracellular loops, and cytoplasmic tail (2, 10, 11, 12, 13, 14, 15, 16). It has previously been shown that the high-affinity binding of hormone occurs primarily through interactions with the first six leucine-rich repeats of the extracellular domain (16). Interestingly, unless a hormone-binding site is disturbed, the hCG binding affinity of the intracellularly retained mutant is comparable to that of the wild-type receptor on the cell surface (2, 10, 11, 12, 13, 14, 15, 16). These observations suggest that, at least for the LHR, the extracellular domain can fold into a conformation compatible with high-affinity hCG binding before its exit from the endoplasmic reticulum. This in contrast to the closely related FSH receptor, where the conformation allowing FSH binding is not achieved until much later in the biosynthetic pathway, and hence no binding activity can be detected in FSH receptor precursors or intracellularly retained FSH receptor mutants (15).
As is seen in this study as well as earlier studies (2, 10, 11, 12, 13, 14, 15, 16), 100%
of the wild-type LHR is not located at the cell surface. Rather,
generally, 60–80% of the binding activity of the wild-type receptor
can be accounted for at the cell surface. The remaining 20–40%
binding activity, which is located intracellularly, is thought to be
due to the binding activity of the precursor forms of the receptor
undergoing maturation and processing. We have defined a mutant receptor
as being retained intracellularly if a significantly larger percentage
of the total receptor is located intracellularly as compared with the
wild-type receptor expressed in the same cell type. Although in some
extreme cases, a mutated LHR is found to be exclusively retained
intracellularly (10, 14), in most cases the mutations cause
intermediate degrees of intracellular retention (2, 11, 12, 13, 15, 16).
Within this latter group, it has been shown that some of these mutants
can be made to fold correctly and be expressed to a larger extent at
the cell surface if the cells are incubated for prolonged periods at
decreased temperatures (16). As shown herein, the hLHR(L608,V609)
mutant falls within the classification of a mutant being partially
retained intracellularly. However, since the hLHR(L608,V609) mutant
present at the cell surface was unable to respond to bound hCG with
increased cAMP, the responsiveness of the cells would not have been
enhanced even if more mutant were expressed at the cell surface at
decreased temperatures. Therefore, we did not examine the temperature
sensitivity of this mutant. It is also important to point out that the
observation that certain mutations of the LHR can cause intracellular
retention is not unique. For example, certain naturally occurring
mutations of the LDL receptor (17), CFTR protein (18, 19, 20), and insulin
receptor (21) result in intracellular retention. Within the superfamily
of G protein-coupled receptors, certain mutations introduced
artificially into the FSH receptor (15) or adrenergic receptors (22)
and some naturally occurring mutations of rhodopsin (23) and the V2
vasopressin receptor (24) have been shown to cause intracellular
retention of the mutant receptors.
Because hLHR(L608,V609) was expressed, albeit at very low levels, at
the cell surface, it was possible to ascertain the functional status of
the mutant cell surface receptor. It was shown that although the mutant
receptor bound hCG with the same high affinity as the wild-type
receptor, the fold increase in cAMP produced in response to hCG was
significantly reduced as compared with cells expressing comparably low
levels of wild-type hLHR. Recently it has been shown that a peptide
corresponding to the juxtacytoplasmic portion of transmembrane helix 6
of the hLHR can activate Gs, demonstrating that this portion of
transmembrane helix 6 has the potential to directly interact with and
activate Gs (25). Whether other transmembrane helices, for example
transmembrane helix 7, can also directly activate Gs is under
investigation. Since the deletion of Leu-608 and Val-609 occurs within
a portion of the seventh transmembrane helix relatively close to the
extracellular surface of the membrane, it is unlikely that the deleted
residues would have been interacting directly with Gs. However, if the
juxtacytoplasmic portion of helix 7 does indeed interact with Gs, then
one could hypothesize that the deletion of Leu-608 and Val-609 might
adversely affect the conformation of the juxtacytoplasmic portion of
the helix. Furthermore, a deletion in a transmembrane helix would be
predicted not only to affect the conformation of that transmembrane
region, but also cause more global conformational changes by virtue of
altering interhelical interactions (26). By analogy with the
ß2-adrenergic receptor (27, 28, 29), it is generally assumed,
although not yet directly demonstrated, that the third intracellular
loop and possibly other intracellular loop regions of the LHR are
required for Gs activation. One can also entertain the possibility,
therefore, of possible conformational changes in critical intracellular
loop regions caused by alterations in interhelical arrangements.
Although the above hypotheses are reasonable, much more needs to be
learned about the mechanism by which the hLHR activates Gs before the
precise consequences of the Leu-608 and Val-609 deletions can be
ascertained.
A homozygous inactivating missense mutation (Ser-616
Tyr) within the
seventh transmembrane helix of the hLHR was first described in a Puerto
Rican prepubertal boy with micropenis, bilaterally descended testes,
and no testosterone response to exogenous hCG (5). This mutation was
later reported in another Puerto Rican boy with micropenis associated
with severe perineoescrotal hypospadias and cryptorchidism (4). This
boy was compound heterozygous with one allele encoding for a mutant
hLHR containing the (Ser-616Tyr) substitution in transmembrane helix
7 and one allele encoding for a mutant hLHR containing a deletion of
exon 8 (a region within the extracellular domain). In 293 cells and
COS-7 cells transfected with a recombinant hLHR harboring the
Ser-616Tyr mutation, it was found that there was a severe
impairment, but not total loss, of hCG-stimulated cAMP production (4, 5). In contrast to the 616 missense mutation in the seventh
transmembrane helix of the LHR that led to micropenis, the
deletion amino acids 608 and 609 resulted in female external genitalia
in a 46,XY male. This finding is consistent with the more severe defect
in receptor expression and signal transduction caused by the deletion
of these two amino acids and, therefore, consistent with complete
failure of the masculinization process.
The familial ovarian resistance to LH has been characterized by normal external genitalia, spontaneous breast and pubic hair development at puberty, menstrual irregularities (secondary amenorrhea, oligoamenorrhea), infertility, enlarged cystic ovaries, and follicular development, at least up to the antral stage. Serum levels of LH and FSH are elevated with a high LH/FSH ratio, normal or low estrogen and androgen levels, and progesterone that never reaches postovulatary levels (5, 30). In the patient described here, bone mineral density was normal, suggesting adequate exposure of the bone to estrogen in this disease. At this time, four distinct inactivating mutations, including the microdeletion described here, were identified in females who were sisters of patients with male pseudo- hermaphroditism due to LCH (5, 8, 9, 30)
Normal pubertal feminization in women with complete ovarian resistance to LH suggests that LH is not essential for female pubertal development (5, 9, 30). Follicular growth and limited estrogen biosynthesis were also observed in the absence of normal LH activity (5, 36). Instead, LH appears essential to increase ovarian steroidogenic capacity and to induce ovulation with subsequent corpus luteum formation. Interestingly, constitutively activating mutations of the LHR appear to have no pathological effect in postpubertal females, suggesting the presence of redundant mechanisms and proper compensation resulting in normal ovarian function despite enhanced LHR bioactivity (31).
Genetic females who are heterozygous for LHR-inactivating mutations, i.e. mothers of patients with LCH, have a normal reproductive phenotype, while women with homozygous inactivating mutations of LHR have severe menstrual irregularities (secondary amenorrhea, oligoamenorrhea) and infertility. Genetic females with mild LHR-inactivating mutations, i.e. sisters of patients with micropenis or sporadic cases, have not been described. We speculate that the phenotype in these women could be manifested by milder forms of menstrual irregularities, polycystic ovaries, or infertility.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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: Pedigree). The
propositus (patient 1) is a 28 yr-old phenotypically female patient,
who was referred for primary amenorrhea. The patient had developed
pubic hair at age 14 yr but exhibited no spontaneous breast development
or menarche. On physical examination, she had an eunuchoid habitus,
absence of breast tissue, and Tanner stage lV pubic hair development.
The external genitalia was female with a normal clitoris and two
separate openings leading to a urethra and a 7-cm long blind-ending
vagina. Gonads measuring 3.0 x 2.5 cm were palpable bilaterally
in the labia majora. The karyotype was 46,XY. Serum basal LH and FSH
concentrations were 46 IU/liter (normal value: 7.7 ± 4.7
IU/liter) and 14 IU/liter (normal value: 6.9 ± 4.9 IU/liter),
respectively. Serum testosterone concentration was 32 ng/dl (normal
men: 240 to 1030 ng/dl) and did not increase after 6000 U of hCG
administration (50 ng/dl). Progesterone, 17-hydroxyprogesterone,
dehydroepiandrosterone, and androstenedione did not accumulate after
hCG stimulation. Psychological evaluation confirmed her female gender
identity. The patient underwent bilateral gonadectomy. Right and left
testes measured 4.5 x 3.5 x 1.0 and 5.0 x 4.0 x
l.5 cm, respectively. Pathology revealed seminiferous tubules
containing Sertoli cells and few germ cells, with moderate thickening
of the basal membrane. Leydig cells were absent in the interstitium,
whereas epididymis and vas deferens were normal. She was treated with
estrogens and developed Tanner stage V breasts. She has had a
satisfactory sexual life. The family history of patient 1 indicated that two other siblings were potential candidates to gonadal resistance. Later, the oldest 46,XX sibling, our patient 2, was evaluated after solicitation. Patient 2 was evaluated at 40 yr of age. She had breast and pubic hair development at age 13 yr. Menarche occurred at age 15 yr, and her menses were always irregular. Menses occurred usually every 60 days, with variable periods of amenorrhea. Despite an active sex life for more than 20 yr with unprotected intercourse, she never became pregnant. Her karyotype was 46,XX. Transvaginal ultrasonography revealed a normal uterus size (volume: 70 ml; normal: 30–90 ml), an enlarged right ovary of 31 ml with a 29 x 22 mm cyst, and a left ovary of 3.3 ml. Serum LH, FSH, estradiol, and progesterone were measured once a week during four consecutive weeks. Serum basal LH and FSH ranged from 15–17 and 8.2–17 IU/liter (normal values during follicular phase: 7.1 ± 3.0 and 10.4 ± 3.4, respectively). The LH/FSH ratio was elevated on all occasions. The serum estradiol concentration ranged from 27–50 pg/ml. Serum progesterone concentrations were below 0.9 ng/ml on all occasions. Serum testosterone, androstenedione, 17-hydroxyprogesterone, and PRL were normal. Bone mass measured by bone densitometry was normal.
DNA Sequencing
Genomic DNA was isolated from peripheral blood samples. Exons
1–11 of the hLHR gene were amplified by the PCR, using different sets
of intronic primers (Table 4).
Amplification was allowed for 30 cycles in a Gene Amp PCR system (9600,
Perkin Elmer Cetus, Norwalk, CT). The PCR products were used to produce
single-stranded DNA, which was purified by filtration through a
Millipore membrane (30,000 NMWL, Ultrafree MC Filters, Bedford, MA).
The DNA was directly sequenced by the dideoxy nucleotide chain
termination method using modified T7-DNA polymerase (United States
Biochemical Corp., Cleveland, OH) in the presence of
-35S-deoxy-ATP. Inner primers that spanned exon 11 of
the LHR gene were used for sequencing, and the reaction products were
run on a 6% polyacrylamide gel. All PCR products were also sequenced
using the ABI PRISM dye terminator reaction kit (Perkin-Elmer Cetus) in
an ABI PRISM 377 automatic DNA sequencer (Perkin-Elmer Cetus).
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Human embryonic kidney 293 cells (ATCC CRL 1573) were maintained at 5% CO2 in a culture medium consisting of DMEM containing 50 µg/ml gentamicin, 10 mM HEPES, and 10% newborn calf serum. Cells were transfected at a 60–80% confluence following the transient transfection procedure of Chen and Okayama (33) except that the overnight precipitation was performed in a 5% rather than 2.5% CO2 atmosphere. After 18–20 h the cells were washed with Waymouth’s MB752/1 media modified with 50 µg/ml gentamicin and 1 mg/ml BSA, after which fresh growth medium was added. The cells were used for experiments 24 h later.
[125I]hCG Binding to Intact Cells
293 cells were plated on gelatin-coated 35-mm wells and
transfected as described above. On the day of the experiment, cells
were cooled on ice for 10 min and then washed two times with cold
Waymouth’s MB752/1 media lacking sodium bicarbonate, but containing 50
µg/ml gentamicin and 1 mg/ml BSA. To determine the maximal binding
capacity, the cells were then incubated overnight at 4 C in the same
media containing a saturating concentration of [125I]hCG
(100 ng/ml final concentration) with or without an excess of unlabeled
hCG (50 IU/ml final concentration). To determine the binding affinity,
the cells were incubated with increasing concentrations of
[125I]hCG in the presence or absence of unlabeled hCG.
The assay was finished by scraping the cells into a small volume of
cold HBSS modified to contain 50 µg/ml gentamicin and 1 mg/ml BSA,
centrifuging (1500 x g, 15 min), and washing the
pellet in 2 ml of the same buffer. Binding affinities were determined
as the concentrations of [125I]hCG yielding half-maximal
binding as calculated by the computer program DeltaPlot. Experiments
with cells expressing the wild-type hLHR confirmed that the binding
affinities calculated in this manner were the same as those derived
from competition binding assays analyzed using the computer program
LIGAND (34, 35).
[125I]hCG Binding to Detergent Extracts
of Cells
Detergent-solubilized cell extracts were prepared using 0.5%
Nonidet P-40 in 150 mM NaCl, 20 mM HEPES, pH
7.4, as described previously (36). Extracts were incubated overnight on
ice with a saturating concentration of [125I]hCG (100
ng/ml final concentration) in the absence or presence of an excess of
unlabeled hCG (50 IU/ml final concentration). The receptor-hormone
complexes were separated from the unbound hormone by vacuum filtration
through polyethylenimine-treated filters (37). To determine the
percentage of receptor on the cell surface, measurements of
[125I]hCG binding to intact cells and to
detergent-solubilized extracts of cells were performed in the same
experiment, both utilizing cells plated on gelatin-coated 35-mm wells.
Since binding to intact cells reflects binding to cell surface
receptors and binding to detergent extracts represents binding to both
cell surface receptors as well as intracellular receptors, the ratio of
binding to intact vs. solubilized cells can be used to
calculate the percentage of receptor at the cell surface.
Measurement of cAMP Production
In the same experiment, 293 cells were assayed both for
[125I]hCG binding to intact cells as well as for
intracellular cAMP production under basal and hCG-stimulated
conditions. Therefore, for any given experiment in which cAMP was
determined, the levels of cell surface expression of receptor were also
determined. 293 cells were plated on gelatin-coated 35-mm wells and
transfected as described above. On the day of the experiment, cells
were washed twice with warm Waymouth MB752/1 media containing 50
µg/ml gentamicin and 1 mg/ml BSA and placed in 1 ml of the same
medium containing 0.5 mM isobutylmethylxanthine. After 15
min at 37 C, a saturating concentration of hCG was added (100 ng/ml
final concentration) or buffer only and the incubation was continued
for 60 min at 37 C. The cells were then placed on ice, the media were
aspirated, and total cAMP was extracted by the addition of 1
N perchloric acid containing 360 µg/ml theophylline and
then measured by RIA. All determinations were performed in
triplicate.
Hormones and Reagents
Purified hCG (CR-127) was provided by the National Hormone and
Pituitary Program of The National Institute of Diabetes and Digestive
and Kidney Diseases, National Institute of Child Health and
Development, and United States Drug Administration. The cDNA for the
hLHR was kindly given to us by Ares Advanced Technology of the
Ares-Serono Group.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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These studies were supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) Grants 96/2040–2 and 96/2020–1 (to A.C.L.) and NIH Grant HD-22196 (to D.L.S.)
Received for publication October 21, 1997. Revision received December 9, 1997. Accepted for publication December 11, 1997.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 U. E. Petaja-Repo, M. Hogue, A. Laperriere, P. Walker, and M. Bouvier Export from the Endoplasmic Reticulum Represents the Limiting Step in the Maturation and Cell Surface Expression of the Human delta Opioid Receptor J. Biol. Chem., April 28, 2000; 275(18): 13727 - 13736. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Angelova, P. Narayan, J. P. Simon, and D. Puett Functional Role of Transmembrane Helix 7 in the Activation of the Heptahelical Lutropin Receptor Mol. Endocrinol., April 1, 2000; 14(4): 459 - 471. [Abstract] [Full Text]
|
|
|
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J. C. Zenteno, P. Canto, S. Kofman-Alfaro, and J. P. Mendez Evidence for Genetic Heterogeneity in Male Pseudohermaphroditism due to Leydig Cell Hypoplasia J. Clin. Endocrinol. Metab., October 1, 1999; 84(10): 3803 - 3806. [Abstract] [Full Text] [PDF]
|
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|
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J. C. Achermann and J. L. Jameson Fertility and Infertility: Genetic Contributions from the Hypothalamic-Pituitary- Gonadal Axis Mol. Endocrinol., June 1, 1999; 13(6): 812 - 818. [Full Text]
|
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|
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S.-M. Wu, K. M. Hallermeier, L. Laue, C. Brain, A. C. Berry, D. B. Grant, J. E. Griffin, J. D. Wilson, G. B. Cutler Jr., and W.-Y. Chan Inactivation of the Luteinizing Hormone/Chorionic Gonadotropin Receptor by an Insertional Mutation in Leydig Cell Hypoplasia Mol. Endocrinol., November 1, 1998; 12(11): 1651 - 1660. [Abstract] [Full Text]
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