A Homozygous Microdeletion in Helix 7 of the Luteinizing Hormone Receptor Associated with Familial Testicular and Ovarian Resistance Is Due to Both Decreased Cell Surface Expression and Impaired Effector Activation by the Cell Surface Receptor

Ana C. Latronico, Yaohui Chai, Ivo J.P. Arnhold, Xuebo Liu, Berenice B. Mendonca and Deborah L. Segaloff

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this report, the genomic DNA was examined from two siblings with gonadal LH resistance. A 46,XY pseudohermaphrodite presented with female external genitalia and his 46,XX sister exhibited menstrual irregularities (oligoamenorrhea) and infertility. Exons 1–11 of the LH receptor (LHR) gene were amplified by the PCR using different sets of intronic primers and were directly sequenced. Sequencing revealed that both individuals carried a deletion of nucleotides 1822–1827, resulting in the deletion of Leu-608 and Val-609 within the seventh transmembrane helix. This mutation was introduced into a recombinant human (h) LHR cDNA. Transfections of 293 cells with hLHR(wt) vs. hLHR({Delta}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({Delta}L608,V609) binds hCG with an affinity comparable to that of the wild-type receptor. However, the cells expressing the hLHR({Delta}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human LH/CG receptor (hLHR) is a member of the G protein-coupled superfamily of receptors with seven transmembrane helices (1, 2). Inactivating mutations of the hLHR gene have been described in males with Leydig cell hypoplasia (LCH), a rare form of male pseudohermaphroditism, characterized by failure of fetal testicular Leydig cell function (3, 4, 5, 6). Affected 46,XY patients present with fetal undermasculinization, ranging from female external genitalia to micropenis, associated with low hCG-stimulated levels of serum testosterone and elevated levels of LH function (3, 4, 5, 6). Seven distinct inactivating mutations of the hLHR gene were described among seven unrelated families with gonadal LH resistance (3, 4, 5, 6, 7, 8).

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. 1Go) and a 46,XX sister with menstrual irregularities and infertility (IV:5 in Fig. 1Go). 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.



View larger version (20K):
[in this window]
[in a new window]
 
Figure 1. Pedigree of the Family with Gonadal LH Resistance

The proband is indicated by the arrow. Closed squares and circles designate the affected males and females, respectively. Subject IV:6 was a 32-yr-old female social sex who has primary amenorrhea, but she was not available for clinical or molecular evaluation.

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sequencing of the hLHR Gene
Manual and automatic sequencing revealed a homozygous deletion of six consecutive nucleotides, corresponding to 1822–1827 within exon 11 of the hLHR gene in both siblings (Fig. 2Go). 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.



View larger version (33K):
[in this window]
[in a new window]
 
Figure 2. Nucleotide Sequence of the hLHR Gene from Two Siblings (46,XY and 46,XX) with Gonadal LH Resistance and from a Normal Control

A homozygous deletion of six consecutive nucleotides (CTGGTT) was found in the hLHR gene of the two affected siblings, resulting in the absence of two consecutive amino acids, Leu-608 and Val-609, within the seventh transmembrane helix of the hLHR.

 
Cell Surface Expression and hCG Binding to Cells Transfected with hLHR({Delta}L608,V609)
To determine the functional consequences of the deletion of Leu-608 and Val-609, a cDNA was constructed encoding for the mutated receptor. The resulting plasmid, hLHR({Delta}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 1Go). 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 2Go, the maximal binding of [125I]hCG to intact cells expressing hLHR({Delta}L608,V609) was only 11% of that observed in intact cells expressing the wild-type hLHR. This decreased cell surface expression of hLHR({Delta}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({Delta}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.


View this table:
[in this window]
[in a new window]
 
Table 1. hCG Binding Affinities to Intact Cells Expressing either the hLHR(wt) or hLHR({Delta}L608,V609)

 

View this table:
[in this window]
[in a new window]
 
Table 2. [125I]hCG Binding to Intact Cells vs. Detergent Extracts of Cells Expressing hLHR(wt) or hLHR({Delta}L608,V609)

 
Additionally, however, the decreased cell surface expression of hLHR(({Delta}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 2Go, 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({Delta}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 3Go 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({Delta}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({Delta}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({Delta}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({Delta}L608,V609) in any of the three experiments. Therefore, the small amount of hLHR({Delta}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.


View this table:
[in this window]
[in a new window]
 
Table 3. Basal and hCG-Stimulated cAMP in Cells Expressing hLHR(wt) vs. hLHR({Delta}L608,V609)

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inactivating mutations of the hLHR gene resulting in decreased levels of cAMP production and impaired LH action cause pseudohermaphroditism or micropenis in 46,XY males and amenorrhea and infertility in 46,XX females (3, 4, 5, 9). Here, we describe a novel homozygous deletion of two consecutive amino acids (Leu-608 and Val-609) in the seventh transmembrane helix of the LHR in a Brazilian family. The affected 46,XY propositus had female external genitalia, whereas a 46,XX sister had irregular menses (oligoamenorrhea) and infertility.

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({Delta}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({Delta}L608,V609) mutant falls within the classification of a mutant being partially retained intracellularly. However, since the hLHR({Delta}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({Delta}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-616->Tyr) 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-616->Tyr 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Subjects
This study was approved by the Ethics Committee of the Division of Endocrinology of Hospital das Clínicas, São Paulo, Brazil. Informed consent was obtained from the patients. The two patients were Creole Brazilian born to consanguineous parents. Their parents were first cousins once removed (Fig. 1Go: 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 4Go). 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 {alpha}-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).


View this table:
[in this window]
[in a new window]
 
Table 4. Primers used in the Study

 
Mutagenesis of the hLHR and Its Transfection into Cells
The template for mutagenesis was the hLHR cDNA kindly donated by Ares Advanced Technology (Ares-Serono Group, Randolph, MA). After subcloning the cDNA into pcDNA3.1/neo, deletion of nucleotides 1822–1827 of the hLHR gene was accomplished by mutagenesis using the PCR overlap extension method (32). The sequence of the entire region that was amplified by PCR was verified by sequencing.

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.


    ACKNOWLEDGMENTS
 
We thank Dr. George P. Chrousos (NIH) and Dr. Mario Ascoli (The University of Iowa) for helpful discussions and critically reading the manuscript. We also thank Ares Advanced Technology (Ares-Seronos Group) for their gift of hLHR cDNA and the staff of the Laboratorio de Pesquisa Clinica Medica I for providing technical support. We also acknowledge the services and facilities supported by the University of Iowa Diabetes and Endocrinology Research Center Grant DK-25295.


    FOOTNOTES
 
Address requests for reprints to: Ana Claudia Latronico, M.D., Hospital das Clínicas, Universidade de São Paulo, Caixa Postal 3671, São Paulo CEP 01060–970, Brazil.

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Minegishi T, Nadamura K, Ibuki Y 1993 Structure and regulation of the LH/CG receptor. Endocrine J 40:275–287
  2. Segaloff DL, Ascoli M 1993 The lutropin/choriogonadotropin (LH/CG) receptor-4 years later. Endocr Rev 14:324–347[Abstract]
  3. Kremer H, Kraaij R, Toledo SPA, Post M, Fridman JB, Hayashida CY, van Reen M, Milgrom E, Ropers H-H, Mariman E, Themmen APN, Brunner HG 1995 Male pseudohermaphroditism due to a homozygous missense mutation of the luteinizing hormone receptor gene. Nature Genet 9:160–164[Medline]
  4. Laue LL, Wu SM, Kudo M, Bourony CJ, Cutler GB, Hsueh AJW, Chan WY 1996 Compound heterozygous mutations of the luteinizing hormone receptor gene in Leydig cell hypoplasia. Mol Endocrinol 10:987–997[Abstract]
  5. Latronico AC, Anasti J, Arnhold IJP, Rapaport R, Mendonca BB, Bloise W, Castro M, Tsigos C, Chrousos GP 1996 Testicular and ovarian resistance to luteinizing hormone caused by homozygous inactivating mutations of the luteinizing hormone receptor gene. N Engl J Med 334:507–512[Free Full Text]
  6. Laue L, Wu SM, Kudo M, Hsueh AJW, Cutler Jr GB, Griffin JE, Wilson JD, Brain C, Berry AC, Grant DB, Chan WY 1995 A nonsense mutation of the human luteinizing hormone receptor gene in Leydig cell hypoplasia. Hum Mol Genet 4:1429–1433[Abstract]
  7. Misrahi M, Meduri G, Pissard S, Bouvattier C, Beau I, Loosfelt H, Jolivet A, Rappaport R, Milgrom E, Bougneres P 1997 Comparison of immunocytochemical and molecular features with the phenotype in a case of incomplete male pseudohermaphroditism associated with a mutation of the luteinizing hormone receptor. J Clin Endocrinol Metab 82:2159–2165[Abstract/Free Full Text]
  8. Stavrou SS, Zhu Y-S, Cai LQ, Katz MD, Ling Q, Herrara C, DeFilho-Ricart M, Imperato-McGinley J, A novel mutation of LH receptor presents as primary amenorrhea in (XX and XY) sisters. Proceedings of the 79th Annual Meeting of the Endocrine Society, 1997, Minneapolis, MN (Abstract P2-507)
  9. Toledo SPA, Brunner GG, Kraaij R, Post M, Dahia PLM, Hayashida CY, Kremer H, Themmen APN 1996 An inactivating mutation of the luteinizing hormone receptor causes amenorrhea in a 46,XX female. J Clin Endocrinol Metab 3850–3854
  10. Thomas D, Rozell TG, Liu X, Segaloff DL 1996 Mutational analyses of the extracellular domain of the full-length lutropin/choriogonadotropin receptor suggest leucine-rich repaets 1–6 are involved in hormone binding. Mol Endocrinol 10:760–768[Abstract]
  11. Wang H, Jaquette J, Collison K, Segaloff DL 1993 Positive charges in a putative amphiphilic helix in the carboxyl-terminal region of the third intracellular loop of the luteinizing hormone/chorionic gonadotropin receptor are not required for hormone-stimulated cAMP production but are necessary for expression of the receptor at the plasma membrane. Mol Endocrinol 7:1437–1444[Abstract]
  12. Wang Z, Wang H, Ascoli M 1993 Mutation of a highly conserved acidic residue present in the second intracellular loop of G-protein-coupled receptors does not impair hormone binding or signal transduction of the luteinizing hormone/chorionic gonadotropin receptor. Mol Endocrinol 7:85–93[Abstract]
  13. Zhu H, Wang H, Ascoli M 1995 The lutropin/choriogonadotropin receptor is palmitoylated at intracellular cysteine residues. Mol Endocrinol 9:141–150[Abstract]
  14. Abell A, Liu X, Segaloff DL 1996 Deletions of portions of the extracellular loops of the lutropin/choriogonadotropin receptor decrease the binding affinity for oLH, but not hCG, by preventing the formation of mature cell surface receptor. J Biol Chem 271:4518–4527[Abstract/Free Full Text]
  15. Rozell TG, Wang H, Liu X, Segaloff DL 1995 Intracellular retention of mutant gonadotropin receptors results in loss of hormone binding activity of the follitropin receptor but not the lutropin/choriogonadotropin receptor. Mol Endocrinol 9:1727–1736[Abstract]
  16. Jaquette J, Segaloff DL 1997 Temperature sensitivity of some mutants of the lutropin/choriogonadotropin receptor. Endocrinology 138:85–91[Abstract/Free Full Text]
  17. Hobbs HH, Russell DW, Brown MS, Goldstein JL 1990 The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Annu Rev Genet 24:133–170[CrossRef][Medline]
  18. Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, O’Riordan CR, Smith AE 1990 Defective intracellular transport and processing of CFTR is the molecular basis for most cystic fibrosis. Cell 63:827–834[Medline]
  19. Denning GM, Ostedgaard LS, Welsh MJ 1992 Abnormal localization of cystic fibrosis transmembrane conductance regulator in primary cultures of cystic fibrosis airway epithelia. J Cell Biol 118:551–559[Abstract]
  20. Denning GM, Anderson MP, Amara JF, Marshall J, Smith AE, Welsh MJ 1992 Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358:761–764[CrossRef][Medline]
  21. Wertheimer E, Barbetti F, Muggeo M, Roth J, Taylor S 1994 Two mutations in a conserved structural motif in the insulin receptor inhibit normal folding and intracellular transport of the receptor. J Biol Chem 269:7587–7592[Abstract/Free Full Text]
  22. Suryanarayana S, von Zastrow M, Kobilka BK 1992 Identification of intramolecular interactions in adrenergic receptors. J Biol Chem 267:21991–21994[Abstract/Free Full Text]
  23. Sung CH, Schneider BG, Agarwal N, Papermaster DS, Nathans J 1991 Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci USA 88:8840–8844[Abstract]
  24. Sadeghi HM, Innamorati G, Birnbaumer M 1997 An X-linked NDI mutation reveals a requirement for cell surface V2R expression. Mol Endocrinol 11:706–713[Abstract/Free Full Text]
  25. Abell AN, Segaloff DL 1997 Evidence for the direct involvement of transmembrane region six of the lutropin/choriogonadotropin receptor in activating Gs. J Biol Chem 272:14586–14591[Abstract/Free Full Text]
  26. Kudo M, Osuga Y, Kobilka BK, Hsueh AJW 1996 Transmembrane regions V and VI of the human luteinizing hormone receptor are required for constitutive activation by a mutation in the third intracellular loop. J Biol Chem 271:22470–22478[Abstract/Free Full Text]
  27. Liggett SB, Caron MG, Lefkowitz RJ, Hnatowich M 1991 Coupling of a mutated form of the human ß2-adrenergic receptor to Gi and Gs. J Biol Chem 266:4816–4821[Abstract/Free Full Text]
  28. Cheung AH, Huang R-RC, Graziano MP, Strader CD 1991 Specific activation of Gs by synthetic peptides corresponding to an intracellular loop of the ß2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation. Cell 67:723–730[Medline]
  29. Okamoto T, Yoshitake, M, Hayashi Y, Inagaki M, Ogata E, Nishimoto I 1991 Identification of a Gs activator region of the ß2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation. Cell 67:723–730[Medline]
  30. Arnhold IJP, Latronico AC, Batista MC, Carvalho FM, Chrousos GP, Mendonca BB 1997 Ovarian resistance to luteinizing hormone: a novel cause of amenorrhea and infertility. Fertil Steril 67:394–397[CrossRef][Medline]
  31. Rosenthal IM, Refetoff S, Rich B, Barnes RB, Sunthornthepvarakul T, Parma J, Rosenfield RL 1996 Response to challenge with gonadotropin-releasing hormone agonist in a mother and her two sons with a constitutively activating mutation of the luteinizing hormone receptor-A clinical research center study. J Clin Endocrinol Metab 81:3802–3806[Abstract]
  32. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR 1989 Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59[CrossRef][Medline]
  33. Chen C, Okayama H 1987 High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752[Medline]
  34. De Lean A, Munson PJ, Rodbard D 1978 Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay and physiological dose-response curves. Am J Physiol 235:E97–E102
  35. Munson PJ, Rodbard D 1980 LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220–239[Medline]
  36. Davis D, Liu X, Segaloff DL 1995 Identification of the sites of N-linked glycosylation on the follicle-stimulating hormone (FSH) receptor and assessment of their role in FSH receptor function. Mol Endocrinol 9:159–170[Abstract]
  37. Roche PC, Bergert ER, Ryan RJ 1985 A simple and rapid method using polyethylenemine-treated filters for assay of solubilized LH/hCG receptors. Endocrinology 117:790–792[Abstract]