title: Four naturally occurring mutations in the human GnRH receptor affect ligand binding and receptor function.
abstract: In the present study, we performed functional analyses of four mutations in the human GnRH receptor (GnRHR) gene, identified in patients with idiopathic hypogonadotropic hypogonadism. These mutations result in amino acid substitutions in the extracellular N-terminal domain (Thr32Ile), second extracellular loop (Cys200Tyr), third intracellular loop (Leu266Arg) and sixth transmembrane helix (Cys279Tyr). Immunocytochemical analysis of cells transfected with HA-tagged GnRHR constructs revealed that all four mutant receptors were present on the cell surface. However, all four mutant receptors failed to exhibit measurable specific GnRH binding and, except for Thr32Ile, any significant inositol phosphate accumulation after GnRH stimulation. In addition, Leu266Arg and Cys279Tyr receptors were unable to stimulate gonadotropin subunit or GnRHR gene promoter activity in response to GnRH. Interestingly, the Cys200Tyr mutant was able to stimulate gonadotropin subunit and GnRHR promoter activity, albeit with a higher EC(50) and a markedly reduced maximal response compared to wild type receptor. The Thr32Ile mutant was also able to stimulate gonadotropin subunit and GnRHR promoters, but with a further significant increase in EC(50). Similarly, this mutant partially retained the ability to activate extracellular signal-regulated kinase 1 and stimulate CRE-luciferase activity with an identical shift in EC(50). Taken together, the studies suggest that the Thr32Ile mutation reduces hGnRHR function primarily by reducing ligand binding affinity, and the Cys200Tyr mutation reduces cell surface receptor expression. All four amino acid substitutions interfered with ligand binding, and affected signal transduction and stimulation of gonadotropin and GnRHR gene expression in response to GnRH.
authors.affiliation: Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 221 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Grégoy Y
authors.initials: GY
authors.lastname: Bédécarrats
authors.forename: Katja D
authors.initials: KD
authors.lastname: Linher
authors.forename: Jo Ann
authors.initials: JA
authors.lastname: Janovick
authors.forename: Milena
authors.initials: M
authors.lastname: Beranova
authors.forename: Faiza
authors.initials: F
authors.lastname: Kada
authors.forename: Stephanie B
authors.initials: SB
authors.lastname: Seminara
authors.forename: P
authors.initials: P
authors.lastname: Michael Conn
authors.forename: Ursula B
authors.initials: UB
authors.lastname: Kaiser
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D006062:Gonadotropins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000032:analysis; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011966:Receptors, LHRH / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D006062:Gonadotropins; D007295:Inositol Phosphates; D008024:Ligands; D008565:Membrane Proteins; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D008156:Luciferases; D020928:Mitogen-Activated Protein Kinases