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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 2 834-843
Copyright © 2003 by The Endocrine Society

Two Common Naturally Occurring Mutations in the Human Gonadotropin-Releasing Hormone (GnRH) Receptor Have Differential Effects on Gonadotropin Gene Expression and on GnRH-Mediated Signal Transduction

Gregoy Y. Bedecarrats, Katja D. Linher and Ursula B. Kaiser

Endocrine-Hypertension Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Ursula B. Kaiser, M.D., Endocrine-Hypertension Division, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: ukaiser{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Studies of naturally occurring human GnRH receptor (GnRHR) mutants may provide a useful approach to dissecting the signal transduction pathways involved in mediating the effects of GnRH. We have analyzed two common mutations in the GnRHR, corresponding to amino acid substitutions Gln106Arg and Arg262Gln, for their effects on the stimulation of gonadotropin subunit and GnRHR gene expression by GnRH. Despite similar impairment of GnRH-stimulated inositol phosphate production, dose-response analyses indicated that Gln106Arg and Arg262Gln both reduced the sensitivity of the FSHß gene promoter to a greater extent than LHß or {alpha}GSU, suggesting the involvement of more than one signaling pathway. Furthermore, although the sensitivities of the LHß and FSHß gene promoters to GnRH were similarly affected by both mutants, {alpha}GSU sensitivity was decreased to a greater extent by Arg262Gln than by Gln106Arg. Similarly, GnRHR gene promoter sensitivity was significantly reduced only by Arg262Gln. To further characterize the differential downstream effects of these mutant GnRHRs, we investigated their effects on additional signal transduction pathways. The mutant receptors differentially affected GnRH-mediated activation of the ERK pathway and GnRH stimulation of cAMP response element-mediated transcription. These results indicate that measurement of inositol phosphate production alone may not be adequate for assessing mutant GnRHR function and additional signal transduction pathways may better reflect physiologically relevant effects. The differential stimulation of LHß, FSHß, and {alpha}GSU gene expression may contribute to the varied phenotypes observed among patients harboring these mutations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN GnRH RECEPTOR (hGnRHR) is a seven-transmembrane G protein-coupled receptor present on the surface of gonadotropes. On binding to the hGnRHR, the hypothalamic decapeptide GnRH stimulates gene expression, synthesis, and release of the gonadotropins, LH, and FSH, from the anterior pituitary gland. Stimulation of the hGnRHR by its ligand induces signaling via an increase in phospholipase C (PLC) activity and results in mobilization of calcium from intra- and extracellular sources and activation of the PKC and MAPK pathways (1). The protein kinase A (PKA)/cAMP pathway may also be activated in response to GnRH (2, 3, 4). Although GnRH-induced inositol phosphate (IP) production via activation of PLC has been used to assess signal transduction in response to GnRH, it is not clear whether activation of this pathway correlates with stimulation of gonadotropin subunit and GnRHR gene expression. The precise mechanisms governing GnRH stimulation of gene transcription have yet to be determined.

Because GnRH plays a pivotal role in mediating reproductive function, the hGnRHR gene was screened for mutations in patients with idiopathic hypogonadotropic hypogonadism (IHH) (5). As a result, an expanding list of naturally occurring mutations in the hGnRHR gene has been identified in association with reproductive abnormalities (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). In vitro functional analyses of these hGnRHR mutants have shown them to alter cellular expression levels, ligand binding, and/or signal transduction. Two common naturally occurring mutations in the hGnRHR, corresponding to an amino acid substitution of arginine for glutamine at position 106 (Gln106Arg) in the first extracellular loop and glutamine for arginine at position 262 (Arg262Gln) in the third intracellular loop, were the first hGnRHR mutations to be identified in a patient with partial IHH (5). Functional analyses of these two mutant hGnRHRs revealed that the Gln106Arg mutation significantly reduced GnRH binding, whereas Arg262Gln had a minimal effect on receptor affinity in vitro. Interestingly, both mutants resulted in a similar decrease in PLC activity, as reflected by a decrease in intracellular IP production in response to GnRH. Subsequently, several patients with IHH, presenting with a wide range of phenotypes, were found to harbor Gln106Arg and Arg262Gln mutations as compound heterozygotes together or in combination with other hGnRHR mutations (5, 6, 8, 10). More recently, a male patient with fertile eunuch syndrome (13) and a female patient with partial IHH (15) were found to be homozygous for the Gln106Arg mutation.

To better understand the relationship between phenotype and genotype in patients harboring the Gln106Arg and/or Arg262Gln mutation(s), it is important to determine mutant receptor function downstream of GnRH binding and IP production. In particular, the effects of the mutant receptors on gonadotropin subunit and GnRHR gene expression in response to GnRH have important pathophysiologic relevance. Furthermore, as partially inactivating hGnRHR mutations, Gln106Arg and Arg262Gln represent useful tools to study pathways of signal transduction in the regulation of gonadotropin subunit and GnRHR gene expression by GnRH.

In the present study, Gln106Arg and Arg262Gln were analyzed for their effects on the GnRH stimulation of gonadotropin subunit and GnRHR gene promoter activity in vitro. Despite a similar reduction in IP response to GnRH elicited by both of these mutant receptors, they had differential effects on GnRH-stimulated gonadotropin subunit and GnRHR gene expression. Therefore, their effects on additional signal transduction pathways were also investigated to determine the mechanisms by which the two mutations may result in these differential effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

All cell culture reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD). COS-7 and GH3 cells were cultured in low-glucose DMEM containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. Cells were incubated at 37 C in a humidified atmosphere of 5% CO2 in air.

Site-directed mutagenesis

Site-directed mutagenesis was used to introduce either Gln106Arg or Arg262Gln into an expression vector encoding the wild-type hGnRHR fused to an N-terminal hemagglutinin (HA) epitope tag generously provided by Dr. Thomas Gudermann (16). In the Gln106Arg hGnRHR mutant, Gln codon CAA was replaced with Arg codon CGA at nucleotide position 372 in the hGnRHR using the primer pair: sense, 5'-GTGGACATTACAGTCCGATGGTATGCTGGAGAG-3', and antisense, 5'-CTCTCCAGCATACCATCGGACTGTAATGTTCCA C-3'. In the Arg262Gln hGnRHR mutant, Arg codon CGG was replaced with Gln codon CAG at nucleotide position 840 in the hGnRHR using the primer pair: sense, 5'-CAATATACCAAGAGCACAGCTGAAGACTCTAAAAATGACG-3', and antisense, 5'-CGTCATTTTTAGAGTCTTCAGCTGTGCTCTTGGTATATTG-3'. Both hGnRHR mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and proper insertion of the mutations was confirmed by bidirectional sequencing.

Immunocytochemistry

COS-7 cells were plated on glass-bottom 35-mm tissue culture dishes (MatTek Corp., Ashland, MA) and transiently transfected by lipofection using the GenePorter transfection reagent (Gene Therapy System, San Diego, CA) with 2 µg of each hGnRHR construct (wild type, Gln106Arg, or Arg262Gln) or a control expression vector (pcDNA3). After 48 h of incubation at 37 C, cells were washed with ice-cold PBS, fixed with freshly prepared 4% formaldehyde solution for 30 min at room temperature, washed twice with PBS, and blocked with PBS/1% BSA for 30 min at room temperature. Cells were incubated at 37 C overnight with 5 µg/ml anti-(HA)-fluorescein antibody (Clone 12CA5, Roche Molecular Biochemicals, Indianapolis, IN) and washed 4 times with PBS. The presence of the wild-type and mutant hGnRHRs on the surface of intact cells was examined under a x40 oil immersion objective using a confocal laser microscope (MRC-1024 multiphoton system, Bio-Rad Laboratories, Inc., Hercules, CA).

Receptor-binding assay

COS-7 cells were plated in 60-mm tissue culture dishes and transiently transfected by lipofection with 3 µg/well of each hGnRHR construct or pcDNA3. After a 48-h incubation at 37 C, cells were washed with DMEM and 0.1% BSA and incubated for 90 min at room temperature with 100,000 cpm 125I-Buserelin, generously provided by Dr. P. Michael Conn (17), and increasing concentrations (10-12 to 10-6 M) of unlabeled GnRH (Sigma, St. Louis, MO). Cells were washed twice with ice-cold PBS and lysed with 2 ml of 0.2 M NaOH, 0.1% SDS. The protein content in the cell lysates was calculated (Coomassie Plus protein assay reagent, Pierce Chemical Co., Rockford, IL), and radioactivity was measured in a {gamma} counter. The dissociation constant (Kd) and maximum binding (Bmax) were calculated based on nonlinear regression of homologous competition-binding analysis using Prism 3.0 (GraphPad Software, Inc., San Diego, CA). All assay points were performed in triplicate, and each experiment was repeated at least three times.

IP assay

The protocol used for measuring total IP production has been described previously (18). Briefly, COS-7 cells were transiently transfected by electroporation with 2 µg/well of each hGnRHR construct or pcDNA3 and plated into 6-well culture plates. After a 24-h incubation at 37 C, medium was replaced with 1 ml inositol-free DMEM for 2 h and then 1 ml of the same medium containing 2 µCi myo-(2-3H) inositol (NEN Life Science Products, Boston, MA), followed by the addition of 10 mM LiCl 15 min later. After an additional 16-h incubation at 37 C, cells were stimulated with increasing concentrations of GnRH (10-11 to 10-6 M) for 45 min. Time-course studies were performed, which indicated that GnRH induction of IP accumulation was maximal at 45 min. Cells were extracted 2 times for 30 min on ice with 20 mM formic acid, and lysates were neutralized to pH 7.5 with 7.5 mM HEPES and 150 mM KOH. After centrifuging for 2 min at 14,000 x g, the protein content in the lysates was measured and the supernatants were loaded onto Ag-X8 resin anion exchange columns (Bio-Rad Laboratories, Inc.), previously equilibrated with 2 ml of 1 M NaOH, 2 ml of 1 M formic acid, and 5 x 5 ml ddH2O. The columns were washed with 5 ml ddH20, and then 5 ml of 5 mM borax, 60 mM sodium formate, and IPs were extracted with 3 ml of 0.9 M ammonium formate, 0.1 M formic acid. The incorporation of radioactivity in the eluates was measured in a scintillation counter, and each sample was corrected for protein content. All assay points were performed in triplicate, and each experiment was repeated at least three times.

Luciferase assays

The reporter constructs used were generated by fusing -797/+5 of the rat LHß gene, -2000/+698 of the rat FSHß gene, -846/0 of the human {alpha}GSU gene, and -1164/+62 of the mouse GnRHR gene to the firefly luciferase (Luc) cDNA, as previously described (19, 20, 21). The cAMP response element (CRE) reporter construct contains four CREs upstream of luciferase (Stratagene). GH3 cells were transiently transfected by electroporation with 2 µg/well of each hGnRHR construct or pcDNA3, 2 µg/well of either {alpha}GSU-Luc, FSHß-Luc, LHß-Luc, or GnRHR-Luc, or 1 µg/well of CRE-Luc, and 1 µg/well of the Rous sarcoma virus (RSV)-ß-galactosidase vector. Cells were then seeded into 6-well tissue culture plates, incubated for 48 h at 37 C and stimulated for 4 h [based on time course studies indicating that this time point gave maximal GnRH-stimulated Luc activity (22)] with increasing concentrations of GnRH (10-11 to 10-5 M). Cells were washed 2 times with ice-cold PBS and lysed with 125 mM Tris-HCl, 0.5% Triton. Following centrifugation at 14,000 x g at 4 C, Luc and ß-galactosidase activities were measured in the supernatants. Luc activity was normalized for ß-galactosidase activity to correct for transfection efficiency, and results were expressed as fold of unstimulated samples. All assay points were performed in triplicate, and each experiment was repeated at least three times.

Western blot analysis of ERK-1 phosphorylation

GH3 cells were transiently transfected with 2 µg/well of each hGnRHR construct or pcDNA3 and 2 µg/well of an HA-tagged wild-type ERK-1 expression vector, generously provided by Dr. Melanie Cobb (23). After a 24-h incubation at 37 C, cells were incubated for an additional 24 h in serum-free DMEM stabilized with 0.1% BSA, followed by stimulation with increasing concentrations of GnRH (10-10 to 10-5 M) for 10 min. Time-course studies indicated that GnRH-induced phosphorylation of ERK-1 was maximal at 10 min. Cells were lysed on ice with RIPA buffer (1 x PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS) containing 0.1 mg/ml phenylmethylsulfonyl fluoride, 30 mg/ml aprotinin, and 1 mM sodium orthovanadate, and sonicated for 20 sec. Following centrifugation at 14,000 x g at 4 C, the protein content in the supernatant was measured, 40 µg denatured protein/well was loaded onto 12% polyacrylamide gels, and electrophoresis was carried out according to standard protocols. Proteins were transferred onto polyvinylidene fluoride membranes (Immobilon-P, Millipore Corp., Bedford, MA), which were blocked overnight at 4 C with Blotto [1 x Tris-buffered saline (TBS), 1% milk, 1% BSA, 0.05% Tween-20]. Membranes were incubated with an anti-p-ERK antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in Blotto for 2 h at 37 C and washed 3 x 10 min with TBS, 0.05% Tween-20 and 1 x 10 min with TBS. A subsequent incubation was carried out for 1 h at 37 C with a monoclonal horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) in Blotto and the appropriate additional washes were performed. Membranes were exposed onto film following chemiluminescent detection (Santa Cruz Biotechnology, Inc.). After strip washing (Pierce Chemical Co.), membranes were reprobed with anti-(HA)-HRP antibody (Roche Molecular Biochemicals) in Blotto for 1 h at 37 C to correct for transfection efficiency and protein loading. Corrected results were expressed as fold of unstimulated samples based on densitometric analysis of the scanned film. Each experiment was repeated at least three times.

Computational and statistical analysis

The data for each set of experiments were subjected to nonlinear regression analysis and the ED50 for each study was calculated using Prism 3.0 (GraphPad Software, Inc.). All statistical analyses were carried out using Instat 3.0 (GraphPad Software, Inc.) and based on nonparametric unpaired t tests (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell surface expression of mutant hGnRHRs

To ensure that the mutant hGnRHRs were expressed appropriately, COS-7 cells were transiently transfected with the hGnRHR constructs and analyzed by immunocytochemistry for expression of the wild-type and mutant receptors on the surface of fixed, intact cells. The wild-type hGnRHR expression vector was used as a positive control, and pcDNA3 was used as a negative control. All hGnRHR expression vectors included an HA epitope tag at the amino-terminus to facilitate detection of the receptors in the absence of an effective anti-GnRHR antibody. Confocal fluorescent microscope images were obtained after incubating the transfected cells with an anti-HA antibody conjugated to fluorescein (Fig. 1Go). Fluorescence was detected at the periphery of cells transfected with the wild-type hGnRHR, as well as the Gln106Arg and Arg262Gln mutants, but not on the surface of cells transfected with pcDNA3. The pattern of fluorescence was similar for all hGnRHRs, suggesting normal cellular expression and trafficking to the membrane. Although the possibility that the presence of the N-terminal HA tag may influence cell surface expression or ligand binding cannot be excluded entirely, no effects on wild-type hGnRHR have been observed (16).



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  		Figure 1. Wild-type, Gln106Arg, and Arg262Gln hGnRHRs are present on the cell surface. COS-7 cells were transiently transfected with the hGnRHRs fused to an HA tag or the empty control vector pcDNA3. The presence of the wild-type and mutant receptors was detected on the cell surface of fixed intact cells after overnight incubation with an anti-(HA)-fluorescein antibody. Each image is a superposition of confocal and optic transmission and is representative of three separate experiments.

 
Receptor-binding characteristics

To measure GnRH binding, COS-7 cells were transfected with each hGnRHR construct or pcDNA3. For displacement analysis, cells were incubated with 100,000 cpm 125I-Buserelin and increasing concentrations of unlabeled GnRH. After nonlinear regression of homologous competition binding analysis, the Kd and Bmax were calculated for the wild-type, Gln106Arg, and Arg262Gln hGnRHR constructs (Table 1Go). No binding to GnRH was observed for pcDNA3. No significant differences were observed between wild-type and Arg262Gln GnRHR for either affinity or receptor number. Because of the marked reduction in binding observed for Gln106Arg, it was not possible to accurately calculate the Kd and Bmax. These findings are consistent with those previously reported for these mutants (5, 6).


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  		Table 1. Kd and Bmax, and ED50 of GnRH-stimulated IP production, gonadotropin subunit and GnRHR gene promoter activity, and CRE and ERK-1 activation by wild type, Gln106Arg, and Arg262Gln hGnRHRs

 
IP accumulation in response to GnRH

To confirm that the hGnRHR constructs used in this study retained partial ability to activate intracellular signal transduction pathways, IP production was measured in transiently transfected COS-7 cells in response to increasing doses of GnRH. Wild-type hGnRHR was able to mediate a 10-fold stimulation of IP accumulation, with an ED50 of 2.5 nM GnRH (Fig. 2Go, Table 1Go). Both mutations caused shifts in the dose-response curves to the right, resulting in similar increases in the ED50, by approximately 7-fold, compared with wild-type hGnRHR, again consistent with previous reports (5, 6). No stimulation of IP production was observed for pcDNA3, used as negative control. Similar results were obtained for GnRH-mediated IP production of these mutants using transiently transfected GH3 cells (data not shown).



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  		Figure 2. Stimulation of GnRH-mediated IP production by wild-type, Gln106Arg, and Arg262Gln hGnRHRs. COS-7 cells were transiently transfected with hGnRHRs, incubated with 2 µCi myo-(2-3H) inositol, stimulated with increasing concentrations of GnRH, and total IPs were measured. GnRH dose-response curves of IP production corrected for protein content are depicted and were used to calculate the ED50 for each of the hGnRHRs (see Table 1Go). Results are expressed as percentage of the maximum stimulation for each hGnRHR (mean ± SE of three representative independent experiments, each performed in triplicate). Open circles represent wild-type, closed circles Gln106Arg, and triangles Arg262Gln hGnRHRs.

 
Differential effects of the mutant GnRHRs on stimulation of gonadotropin subunit gene promoter activity by GnRH

The signal transduction pathways by which GnRH stimulates gonadotropin biosynthesis have not yet been fully elucidated. It is not known whether IP induction correlates well with activation of gonadotropin subunit gene expression. Therefore, the ability of GnRH to stimulate gonadotropin subunit gene expression in cells expressing the mutant hGnRHRs was measured.

GH3 cells were transiently cotransfected with the hGnRHR constructs or pcDNA3, a Luc reporter fused to the LHß, FSHß, or {alpha}GSU promoters, and an RSV-ß-galactosidase vector. When COS-7 cells were used for these experiments, no stimulation of gonadotropin subunit or GnRHR gene promoter activity by GnRH was observed, even for the wild-type hGnRHR (data not shown). In contrast, GH3 cells have been shown previously to support gonadotropin subunit and GnRHR gene promoter activity and responsiveness to GnRH (21, 23). As noted above, similar effects of the mutant hGnRHRs on GnRH-mediated IP production were observed in both GH3 and COS-7 cells, thereby validating the comparison of the two models.

Dose-response analyses of GH3 cells transfected with LHß-Luc, FSHß-Luc, or {alpha}GSU-Luc are presented in Fig. 3Go. The ED50 for each gonadotropin subunit and receptor construct tested is summarized in Table 1Go. For LHß-Luc, the two mutations caused an 8.8- and 8.1-fold increase in the ED50 (i.e. a reduction in the sensitivity to GnRH) for Gln106Arg and Arg262Gln, respectively (Fig. 3AGo, Table 1Go). Thus, both mutant receptors reduced GnRH-stimulated LHß gene promoter activity to the same extent and in a manner similar to the effects on GnRH-stimulated IP production.



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  		Figure 3. Differential stimulation of gonadotropin subunit and GnRHR gene promoters by wild-type, Gln106Arg, and Arg262Gln hGnRHRs. GH3 cells were transiently cotransfected with hGnRHRs, RSV-ß-galactosidase, and reporter constructs (A) LHß-Luc, (B) FSHß-Luc, (C) {alpha}GSU-Luc, and (D) GnRHR-Luc. Forty-eight hours after transfection, cells were stimulated with increasing concentrations of GnRH for 4 h, and cell extracts were assayed for Luc activity and normalized for ß-galactosidase activity. GnRH dose-response curves of gonadotropin subunit and GnRHR gene promoter activity are depicted and were used to calculate the ED50 for each of the hGnRHRs (see Table 1Go). Results are expressed as percentage of the maximum stimulation for each hGnRHR (mean ± SE of three independent experiments, each performed in triplicate). Open circles represent wild-type, closed circles Gln106Arg, and triangles Arg262Gln hGnRHRs.

 
Both mutant receptors decreased the sensitivity of the FSHß subunit gene promoter to GnRH, by 14- and 15-fold for Gln106Arg and Arg262Gln, respectively, compared with the wild-type receptor (Fig. 3BGo and Table 1Go). Although both mutants reduced the sensitivity to GnRH to a similar extent, the effect was greater than that on GnRH-stimulated IP production and LHß gene promoter stimulation. Interestingly, in these studies, the FSHß subunit gene promoter was approximately 5-fold more sensitive to GnRH in the presence of the wild-type receptor than either the LHß or {alpha}GSU gene promoters.

In the case of the {alpha}GSU gene promoter, there was a 3.8-fold reduction in the sensitivity to GnRH for Gln106Arg relative to the wild-type receptor and a 10.6-fold reduction for Arg262Gln (Fig. 3CGo, Table 1Go). Interestingly, the Arg262Gln mutation had greater effects on the dose-response curve for GnRH-mediated {alpha}GSU-Luc stimulation than did the Gln106Arg mutation. The two mutants have differential effects on {alpha}GSU, distinct from the parallel effects of these two mutants on GnRH-stimulated IP production and LHß and FSHß gene promoter activity.

Stimulation of GnRHR gene promoter activity by GnRH

Because GnRH has been shown to regulate the expression of its own receptor (24), differences in GnRHR gene promoter stimulation by the hGnRHR mutants may result in changes in cell surface receptor number in vivo. Experiments were therefore conducted to investigate whether the two hGnRHR mutants had effects on stimulation of expression of the GnRHR gene by GnRH. GH3 cells were transiently cotransfected with the hGnRHR constructs or pcDNA3, a luciferase reporter fused to the mouse GnRHR gene promoter (GnRHR-Luc), and an RSV-ß-galactosidase vector. The resulting dose-response curve is presented in Fig. 3DGo and the ED50 in Table 1Go. The GnRHR gene promoter sensitivity to GnRH was modestly reduced by 2.2-fold for Gln106Arg (i.e. the ED50 was increased), and more significantly by 3.7-fold for Arg262Gln. Like the differential effects on {alpha}GSU, these two mutant hGnRHRs had differential effects on the dose-response curves for GnRH stimulation of GnRHR gene promoter activity.

GnRH stimulation of ERK activity

The differential effects of Gln106Arg and Arg262Gln on gonadotropin subunit and GnRHR gene promoter activity suggested that these two mutant receptors might differentially affect signal transduction pathways by which GnRH activates gene expression. These distinct effects could be explained only partially by the identical effects of both mutant receptors on GnRH-stimulated IP production and suggested that additional signal transduction pathways may be involved. GnRH has been shown to stimulate ERK activity, and this MAPK pathway has been suggested to play a role in transcriptional activation of the {alpha}GSU gene promoter (25, 26) and increases in LHß protein (4, 27). We therefore sought to determine the effects of Gln106Arg and Arg262Gln on GnRH stimulation of the ERK pathway.

Because endogenous levels of phosphorylated ERK were found to mask the effects of GnRH-mediated ERK activation in our transient transfection paradigm, GH3 cells were transiently cotransfected with each of the hGnRHR constructs or pcDNA3 and an HA-tagged wild-type ERK-1 expression vector. Cells were stimulated with increasing concentrations of GnRH, followed by Western blot analysis. The dose-dependent activation of ERK-1 by GnRH for the wild-type and mutant receptors, as well as the lack of GnRH-stimulated ERK activity for the negative control pcDNA3, are depicted in Fig. 4AGo. Densitometric analysis of the band intensities was performed for phosphorylated HA-ERK-1 relative to total HA-ERK-1, which was used to correct for transfection efficiency and protein loading. These data were used to generate dose-response curves (Fig. 4BGo) and calculate the ED50 for ERK-1 activation for each receptor construct (Table 1Go). For Gln106Arg, the ED50 was 2.7-fold higher than for wild-type hGnRHR, whereas Arg262Gln resulted in a 5.1-fold increase in the ED50. Thus, Gln106Arg and Arg262Gln differentially affected the ED50 for GnRH stimulation of ERK activity in a manner parallel to the effects of these mutations on {alpha}GSU and GnRHR gene promoter activity.



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  		Figure 4. Differential effects of wild-type, Gln106Arg, and Arg262Gln hGnRHRs on phosphorylation of ERK-1. GH3 cells were transiently cotransfected with a HA-tagged ERK-1 expression vector and hGnRHRs or the empty control vector pcDNA3. Cells were serum-starved for 24 h, followed by stimulation with increasing concentrations of GnRH for 10 min and Western blot analysis. A, The dose-dependent activation of ERK-1 by GnRH for the wild-type and mutant hGnRHRs. An anti-p-ERK antibody reacted with transfected HA pERK-1, endogenous pERK-1, and pERK-2. To correct for transfection efficiency and protein loading, membranes were reprobed with an anti-(HA)-HRP antibody, which was specific for transfected HA ERK-1. B, Dose-response curves of HA ERK-1 activation for wild-type, Gln106Arg, and Arg262Gln. These curves were used to calculate the ED50 for each of the hGnRHRs (see Table 1Go). Results are derived from the densitometric analysis of three independent experiments and are expressed as percentage of the maximum stimulation for each hGnRHR (mean ± SE). Open circles represent wild-type, closed circles Gln106Arg, and triangles Arg262Gln hGnRHRs.

 
GnRH stimulation of CRE-Luc activity

It has been suggested that GnRH stimulation of cAMP-dependent pathways may play a role in regulating gonadotropin subunit gene expression (28) and LHß protein levels (4). Stimulation of CRE-Luc has been used as a marker for induction of the cAMP pathway. To measure CRE-mediated transcription, GH3 cells were transiently transfected with the hGnRHR constructs or pcDNA3, a CRE-Luc reporter construct, and an RSV-ß-galactosidase vector. Cells were stimulated with increasing concentrations of GnRH, and dose-response curves were generated (Fig. 5Go). The calculated ED50 for GnRH-stimulated activity was 2.7-fold higher for the Gln106Arg mutant, compared with the wild-type hGnRHR, whereas Arg262Gln resulted in a 6.7-fold increase in the ED50 (Table 1Go). Thus, Gln106Arg and Arg262Gln had differential effects on GnRH stimulation of CRE-dependent transcription, mirroring the patterns observed for GnRH-mediated {alpha}GSU and GnRHR gene promoter stimulation and ERK activation.



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  		Figure 5. Activation of CRE-Luc by wild-type, Gln106Arg, and Arg262Gln hGnRHRs. GH3 cells were transiently cotransfected with hGnRHRs, RSV-ß-galactosidase, and a CRE-Luc reporter construct. Forty-eight hours after transfection, cells were stimulated with increasing concentrations of GnRH for 4 h, and cell extracts were assayed for Luc activity and normalized for ß-galactosidase activity. The GnRH dose-response curves of CRE-Luc activity were used to calculate the ED50 for each of the hGnRHRs (see Table 1Go). Results are expressed as percentage of the maximum stimulation for each hGnRHR (mean ± SE for three independent experiments, each performed in triplicate). Open circles represent wild-type, closed circles Gln106Arg, and triangles Arg262Gln hGnRHRs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have analyzed the Gln106Arg and Arg262Gln mutations in the hGnRHR for their effects on GnRH stimulation of gonadotropin subunit and GnRHR gene expression. In addition, effects on signal transduction pathways were investigated to determine the mechanisms by which the two mutations may alter GnRH stimulation of gonadotropin subunit and GnRHR gene promoter activity.

Because of a higher transfection efficiency, we performed binding assays and IP analyses in COS-7 cells. This cell line has also been used by other groups in previous characterizations of mutant hGnRHRs for the calculation of Kd using either whole cells or membrane fragments, as well as for measurement of IP levels (5, 6, 7, 8, 11, 29). Subsequent experiments measuring gonadotropin subunit gene promoter activity were performed in GH3 cells because of the lack of response to GnRH in COS-7 cells. We therefore repeated the IP analyses using GH3 cells, and although the magnitude of the increase in IP accumulation following GnRH stimulation was less than in COS-7 cells, the difference in ED50 between the wild-type and mutant receptors was the same in the two cell lines, validating their comparison.

Interestingly, despite causing a similar reduction in the sensitivity of GnRH-mediated IP production, the Gln106Arg and Arg262Gln hGnRHR mutants resulted in distinct, differential sensitivities of the gonadotropin subunit genes to GnRH. Before testing the mutant receptors, we first noted that for the wild-type hGnRHR, the FSHß subunit gene promoter was 5-fold more sensitive to GnRH stimulation than either the LHß or {alpha}GSU gene promoters. This finding is consistent with a previous observation in vivo that the amount of GnRH required to induce FSH secretion is less than for LH (30). In addition, in hypophysectomized rats that have the pituitary gland transplanted beneath the kidney capsule and are treated with GnRH, plasma concentrations of FSH have been reported to increase before those of LH (31). The greater sensitivity of the wild-type FSHß gene promoter to GnRH suggests that either the FSHß gene is more sensitive to activation of intracellular pathways than the LHß and {alpha}GSU genes or that different signaling pathways are involved.

Subsequent dose-response analyses of the mutant receptors revealed that both Gln106Arg and Arg262Gln reduced the sensitivity of the FSHß subunit gene promoter to GnRH to a greater extent than LHß or {alpha}GSU. Again, this could be due to differential sensitivity of the gonadotropin subunit genes to a reduction in the dose-dependent activation of a common signal transduction pathway. However, given the greater sensitivity of the FSHß gene to low doses of GnRH, it would be expected that transcriptional activation of this gene would be less affected by the impairment of a shared signaling pathway, rather than more affected. This would favor the hypothesis that the differential responses of the gonadotropin subunit genes to GnRH may involve more than one signaling pathway.

In addition, Gln106Arg and Arg262Gln elicited distinct effects on GnRH-stimulated {alpha}GSU gene promoter activity. Both mutants reduced the sensitivity of the {alpha}GSU gene to GnRH relative to the wild-type hGnRHR, but the shift in the ED50 was more dramatic for the Arg262Gln hGnRHR mutant. The two mutant receptors had differential effects on {alpha}GSU gene activation by GnRH despite their identical effects on GnRH-mediated IP production. In contrast, the two mutants similarly affected the sensitivity of the LHß and FSHß subunit genes to GnRH. The {alpha}GSU gene appears to be linked to signaling pathways that are distinct from either LHß or FSHß gene activation, which in turn are likely to be governed by different mechanisms. Clearly, measurement of IP production alone is not adequate in determining mutant receptor function in a physiologically relevant context. Signaling pathways other than via PLC most likely play an important role in mediating the differential downstream effects elicited by the mutant receptors described here, warranting more detailed analyses.

Similar to the effects on {alpha}GSU gene activation, the Gln106Arg and Arg262Gln hGnRHR mutations also caused a differential reduction in the sensitivity of the GnRHR gene promoter to GnRH, in this case reaching statistical significance only for the Arg262Gln mutation. The response of pituitary gonadotropes to GnRH correlates directly with the concentration of GnRHRs on the cell surface, which is mediated in part at the level of GnRHR gene expression. Because GnRH itself regulates the expression of its own receptor (21), failure of GnRH to stimulate GnRHR gene promoter activity would be predicted to result in a lack of homologous up-regulation of the receptor by pulsatile GnRH, contributing to a reduced number of GnRHRs on the cell surface of gonadotropes. This, in turn, may further compound GnRH resistance in vivo.

In this study, we have not addressed the possible effects of the mutations on cell surface expression. It is possible that in addition to their effects on the dose-response curves and ED50 values, the Gln106Arg and Arg262Gln mutations may also affect rates of synthesis and/or degradation of the receptor, resulting in changes in the levels of GnRHR. These considerations are particularly relevant in the light of recent reports suggesting that some mutant GnRH receptors are expressed at reduced levels and either structural modifications of such mutants or the use of a membrane permeable nonpeptide hGnRH antagonist can partially restore ligand binding and stimulation of IP production (29, 32, 33). In our ligand-binding assay, measurement of Bmax confirmed that transfected cells expressed equivalent amounts of wild-type and Arg262Glu hGnRHRs. Although we were not able to quantitate Bmax for Glu106Arg, given the low level of binding detected for this receptor, confocal microscopy analyses indicated a qualitatively similar fluorescence pattern at the periphery of cells between wild-type and Glu106Arg receptors. Nonetheless, our current studies have focused on the effects of Gln106Arg and Arg262Gln on ED50 values for GnRH activation of signal transduction pathways and gene transcription. Although changes in receptor number may influence the absolute response to a specific concentration of GnRH, as well as the maximal response, the ED50 values would be expected to remain unchanged. ED50 values have been shown to be independent of receptor number (34, 35, 36) but rather would be predicted to reflect changes in affinity of the receptor for ligand as well as coupling of the receptor to effectors (e.g. G proteins). In particular, the potency of a GnRH agonist in stimulating IP production in GH3 cells has been shown to be independent of GnRHR concentration (37). We have also performed GnRHR analyses for the wild-type, Glu106Arg, and Arg262Glu GnRHRs that indicate that the differential effects of the mutant receptors on sensitivity of the {alpha}GSU gene promoter to GnRH are independent of receptor concentrations (data not shown). Nonetheless, the possible effects of the Gln106Arg and Arg262Gln mutations on receptor number or turnover are an avenue for future exploration.

Although no effect of the presence of the HA tag has been observed on the cell surface expression or function of the wild-type hGnRHR (16), we cannot entirely exclude an effect of the HA tag on mutant receptor function. In this regard, it is noteworthy that the effects of the Gln106Arg and Arg262Gln mutations on ligand binding and IP production in our studies are consistent with those previously reported (using receptors without HA tags) (5, 6). Furthermore, as above, any potential effect of the HA tag on cell surface expression of the mutant receptors would not be expected to affect ED50 values.

To determine whether additional signal transduction pathways other than via IP production, which may be involved in GnRH stimulation of gonadotropin subunit and GnRHR gene expression, were differentially affected by the Gln106Arg and Arg262Gln mutant receptors, we investigated GnRH stimulation of ERK phosphorylation and CRE-dependent transcription. The ERK/MAPK pathway has been suggested to play a role in GnRH-mediated transcriptional activation of the {alpha}GSU and potentially LHß genes (25) as well as in FSHß gene (38) and LHß protein expression (4, 27). GnRH has been shown to activate the ERK cascade in primary pituitary cell cultures and the {alpha}T3–1 and LßT2 gonadotrope cell lines (26, 27, 39, 40). Both basal and GnRH-stimulated activities of the {alpha}GSU gene appear to be mediated through the ERK pathway (26, 41, 42). ERK or other MAPKs also appear to play a role in GnRH regulation of LHß gene expression (40, 43). Our results demonstrate that the Gln106Arg and Arg262Gln mutant hGnRHRs differentially affect GnRH activation of the ERK pathway. Both mutant hGnRHRs reduced the sensitivity of ERK activation by GnRH, but the Arg262Gln hGnRHR mutation evoked a greater shift in the ED50 than Gln106Arg, mirroring the pattern of shifted ED50 observed for {alpha}GSU and GnRHR gene promoter activity in response to GnRH. Interestingly, although GnRH-mediated activation of ERK has been reported to occur downstream of protein kinase C (26, 43, 44), the two mutant receptors studied here had differential effects on ERK activation by GnRH despite their identical effects on GnRH-mediated IP production. Other signal transduction pathways, including calcium influx and epidermal growth factor receptor tyrosine kinase, also play a role in GnRH-mediated ERK activation (45, 46) and may contribute to the differential effects of Gln106Arg and Arg262Gln on the ERK pathway.

Several reports have suggested that GnRH stimulation of cAMP-dependent pathways may also contribute to the regulation of the gonadotropin subunit genes by GnRH (2, 3). Cyclic AMP has been shown to increase LHß and {alpha}GSU mRNA levels in primary rat pituitary cell cultures in a nonadditive manner with GnRH, and reduction of intracellular cAMP levels results in attenuation of GnRH-stimulated gonadotropin subunit mRNA levels and gonadotropin secretion, suggesting that PKA pathways may contribute to GnRH-mediated stimulation of gonadotropin subunit gene expression (28, 47). In addition, GnRHR has been shown to couple to G{alpha}s as well as G{alpha}q in LßT2 cells, and stimulation with GnRH resulted in an increase in cAMP levels (4). Specific blockade of the G{alpha}s pathway resulted in reduced ERK activation and a reduction in c-fos and LHß proteins (4). We have used GnRH stimulation of CRE-Luc as a general marker for induction of the cAMP pathway in the analysis of the hGnRHR mutants. For CRE-dependent transcription, the effects of the Gln106Arg and Arg262Gln mutants paralleled the pattern of loss in GnRH sensitivity observed for GnRH stimulation of {alpha}GSU and GnRHR gene promoter activities, with the Arg262Gln hGnRHR mutation again causing a greater shift in the ED50 than Gln106Arg. Our data therefore suggest that cAMP-mediated pathways are also candidates for mediating differential effects of Gln106Arg and Arg262Gln. It should be noted, however, that CRE is responsive to other pathways in addition to cAMP/PKA. Of particular note, phosphorylation and activation of CRE-binding protein B have been reported to occur downstream of MAPK activation (48). It seems likely that several signal transduction pathways may be converging via a complex system of cross-talk to orchestrate regulation of gonadotropin subunit and GnRHR gene expression.

Use of the two common naturally occurring mutations for in vitro studies may help to clarify how structural changes in the hGnRHR contribute to the considerable range of phenotypes observed among patients harboring these partially inactivating mutations. The Gln106Arg and Arg262Gln hGnRHR mutations have been previously identified in several patients with varying degrees of IHH. Recently, a male patient with fertile eunuch syndrome was found to be homozygous for the Gln106Arg hGnRHR mutation (13). In addition, a female patient homozygous for the Gln106Arg hGnRHR mutation achieved a spontaneous pregnancy (15). In the reported male patient, basal levels of both gonadotropins were within the normal range. In response to a single injection of GnRH (100 µg), gonadotropins increased differentially, with a greater increase in LH than FSH. Similarly, pulsatile GnRH administration over 7 d induced a response only in LH, with no increase in FSH (13). These in vivo responses are consistent with our in vitro observations that the Gln106Arg mutation reduced the sensitivity of the FSHß gene to GnRH to a greater extent than LHß or {alpha}GSU. As suggested by Pitteloud et al. (13), the lack of FSH response to GnRH also may be due to the relatively high circulating levels of inhibin B observed in this male patient.

To date, the Arg262Gln mutation has not been observed in a homozygous state in patients. However, several compound heterozygotes harboring both the Gln106Arg and Arg262Gln mutations have been reported. Although some variability in the phenotypes of these compound heterozygotes has been noted, most likely resulting from the influences of gender and other endocrine inputs on reproductive control, these patients consistently have more severe IHH phenotypes than the relatively mild clinical manifestations noted in the Gln106Arg homozygotes. Differences in the severity of phenotype would have been surprising based on evaluating the IP responses to GnRH alone but may be explained by the differential reduction in sensitivity to GnRH of the two mutant hGnRHRs for stimulation of {alpha}GSU. Furthermore, GnRH stimulation of GnRHR gene expression is also reduced to a greater extent by Arg262Gln than by Gln106Arg. This can be expected to reduce gonadotrope cell surface GnRHR numbers, which would further compound resistance to GnRH stimulation of gonadotropins in such compound heterozygotes, compared with the Gln106Arg homozygotes.

In summary, we have analyzed two common naturally occurring mutations in the hGnRHR, Gln106Arg and Arg262Gln, for their effects on the GnRH stimulation of gonadotropin subunit and GnRHR gene promoter activity in vitro. Despite similar reductions in the IP response to GnRH elicited by both mutant receptors, differential effects on GnRH-stimulated gonadotropin subunit and GnRHR gene expression were observed, suggesting either different effects on a common signaling pathway, or, more likely, distinct effects on different signal transduction pathways. Indeed, further studies elucidated differential effects of the mutant receptors on GnRH-stimulated ERK activation and CRE-mediated transcription. Clearly, measurement of IP production alone, which has been used widely to assess the signaling capacity of hGnRHR mutants, is not adequate for determining mutant receptor function in a physiologically relevant context. The differential effects on GnRH-stimulated LHß, FSHß, and {alpha}GSU subunit gene expression may account for some of the phenotypic variation observed in patients with IHH caused by mutations in the hGnRHR. Such naturally occurring mutants serve as useful tools for dissecting signaling pathways involved in mediating the differential effects of GnRH on gonadotropin subunit and GnRHR gene expression.


   Acknowledgments
 
We thank Jo Ann Janovick and P. Michael Conn (Oregon Health Sciences University, Beavertown, OR) for generously providing us with radiolabeled Buserelin, and Thomas Gudermann (Free University of Berlin, Berlin, Germany) for providing us with the wild-type hGnRHR vector. We thank Dr. Armen Tashjian for helpful discussions.


   Footnotes
 
This work was supported by National Institute of Child Health and Human Development/NIH through cooperative agreement U54-HD28138 as part of the Specialized Cooperative Centers Program in Reproduction Research (to U.B.K.), the George W. Thorn Center (to U.B.K.), and the Lalor Foundation (to G.Y.B.).

Abbreviations: Bmax, Maximum binding; CRE, cAMP response element; HA, hemagglutinin; hGnRHR, human GnRH receptor; HRP, horseradish peroxidase; IHH, idiopathic hypogonadotropic hypogonadism; IP, inositol phosphate; Kd, dissociation constant; Luc, luciferase; PKA, protein kinase A; PLC, phospholipase C; RSV, Rous sarcoma virus; TBS, Tris-buffered saline.

Received May 24, 2002.

Accepted November 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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