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Mutation Ala¹⁷¹Thr Stabilizes the Gonadotropin-Releasing Hormone Receptor in Its Inactive Conformation, Causing Familial Hypogonadotropic Hypogonadism

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.

Mutations of the GnRH receptor have been recognized as a cause of familial gonadotropin deficiency. We here identify and functionally characterize a novel human GnRH receptor variant bearing an Ala¹⁷¹Thr substitution located at transmembrane helix 4 (TMH4). The affected kindred displays severe hypogonadotropic hypogonadism. After in vitro expression in human embryonic kidney 293T cells, the Ala¹⁷¹Thr mutant GnRH receptor exhibited a lack of phospholipase C activity in signal transduction. Specific receptor binding of ¹²⁵I-labeled GnRH ligand was undetectable in Ala¹⁷¹Thr GnRH receptor-transfected cells. Molecular modeling and dynamic simulation of the Ala¹⁷¹Thr GnRH receptor suggests the introduction of a stable hydrogen bond between residue Thr¹⁷¹ and Tyr¹¹⁹ side-chains at a distance of 2 Å. Although spatially distant from the GnRH ligand-binding site, this hydrogen bond impedes conformational mobility of the TMH3 and TMH4 domains required for sequential ligand binding and receptor activation, thus stabilizing the GnRH receptor in its inactive conformation. Receptor structure modeling and functional data provide a comprehensive molecular view of how mutation Ala¹⁷¹Thr causes a complete loss of GnRH receptor function.

This work was supported in part by a European Society of Pediatric Endocrinology Research Fellowship (to B.K.) sponsored by Novo Nordisk A/S, and by Zentaris AG (Frankfurt, Germany; to R.K.).

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.

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