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INSERM, U-135, Unité de Recherches Hormones Gènes et Reproduction, Hôpital de Bicêtre (B.K., M.M., E.M., N.d.R.), Le-Kremlin-Bicêtre, 94270 Paris, France; Pediatric Endocrinology, University Children’s Hospital (B.K.), and Division of Endocrinology, Department of Internal Medicine (W.K., L.L.), University of Ulm, D-89075 Ulm, Germany; and Institute of Molecular Pharmacology (R.K.), D-13125 Berlin, Germany
Address all correspondence and requests for reprints to: Dr. Nicolas de Roux, INSERM, U-135, Hôpital de Bicêtre, 78 rue du Général Leclerc, F 94270 Le-Kremlin-Bicêtre, France. E-mail: nicolas.deroux{at}bct.ap-hop-paris.fr.
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Abstract |
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Introduction |
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In vitro studies of naturally occurring human GnRHR mutations (1, 2, 3, 4, 5, 6, 7, 8, 9) have provided new insight into GnRHR function. Similarly, experimental GnRHR variants have been generated by site-directed mutagenesis to study the contribution of single amino acid residues to receptor structure and function (14, 15). However, the structural basis for altered receptor activation in naturally occurring human GnRHR mutations remained mostly unclear.
We therefore used a molecular modeling system based on the crystal structure of rhodopsin (16) to characterize the novel transmembrane substitution Ala171Thr of the GnRHR. Our functional in vitro studies and molecular modeling data show that the loss of GnRHR function is caused by the introduction of an excess hydrogen bond between TMH domains 3 and 4, resulting in disrupted conformational mobility, ligand binding, and signal transduction, leading to clinical hypogonadotropic hypogonadism.
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Subjects and Methods |
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The index patient (subject II-1 in Fig. 1
), a 19-yr-old Caucasian man from southern Germany, presented with typical signs and symptoms of hypogonadism, including a puerile voice, absence of pubic and axillar hair, microphallus (10 mm extended length), and testicular volume less than 1 ml each. Moderate bilateral gynecomastia was detectable. His height was 181 cm, and his weight was 104 kg. The bone age, as radiologically determined by the method of Greulich-Pyle, was retarded (15.0 yr).
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, aged 21 yr) showed typical signs and symptoms of hypogonadism. He never shaved. As child he had undergone orchidopexy because of cryptorchidism. His height and weight were 189 cm and 90 kg, respectively. Physical examination revealed the absence of axillary and pubic hair, microphallus, hypoplastic scrotum, and small testes (1 ml) as well as bilateral gynecomastia. The right and left mammary glands measured 4 x 4 cm and 4 x 3 cm by mammography, respectively. Both brothers had a normal sense of smell, and magnetic resonance imaging showed a normal hypothalamic pituitary region and olfactory bulbs. They both had a normal karyotype (46,XY), and further endocrinological work-up was normal. Both patients currently receive 250 mg testosterone enanthate, im, every 4 wk. All endocrine tests were performed before androgen replacement.
The patients’ mother (subject I-2 in Fig. 1, aged 47 yr) and father (subject I-1, aged 49 yr) were phenotypically normal, without clinical or laboratory evidence for endocrine or other disorders. They do not have other children. Informed consent was obtained from all individuals before analyses, and studies were performed in accordance with the Declaration of Helsinki.
Hormone assays
Serum LH and FSH were determined by RIA (Vitros Immunodiagnostic FSH/LH Assays, Amersham Pharmacia Biotech, Little Chalfont, UK), with a sensitivity 0.5 mIU/ml. Serum total testosterone and estradiol were measured by RIA (Diagnostic System Laboratories, Inc., Sinsheim, Germany).
GnRHR sequence analysis
Automated sequencing of the human GnRHR gene (GenBank accession no. L03380) in patients and relatives was performed as previously described (1, 2) using genomic DNA from peripheral blood lymphocytes (DNeasy, kit, QIAGEN, Hilden, Germany) as template.
GnRHR expression constructs
We have previously described the plasmids coding for wild-type (WT) human GnRHR (1, 2). For functional in vitro studies of the novel GnRHR mutation (Ala171Thr), the substitution in nucleotide 511 was introduced by site-specific PCR mutagenesis and ligated into the PSG5 expression vector (Stratagene, La Jolla, CA). Subsequently, WT and mutated GnRHR cDNA were subcloned into the EcoRI/BamHI sites of pcDNA3.1 (Invitrogen, Cergy Pontoise, France). These vectors were named pcDNA-GnRHR-WT and A171T.
A chimeric GnRHR fused to the green fluorescent protein (GFP) at its C-terminus was constructed by PCR. First, the total coding sequence of the GnRHR with the 3'-untranslated sequence from the pcDNA-GnRHR-WT vector was subcloned into the enhanced GFP (EGFP) promoter-N2 vector at EcoRI and KpnI sites. This vector was named pGnRHR-3'UT-GFP. To remove the stop codon and the 3'-untranslated sequence, the DNA was amplified by PCR with a primer located upstream of the unique PstI site and a 36-bp primer (5'-TCCCCCGGGCAGAGAAAAATATCCATAGA-TAAGTGG-3') that eliminates the stop codon and creates a new SmaI site. This PCR product was subcloned in place of the 3'-untranslated sequence at PstI and SmaI sites of pGNRHR-3'UT-EGFP. The construct (pGnRHR-EGFP) contains the PGIHRPVAT protein sequence as a spacer between the last amino acid of the GnRHR and EGFP. To construct the Ala171Thr-GnRHR-EGFP vector, an EcoRI-PshAI fragment from the pcDNA-Ala171Thr-GnRHR vector was substituted for an EcoRI-PshAI WT fragment in the pGNRHR-EGFP vector. Plasmid sequences were confirmed by bidirectional DNA sequencing.
Cell culture and in vitro transfection
Receptor binding and signal transduction studies were performed in human embryonic kidney cells (HEK) 293T cells, grown in DMEM/10% FCS/1% penicillin-streptomycin. Cells (1.2 x 105/well) were seeded in 12-well dishes previously coated with fibronectin (20 ng/µl; Sigma-Aldrich, St. Louis, MO). Transient transfection was performed 24 h after plating using FuGENE6 transfection reagent (Roche, Mannheim, Germany). Equal transfection efficiencies were ascertained by cotransfection with pcDNA3.1 plasmid expressing the lacZ cDNA, and total protein contents in cell lysates were determined to be equal. Transfections were performed in triplicate, and each independent experiment was repeated at least once.
Hormone-induced PLC activity
The accumulation of inositol phosphates in transfected HEK 293T cells exposed to various concentrations of GnRH (Sigma-Aldrich) was measured to compare signal transduction of mutant and WT receptors (1, 2). Twenty-four hours after transfection, culture medium was replaced by inositol-free DMEM supplemented with labeled inositol (2 µCi/well myo-[2-3H]inositol, 17.0 Ci/mmol, TRK911, Amersham Pharmacia Biotech). Twenty-four hours later, cells were incubated with 10 mM LiCl for 10 min and stimulated with increasing amounts of GnRH at 37 C for 30 min. Cells were lysed with 10 mM formic acid and centrifuged at 14,000 x g. Supernatants were neutralized with 5 mM NH3 and loaded onto a 0.8-ml AG-X8 resin anion exchange column as previously described (1). After washing with 5 and 40 mM ammonium formate, pH 5, inositol phosphates were eluted with 2 M ammonium formate pH 5. Radioactivity was determined by scintillation counting. PLC activity was expressed as the percentage of maximal inositol phosphate accumulation obtained for WT receptor activation.
Ligand binding by mutant GnRHR
For receptor binding studies, HEK 293T cells were transfected with WT or mutant GnRHR cDNA as described above. Cells were incubated with 1 x 105 cpm/well [125I]GnRH-A (pGlu-His-Trp-Ser-[125I]Tyr-DAla-Leu-Arg-Pro-NHEt, 2442 Ci/mmol; provided by Dr. Dugave, Gif-sur-Yvette, France) in the presence of increasing concentrations (10-12–10-6 M) of unlabeled GnRH (Sigma-Aldrich) for 3 h in DMEM containing 0.3% BSA/25 mM HEPES, pH 7.4. After washing to remove unbound GnRH ligand, cells were lysed with 1% Triton/100 mM NaOH. GnRH agonist ligand affinity and the concentration of GnRHR sites on the cell surface were calculated with PRISM software (version 3.0, GraphPad Software, Inc., San Diego, CA). Binding studies were performed twice in triplicate.
Cellular localization of GnRHR
For confocal analysis, DNA transfection was performed in 60-mm dishes with 1 µg pGNRHR-EGFP vector. Twenty-four to 48 h after transfection, 50,000 cells were plated in DMEM-10% FCS without phenol red on poly-D-lysine (Sigma-Aldrich)-coated glass coverslips, rinsed twice with water, and allowed to dry for 2 h before plating the cells. The cells were allowed to attach at 37 C for 24 h. After washing twice with PBS at 4 C, cells were fixed by 3% formaldehyde in PBS for 15 min at room temperature. Coverslips were then mounted on a slide with fluorescent mounting medium (DAKO Corp., Carpenteria, CA) before confocal microscopy analysis. Confocal analysis was performed with a Zeiss LSM 410 confocal system (Leitz, Wetzlar, Germany).
Molecular modeling of GnRHR structure
The starting structure of the transmembrane bundle of the human GnRHR was based on the crystal structure of bovine rhodopsin as template (16). The extra- and intracellular domains of human GnRHR were designed by combination of de novo homology modeling based on three-dimensional structures of sequence elements using the Brookhaven database and C
-positions derived from the rhodopsin structure for more conserved sequences. Two disulfide bridges were included into the model: the highly conserved disulfide bridge C114-C196 located at the same position as in rhodopsin (16), and the disulfide bridge C14-C200 stabilizing the antiparallel ß-strands formed by the second extracellular loop and the N terminus (17). After refinement of the receptor structure (15), the Ala171Thr mutation was introduced using the Biopolymer module of Sybyl6.6 (Tripos, Inc., St. Louis, MO). To avoid van der Waals clashes, the Ala171Thr mutant receptor was minimized again using the sander module of AMBER5.0 (18) up to an energy gradient less than 0.05.
To study structural effects of the receptor mutation, a molecular dynamic simulation was performed using the sander module of AMBER5.0 (18) (500 psec; 300 K; step width, 2 fsec) with the bond-shaking option for all hydrogen-containing bonds and harmonic restraint on all C atoms of 1 kcal/mol/Å-2, except for residues 169–176, thus allowing conformational changes in the region of mutation. After equilibration of 100 psec, the molecular trajectory was finally analyzed.
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Results |
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Hormone analysis showed low baseline values for LH, FSH, and testosterone in both affected patients (Fig. 1C), confirming severe hypogonadotropic hypogonadism. Thrity minutes after administration of synthetic GnRH (100 µg, iv; Gonadorelin, Roche), plasma LH and FSH levels increased from 0.3 to 6.8 mU/ml and from 1.4 to 3.82 mU/ml, respectively, in patient II-1. Baseline values for TSH, T4, T3, PRL, GH, IGF-I, ACTH, and cortisol were normal in both brothers (data not shown). In addition, pituitary function was assessed in patient II-1 by simultaneous iv administration of synthetic CRH (100 µg), GHRH (100 µg), and TRH (200 µg), showing normal dynamic responses for ACTH, GH, and TSH (data not shown).
Identification of GnRHR gene mutations
Sequencing of the GnRHR coding region revealed two heterozygous mutations in both patients (Fig. 1, A and B), encoded in the first exon. The first mutation, Ala171Thr (GCA to ACA, nucleotide 511) results in the replacement of alanine by threonine at position 171, located in TMH4 of the GnRHR. The second mutation, Gln106Arg (CAA to CGA, nucleotide 317) leads to the exchange of glutamine (Gln) by arginine (Arg) at position 106, located in the first extracellular loop of the GnRHR. Ala171Thr represents a novel GnRHR mutation, and Gln106Arg was the first human GnRHR mutation described (1), subsequently also reported by other groups (2, 6, 7, 8, 9).
Both patients (subjects II-1 and II-2) were compound heterozygous for Ala171Thr and Gln106Arg. In contrast, their healthy parents were heterozygotes for a single mutation (Fig. 1), carrying either Ala171Thr (father, subject I-1) or Gln106Arg mutation (mother, subject I-2). This finding is in accordance with their clinical status, showing normal sexual development and function as well as normal baseline values for LH, FSH, testosterone, and estradiol.
The Gln106Arg mutation was associated with a silent polymorphism (C to T transition) at nucleotide 453 encoding for the serine residue at position 151 situated in the second intracellular loop in all family members. The segregation of this silent polymorphism with the Gln106Arg mutation was found in all individuals harboring this mutation in our population (de Roux, N., unpublished results) as well as in other French (19) and Brazilian patients (9).
Signal transduction through the mutant GnRHR
The functional relevance of the novel Ala171Thr GnRHR mutation on agonist-induced signal transduction was studied in HEK 293T cells transiently expressing WT or mutated GnRHR. Hormone-induced PLC activity was analyzed in transfected cells by measuring intracellular inositol phosphate accumulation. A significant (5-fold) increase in intracellular inositol phosphate accumulation was observed in the WT GnRHR for 10-7 M GnRH compared with the basal level (Fig. 2). The GnRH concentration required to yield a half-maximal increase in inositol production was 6 x 10–10 M.
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). Thus, mutation Ala171Thr severely impairs ligand-induced GnRHR signaling of the PLC pathway. Ligand binding to Ala171Thr-mutant GnRHR
The loss of Ala171Thr GnRHR signaling may potentially result from altered ligand-receptor interaction. To test this hypothesis, competitive binding experiments were performed in HEK 293T cells transfected with WT or Ala171Thr-mutant GnRHR cDNA. Increasing concentrations of unlabeled GnRH ligand elicited a progressive displacement of labeled [125I]GnRH agonist from WT GnRHR (Fig. 3), indicating specific ligand-receptor binding. The inhibition constant (Ki) for GnRH to the WT receptor calculated from the observed specific binding was 3 x 10-9 M. No specific radioactivity was bound by untransfected HEK 293T cells as the control (data not shown). In striking contrast, cells transfected with Ala171Thr GnRHR did not display any specific [125I]GnRH agonist binding (Fig. 3), suggesting a complete failure of the mutant GnRHR to bind its natural ligand.
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To study cell surface expression of the Ala171Thr mutated receptor, we fused EGFP to the C-terminus of the GnRHR and performed confocal microscopy on transiently transfected HEK293T cells. In Fig. 4, A and B, the fluorescence resulting from the GnRHR-GFP fusion protein is seen as a green signal. Both receptors show similar localization at the plasma membrane of the HEK293T transfected cells. Thus, the Ala171Thr mutation does not disturb cell surface expression of the GnRHR.
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To account for the observed loss of GnRHR function at the structural level, molecular modeling experiments (15) were performed. Models of the three-dimensional Ala171Thr GnRHR protein structure as well as simulation of receptor-specific dynamic conformational changes were developed in the AMBER force field (18).
Figure 5B shows the distribution of the Thr171-Tyr119 (TMH3) distance during the last 400 psec of molecular dynamic simulation of the Ala171Thr mutant. It is clearly demonstrated that in the majority of cases the distance is close to 2 Å, indicating a stable hydrogen bond between the Thr171 hydroxyl group and the backbone carbonyl oxygen of Tyr119. This interaction stabilizes the orientation of the side-chain methyl group toward Leu122. The model with the lowest potential energy (Fig. 5A) shows that the Tyr119 backbone carbonyl forms hydrogen bonds to both backbone NH of Phe123 and the Thr171 side chain. The hydrogen bond between TMH3 and TMH4 introduced by the mutation stabilizes the interaction of Lys121 with Asp98 and Glu90 in TMH2, which is typical for the inactive receptor state and makes the mutant receptor unable to bind the natural hormone ligand.
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Discussion |
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Both affected male patients had severe hypogonadism and gynecomastia at initial presentation. Although gynecomastia indicates imbalance of the normal molar ratio of androgens to estrogen, it is rarely seen in patients with Kallmann syndrome and idiopathic hypogonadotropic hypogonadism (20). In hypogonadotropic patients due to inactivating mutations of the GnRHR gene, gynecomastia appears to be more frequent or more severe than in patients with Kallmann syndrome or patients without GnRHR mutation. Gynecomastia has been described in 7 of 16 male patients carrying GnRHR mutations (5, 8, 21), including the subjects of this report. The genes of the GnRHR and GnRH are expressed in human breast tissue (22), and GnRH agonists inhibit the growth of breast tumor cells in culture (23, 24). It is conceivable that GnRH may have an autocrine or paracrine inhibitory effect on breast cell growth (22) disrupted by the GnRHR mutation.
Patients carrying GnRHR mutations present a wide phenotypic spectrum, ranging from normal testicular volumes (21) and spontaneous spermatogenesis (1) to complete lack of pubertal development (4, 9). Functional in vitro characterization of these mutations distinguishes between substitutions with complete or partial loss of function. Although we have shown that the phenotype may vary between siblings carrying the same GnRHR mutation (2), in vitro GnRHR function is commonly correlated with the clinical severity of hypogonadism. The majority of described patients are compound heterozygotes for two different GnRHR mutations. Comparison of these patients with homozygous patients shows that their phenotype and the response to high doses of GnRH are predominantly determined by the GnRHR variant with the less severe loss of function.
Although many of these mutations have been characterized regarding receptor binding and signal transduction, GnRHR protein structure with naturally occurring mutations has not been studied in detail. In our work we therefore used a molecular modeling approach to analyze the functional effects of a novel human GnRHR mutation. Based on the crystal data of rhodopsin (16) as a template for other GPCR and experimental results of the GnRHR (15), we constructed a three-dimensional molecular model of the mutant Ala171Thr GnRHR. Our data predict the introduction of a stable hydrogen bond between Thr171 in TMH4 and Tyr119 in TMH3. Ligand binding to the GnRHR involves interaction between His2 of GnRH and Lys121 in TMH3 (15, 25). Interhelical interaction of Lys121 with Asp98 in TMH2 contributes to the structure of the ligand binding pocket, while Asp98 stabilizes the hormone-receptor complex by interaction with His2 (26). Hormone binding requires movement of the side-chain of Lys121 from interactions with residues Glu90 and Asp98 in TMH2 in the inactive state toward TMH7 to interact with the agonist (27). In our molecular model, these conformational changes in TMH3 are prevented by introduction of the stable hydrogen bond between TMH4 and TMH3 in the mutant Ala171Thr receptor. This hydrogen bond stabilizes the interactions of Lys121 with residues in TMH2, which is typical for the inactive receptor state. As further helix-helix interactions of TMH3 are unlikely in the inactive state (16), the fixed position between TMH4 and TMH3 by Thr171 thus prevents conformational mobility of TMH3 necessary for ligand binding and GnRHR activation.
This concept was confirmed by our experimental data, showing loss of ligand binding and subsequent loss of G protein-coupled activation of PLC of the mutant Ala171Thr GnRHR. Although not part of the putative peptide-binding site, residue Ala171 thus seems critical for agonist binding. Similar results were reported for another naturally occurring mutation of the GnRHR gene located in TMH4, substitution S168R, that completely abrogates ligand binding and signal transduction, although this residue is not directly involved in ligand binding (4). It is noteworthy that residue Ala171 is highly conserved in GnRHRs within species and other human GPCR (11). This unpolar and uncharged amino acid may contribute to the flexibility of the receptor for conformational changes between different intermediate receptor states. Recently, evidence was shown that interhelical hydrogen bonds may stabilize the inactive state of glycoprotein hormone receptors (28). Additional hydrogen bonds within TMH6 or between TMH6 and TMH7 of the TSH receptor fix the receptor in the inactive state (29). The release of this hydrogen bond leads to constitutive activation of the TSH receptor as well as the LH receptor (29).
Gene mutations may potentially affect GnRHR function through a variety of posttranscriptional molecular mechanisms, including decreased mRNA stability, protein misfolding or instability, and deficient receptor targeting to the cell surface. We have used WT or mutant GnRHRs fused at the C terminus with GFP to study cell surface expression of the mutated receptor. The fluorescence intensity and pattern were similar in cells transfected with WT and Ala171Thr mutant. The overlay of the GFP labeling with the fluorescent Con A cell surface labeling indicates normal cell surface expression of the mutated receptor. A summary of the effects of various GnRHR mutations on its membrane localization has recently been published (30).
Our model supports the concept of sequential ligand binding and conformational mobility until the receptor has been stabilized in the active state (31). In this model the highest affinity for the ligand is observed in the active state. Therefore, all mutations impeding conformational changes in the receptor toward the active state should decrease the binding affinity. The movement of TMH3 was first proposed for rhodopsin and then extended to other GPCR (32). An additional hydrogen bond between TMH3 and TMH4 in the mutant Ala171Thr GnRHR may cause a shift of the equilibrium toward the inactive state.
In conclusion, we describe a novel, naturally occurring GnRHR mutation, Ala171Thr, and propose a mechanism explaining how agonist binding and receptor signaling are prevented by residues distant from the hormone ligand binding pocket. Our data emphasize the importance of dynamic conformational changes in the GnRH ligand-receptor interaction. Receptor structure modeling and functional data contribute to a comprehensive molecular view of GnRHR mutations as a cause of familial hypogonadotropic hypogonadism.
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Acknowledgments |
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Footnotes |
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Abbreviations: ECL, Extracellular loop; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; GnRHR, GnRH receptor; GPCR, G protein-coupled receptor; PLC, phospholipase C; TMH, transmembrane helix; WT, wild type.
Received January 7, 2002.
Accepted January 7, 2003.
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