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Research Unit in Developmental Biology (G.M.-N., D.S., J.P.M.), Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, México, 06725 México; Research Unit in Reproductive Medicine (A.U.-A.), Hospital de Ginecobstetricia "Luis Castelazo Ayala," Instituto Mexicano del Seguro Social, México, 01090 México; and Oregon Regional Primate Research Center (J.A.J., A.U.-A., P.M.C.) and Department of Physiology and Pharmacology (P.M.C.), Oregon Health Sciences University, Beaverton, Oregon 97006
Address all correspondence and requests for reprints to: Guadalupe Maya-Núñez, M.S., Unidad de Investigación Médica en Biología del Desarrollo, Coordinación de Investigación Médica, Avenida Cuauhtémoc 330, Apartado Postal 73-032, Colonia Doctores, C. P. 06725, México, D. F., Mexico. E-mail: . mayanune{at}yahoo.com
Abstract
GnRH regulates the synthesis and release of pituitary gonadotropins. Mutations in the human GnRH receptor (hGnRHR) gene have been reported in families with hypogonadotropic hypogonadism. Our group recently described a novel homozygous E90K mutation of the hGnRHR in two siblings with the complete form of hypogonadotropic hypogonadism. In the present study, mutational analysis of the E90K substitution was performed to assess the functional role of this particular residue, which is located in the second transmembrane helix of the hGnRHR. Although E90 is highly conserved in all other known mammalian GnRH receptors, this residue has not been previously implicated in GnRH binding and/or GnRHR activation. Transient expression of the mutant E90K receptor in COS-7 cells resulted in a virtual abolition of GnRH agonist binding and agonist-stimulated phosphoinositide turnover, initially suggesting that E90 may be essential for GnRH binding. Furthermore, incubation with 1 µM of different GnRH agonists (D-Trp6-GnRH, GnRH, leuprolide, Catfish-1 GnRH, Catfish-2 GnRH, D-Lys6-Pro9-EA-GnRH, DesGly10-GnRH, D-Trp6-Pro9-EA-GnRH, Buserelin, and D-Lys6-GnRH) or antagonists (Antide and "Nal-Arg") did not result in elevated inositol phosphate production from cells expressing the E90K mutant. To examine the role of a site known to suppress hGnRHR function, mutants with deletion of K191 (
K191) from the hGnRHR and/or addition of catfish GnRHR intracellular carboxyl-terminal tail (cfCtail) to hGnRHR were prepared. Exposure to the GnRH analog Buserelin resulted in a significant increase in total inositol phosphate production in cells expressing the hGnRHR-cfCtail, hGnRHR(K191) and hGnRHR(K191)-cfCtail. Activation of intracellular signaling in response to Buserelin was restored by deletion of K191 from the E90K mutant receptor but minimally by addition of the catfish GnRHR carboxyl-terminal tail. There were no significant differences in total inositol phosphate production between the chimeric receptors bearing the K191 or the E90K/K191 modifications. All but the (E90K) and (E90K)-cfCtail altered receptors were membrane expressed as disclosed by Western blot analysis of epitope-tagged receptors. This study provides evidence that the E90K mutation impairs hGnRHR-effector coupling. The observation that sequence modifications that enhance surface expression of the receptor restore function, presents the possibility that loss of surface expression may underlie the severe phenotype exhibited by hypogonadotropic hypogonadism patients bearing this mutational defect.
GONADOTROPIN-RELEASING HORMONE plays a key role in regulating reproductive function. This decapeptide is produced by specialized neurons located in the mediobasal hypothalamus, whose axons project to the median eminence into which GnRH enters the portal circulation and interacts with a specific receptor on pituitary gonadotrope cells to stimulate synthesis and release of gonadotropins. Sequence analysis of the GnRH receptor (GnRHR) suggests a seven-transmembrane domain motif characteristic of the G protein-coupled receptors superfamily (1). The mammalian GnRHR is unique among the thousands of members of this family of receptors in that it lacks the carboxyl- terminal domain. The human GnRHR (hGnRHR) is preferentially coupled to the Gq/11 protein; after GnRH binding, the activated GnRHR-Gq/11 protein system stimulates the activity of the membrane-associated enzyme phospholipase Cß, which in turn promotes inositol 1,4,5-trisphosphate production and the release of intracellular calcium (2).
Congenital hypogonadotropic hypogonadism (HH) may result from defects in the synthesis or action of GnRH. Although a mutation in the GnRH gene has been reported in the hypogonadotropic hpg/hpg mouse (3), no such mutation has been yet detected in humans. Kallmann’s syndrome is characterized by the association of HH and anosmia (or hyposmia) and is caused by a migration defect that involves the GnRH neuronal system (4). The gene for the X-linked form of Kallmann’s syndrome has been mapped to chromosome Xp22.3 (5, 6), and several mutations have been described to date (7, 8, 9, 10). Mutations of the AHC gene (also in Xp22.3), which result in congenital adrenal hypoplasia and HH, have also been described (11).
Alterations in the hGnRHR function constitute a rare cause of HH (12, 13, 14, 15, 16, 17). To date, eight different hGnRHR mutations causing either complete or partial HH have been described; although these mutations are distributed along the entire coding sequence, they have been mainly localized within transmembrane domains 3 to 5 (12, 13, 14, 15, 16, 17, 18). These mutations affect ligand binding, receptor expression at the cell surface, and/or signal transduction and included a wide spectrum of phenotypes, from partial to complete hypogonadism (18). More recently, our group described a novel homozygous E90K mutation in the second transmembrane helix of the hGnRHR in two siblings with a complete form of HH; both patients exhibited sexual infantilism, extremely low serum gonadotropin concentrations, and lack of response to supraphysiological doses of exogenous GnRH (19). In the present study, we examined the effects of this mutation on hGnRHR function. Mutant and chimeric hGnRH receptors were prepared, expressed in COS-7 cells, and assessed for GnRH agonist-stimulated intracellular signal transduction. The results demonstrate that the E90K mutation completely abolished hGnRHR expression at the cell surface and that the E90 residue has a prominent role in receptor function.
Materials and Methods
Materials
Natural sequence GnRH was provided by the NIDDK’s National Hormone and Peptide Program through Dr. A. F. Parlow (Torrance, CA). The GnRH agonist (GnRHa), Buserelin (D-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH), was a kind gift from Hoeschst-Roussel Pharmaceuticals (Somerville, NJ); Leuprolide (DLeu6-Pro9-des-Gly10-ethylamide-GnRH) was from TAP Pharmaceuticals, Inc. (Deerfield, IL); Antide ([N-Ac-DNal1-DpCl-Phe2-DPal3-Lys(Nic)5-DLys(Nic)6-Lys(iPr)8-DAla10]GnRH) and "Nal-Arg" ([NAc-DNal1-DpCl-Phe2-DPal3,Arg5-DGlu6-DAla10]GnRH) were provided by the NIH Contraceptive Development Branch (Bethesda, MD); DLys6-Pro9-ethylamide-GnRH and Tyr0-calcitonin-gene related peptide (Tyr0-CGRP) were purchased from Peninsula Laboratories, Inc. (San Carlos, CA) and D-Trp6-GnRH as well as des-Gly10-GnRH were from Bachem (Torrance, CA). Catfish-1 and -2 GnRH were kind gifts from Dr. Jean Rivier (Salk Institute, La Jolla, CA). The expression vector pcDNA3.1 was purchased from Invitrogen (San Diego, CA). DMEM, OPTI-MEM, lipofectamine, and PCR reagents were purchased from Life Technologies, Inc. (Grand Island, NY). Restriction enzymes, modified enzymes, and competent cells for subcloning were purchased from Promega Corp. (Madison, WI). The Endofree maxiprep kit was purchased from QIAGEN (Valencia, CA). Other reagents were of the highest degree of purity available from commercial sources, unless otherwise noted.
Vector construction
The wild-type (WT) hGnRHR cDNA in pcDNA3 was subcloned into pcDNA3.1 at KpnI and XbaI restriction enzymes sites. The E90
K90 (GAG to AAG) mutation was constructed by overlap extension PCR (20). Deletion of K191 (K191) from the hGnRHR [hGnRHR(K191)] and the chimeric receptor (hGnRHR-cfCtail) containing the hGnRHR and the intracellular carboxyl-terminus (Ctail) of the catfish (cf) GnRHR were constructed as previously described (21). An amino-terminal hemagglutinin (HA) epitope tag (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) was inserted before the first Met residue of the WT and altered hGnRHRs by the PCR. The 80 oligonucleotides primer sequence included the KpnI restriction enzyme sequence, the Kozak consensus sequence, an ATG start codon, the HA-tag sequence, and a Ser-Thr-Gly spacer sequence before the ATG start site of the hGnRHR. All cDNAs were digested with KpnI and XbaI and subcloned into the expression vector pcDNA3.1. The identity of all constructs and the correctness of the PCR-derived sequences were verified by Dye Terminator cycle sequencing according to the manufacturer’s instructions (Perkin-Elmer Corp., Foster City, CA). For transfection, large-scale plasmid DNAs were prepared using an Endofree maxiprep kit (QIAGEN). The purity and identity of the amplified plasmid DNAs were further verified by restriction enzyme analysis.
Transient transfection of COS-7 cells
Wild-type hGnRHR and altered receptors were transiently expressed in COS-7 cells. Cells were maintained in growth medium (DMEM) containing 10% FCS (Life Technologies, Inc.) and 20 µg/ml gentamicin (Gemini Bioproducts, Calabasas, CA) in a 5% CO2 humidified atmosphere at 37 C. One thousand cells/well were seeded in 24-well plates (Costar, Cambridge, MA). Twenty-four hours after plating, the cells were transfected with 0.8 µg DNA per well using 2 µl lipofectamine in 0.25 ml OPTI-MEM and 5 h later, 0.25 ml of DMEM containing 20% FCS calf serum were added to each well. Twenty-four hours after the start of transfection, the medium was replaced with fresh DMEM, and the cells were allowed to grow for another 24 h before treatment. For GnRHR binding, the same transfection procedure was followed except that 20 µg plasmid DNA and 50 µl lipofectamine were used to transfect 60–80% confluent cells in 75-cm2 flasks (Costar).
Measurement of inositol phosphates (Ip) production
Quantification of Ip production was performed as described previously (22). Briefly, 48 h after the start of transfection, transiently transfected cells were washed twice with DMEM containing 0.1% BSA and intracellular inositol lipids were labeled in inositol-free DMEM supplemented with 4 µCi/ml [3H]myo-inositol for 18 h at 37 C. After the preloading period, cells were washed twice with DMEM (inositol free) containing 5 mM LiCl and incubated for 2 h at 37 C in the absence or presence of the indicated doses of Buserelin dissolved in 0.5 ml DMEM (inositol free)-LiCl. At the end of the incubation period, medium was removed, and 1 ml 0.1 M formic acid was added to each well. Cells were then frozen and thawed to disrupt the cell membranes. Inositol phosphate accumulation was measured by Dowex anion exchange chromatography and liquid scintillation spectroscopy, as previously described (22).
Receptor binding
Intact cell GnRHR binding was assessed over a range of concentrations of [125I]Buserelin, prepared as reported previously (23) in DMEM/0.1% BSA. Seventy-two hours after the start of transfection, cells were scraped and resuspended in warm DMEM/0.1% BSA. Cells were then pelleted and washed twice with ice-cold DMEM/0.1% BSA. A total of 100 µl of the cell suspension (
106 cells) was added to each tube, and the assay was allowed to reach equilibrium (3 h) at 4 C at a final volume of 150 µl. Binding was determined by overlayering each sample on 2 ml of DMEM/0.3 M sucrose at 4 C and centrifugation at 2000x g for 10 min at 4 C in a Sorvall SM-24 rotor. The supernatant fraction was then aspirated and the cell pellet resuspended in 1 ml PBS. Radioactivity was determined using a 10-channel 
counter (Packard Bioscience Co., Downers Grove, IL).
Western blot analysis of WT and mutant GnRHRs
Transfected cells (in 75-cm2 cell culture flasks) were washed with PBS prior to harvesting and homogenization in lysis buffer (50 mM HEPES, 0.2 mM EDTA, 1 mM DTT, and protease inhibitors cocktail 1x). The homogenate was centrifuged for 5 min at 500x g and 4 C and the resulting supernatant was centrifuged at 100,000x g for 40 min at 4 C to obtain the membranes. Samples were electrophoresed under reducing conditions on 7.5% polyacrylamide gels and electroblotted onto nitrocellulose membranes (Hybond C+, Amersham Pharmacia Biotech, Buckinghamshire, UK). Blots were blocked for 2 h in blot buffer [20 mM Tris, 500 mM NaCl (pH 7.7), and 10% horse serum] before overnight incubation with the anti-HA antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:200 dilution in blot buffer. Bound antibody was detected using the horseradish peroxidase-conjugated goat antirabbit immunoglobulin kit (Bio-Rad Laboratories, Inc., Hercules, CA).
Statistical analysis
The data shown represent the means ± SEM from triplicate incubations. In all experiments, the SD was typically less than 10% of the corresponding mean. The data were analyzed by one-way ANOVA followed by the Duncan’s multiple range test. P less than 0.05 was considered statistically significant. Each experiment was repeated three or more times.
Results
Characterization of the WT and the E90K-mutated hGnRHRs
Figure 1
shows a schematic representation of the hGnRHR depicting the position of the E90 residue in the second transmembrane domain (TMD). The WT hGnRHR and the hGnRHR bearing the E90K mutation were transiently expressed in COS-7 cells. To compare the cell surface expression level and the structural integrity of these receptors, radioligand-binding assays were performed using the metabolically stable GnRHa Buserelin. These studies showed that hGnRHR(E90K) was unable to bind [125I]Buserelin at the cell surface level (Fig. 2a). A representative dose-response experiment of Buserelin-stimulated Ip production is shown in Fig. 2b. Exposure of cells expressing the WT hGnRHR to GnRHa for 2 h, resulted in a significant dose-dependent increase in Ip turnover. In contrast, Ip production by cells transiently expressing the hGnRHR(E90K) was virtually abolished, compared with that exhibited by cells expressing the WT receptor after exposure to 10-11 to 10-7 M Buserelin.
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). Cells expressing either the GnRHR WT or the hGnRHR(E90K) did not respond to treatment with the GnRH antagonists (Antide and "Nal-Arg," 10-6 M). Stimulation with a non-GnRH-related peptide [calcitonin-gene related peptide (Tyr0-CGRP)] did not modify basal Ip production in cells bearing the WT or the mutant (E90K) hGnRHR. To address the issue of whether the failure in Ip production by the hGnRHR(E90K)-transfected cells was owing to the absence of ligand binding or to disruption of the downstream signaling pathway, transfected cells were stimulated with 20 mM NaF, a G protein activator. Addition of NaF provoked an augmentation in Ip turnover by both the WT receptor- and the hGnRHR(E90K)-transfected cells (Fig. 3).
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In a previous study (21), we showed that deletion of K191 from the hGnRHR or addition of the cfGnRHR intracellular carboxyl-terminal tail to the human receptor significantly increases the expression levels of the modified receptors at the cell surface level. Deletion of K191 and addition of the cfGnRHR intracellular Ctail to the hGnRHR were achieved by overlap extension PCR (20). The chimeric hGnRHR- cfCtail construct was comprised by the 328 amino acids of the hGnRHR WT and by 51 amino acids from the carboxyl-terminus of the cfGnRHR (Fig. 4a). These mutated receptors were used as a template to create chimeric receptors bearing the E90K mutation, which were subsequently expressed in COS-7 cells (Figs. 4, a–f). Basal and Buserelin-stimulated Ip production in cells expressing these modified receptors is shown in Fig. 5. Exposure to 10-7 M Buserelin resulted in a significant, dose-dependent increase in Ip production by COS-7 cells expressing the hGnRHR-cfCtail and the hGnRHR(K191). GnRHa-provoked activation of intracellular signaling was restored by deletion of Lys191 from the E90K mutant receptor [hGnRHR(E90K/K191); Fig. 4d] but not by addition of the cfGnRHR Ctail [hGnRHR(E90K)- cfCtail; Fig. 4b and Fig. 5]. There were no significant differences in total Ip production between the chimeric receptors bearing either the K191 or the E90K/K191 modifications.
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. Western blot analysis of the epitope-tagged receptors yielded a band for all but the (E90K) and (E90K)-cfCtail altered receptors and a faint signal for the hGnRHR(K191)-cfCtail, indicating that the low or absent Ip response of hGnRHR(E90K) and hGnRHR(E90K)-cfCtail was associated with the substantially reduced membrane expression levels of receptor protein.
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In the present study, we recreated and expressed in a heterologous cell system the naturally occurring E90K mutation on the hGnRHR to assess more precisely the impact of this amino acid substitution on GnRHR function. This E90 residue is highly conserved in all other known mammalian GnRH receptors (24) and had not been previously shown to be critical for hGnRHR function despite its location near N87 (which is substituted by a highly conserved aspartic acid in the majority of the G protein-coupled receptors belonging to the rhodopsin/ß-adrenergic superfamily), which pairs with D318 (asparagine in other receptors) in TMD-7, forming a TMD-2/TMD-7 microdomain implicated in receptor expression and interaction with heterotrimeric G proteins (24, 25, 26, 27, 28, 29, 30). With expression of the mutant, no specific binding of [125I]-labeled Buserelin was detectable and the hGnRHR(E90K) was incapable of mediating GnRHa-stimulated Ip production. Further, exposure of COS-7 cells bearing the hGnRHR(E90K) to a number of GnRH analogs having multiple, asymmetrically distributed contact points on the receptor (and thus that may potentially bind distinctly from the WT and mutant receptors) failed to trigger activation of the receptor-G protein system and, consequently, to stimulate Ip production. This observation initially suggested that the disruption of the hydrophobic 
-helix provoked by the E90K mutation altered the configuration of the TMD-2 and thus distorted the binding pocket and/or proper membrane insertion of the mutant receptor.
To examine the role of a site known to suppress hGnRHR function, mutants with deletion of K191 from the hGnRHR and/or addition of cfGnRHR intracellular carboxyl-terminal tail to hGnRHR were prepared and overexpressed in COS-7 cells. Previous studies (31, 32) have shown that deletion of K191 (a residue located in the second extracellular loop of the hGnRHR) from the hGnRHR or addition of the cfGnRHR Ctail to the human receptor significantly increases cell surface receptor expression and decreases the internalization rates of the altered receptors, effects that may be further enhanced by the simultaneous addition of the cfCtail and deletion of K191 (21). Whereas GnRHa-provoked activation of intracellular signaling was restored by deletion of K191 from the E90K mutant receptor, the signaling capacity of the mutant was modestly recovered by the addition of the cfGnRH Ctail. The differences in functional retrieval achieved through the separate inclusion of both modifications into the hGnRHR(E90K) may be owing to the presumptively distinct mechanisms whereby each modification enhances the cell surface expression of the mutant receptor (21). Apparently, deletion of K191 stabilized and restored more efficiently the loss in spatial arrangement of the TMD-2/TMD-7 functional microdomain provoked by the E90 to K90 substitution, suggesting that functional integrity of this microdomain may be a prerequisite for the enhanced cell surface expression of the hGnRHR-cfCtail chimera. This latter possibility may eventually explain the marginal recovery exhibited by the hGnRHR(E90K)-cfCtail chimera, a view that is additionally supported by the finding that the function and membrane expression of this chimeric receptor was completely restored by deleting K191. Finally, the observation that total Ip production by cells expressing the chimeric (C-tailed) receptors bearing the K191 or the E90K/K191 modifications was virtually identical favors the possibility that the loss in receptor binding of the hGnRHR(E90K) most likely resulted from a decrease in membrane receptor density as a consequence of either disruption of trafficking to the membrane or instability and subsequent degradation of the mutant receptor.
Interestingly, deletion of K191 from the hGnRHR conferred slightly elevated basal Ip production to COS-7 cells transiently expressing the altered receptors. This observation strongly suggests that the K191 modification may potentially alter receptor conformation and lead to some degree of ligand-independent receptor isomerization to an active, unconstrained state, a possibility that deserves further study.
In this study, we provided evidence that the E90K mutation in the hGnRHR severely impairs receptor function and that E90 in the TMD-2 may have an important role in cell membrane expression of the hGnRHR. These findings explain the severity in the phenotype found in HH patients bearing the E90K mutation.
Acknowledgments
We thank the NIDDK National Hormone and Pituitary Program and Dr. A. F. Parlow for providing the natural sequence GnRH.
Footnotes
This work was supported by Grants G29790M (to J.P.M.) and 28589N (to A.U.-A.) from the Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico; and by Grants HD19899, RR00163, HD18185, and TW/HD00668 (to P.M.C.) from the NIH (Bethesda, MD). G.M.-N. and D.S. are postgraduate students from the Facultad de Medicina, Universidad Nacional Autónoma de México, México D.F.
Abbreviations: cfCtail, Catfish GnRHR intracellular carboxyl-terminal tail; K191, deletion of K191; GnRHa, GnRH agonist; GnRHR, GnRH receptor; HA, hemagglutinin; hGnRHR, human GnRHR; Ip, inositol phosphates; TMD, transmembrane domain; WT, wild type.
Received March 7, 2001.
Accepted December 21, 2001.
References
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