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Molecular Basis of Hypogonadotropic Hypogonadism: Restoration of Mutant (E⁹⁰K) GnRH Receptor Function by a Deletion at a Distant Site

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.

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