This Article Full Text Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Maya-Núñez, G. Articles by Méndez, J. P. Search for Related Content PubMed PubMed Citation Articles by Maya-Núñez, G. Articles by Méndez, J. P.

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.

This article has been cited by other articles:

				P. M. Conn, A. Ulloa-Aguirre, J. Ito, and J. A. Janovick G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo Pharmacol. Rev., September 1, 2007; 59(3): 225 - 250. [Abstract] [Full Text] [PDF]

				T. T. Leskela, P. M. H. Markkanen, E. M. Pietila, J. T. Tuusa, and U. E. Petaja-Repo Opioid Receptor Pharmacological Chaperones Act by Binding and Stabilizing Newly Synthesized Receptors in the Endoplasmic Reticulum J. Biol. Chem., August 10, 2007; 282(32): 23171 - 23183. [Abstract] [Full Text] [PDF]

				P. E. Knollman, J. A. Janovick, S. P. Brothers, and P. M. Conn Parallel Regulation of Membrane Trafficking and Dominant-negative Effects by Misrouted Gonadotropin-releasing Hormone Receptor Mutants J. Biol. Chem., July 1, 2005; 280(26): 24506 - 24514. [Abstract] [Full Text] [PDF]

				C. Castro-Fernandez, G. Maya-Nunez, and P. M. Conn Beyond the Signal Sequence: Protein Routing in Health and Disease Endocr. Rev., June 1, 2005; 26(4): 479 - 503. [Abstract] [Full Text] [PDF]

				A. Leanos-Miranda, A. Ulloa-Aguirre, J. A. Janovick, and P. M. Conn In Vitro Coexpression and Pharmacological Rescue of Mutant Gonadotropin-Releasing Hormone Receptors Causing Hypogonadotropic Hypogonadism in Humans Expressing Compound Heterozygous Alleles J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 3001 - 3008. [Abstract] [Full Text] [PDF]

				S. P. Brothers, A. Cornea, J. A. Janovick, and P. M. Conn Human Loss-of-Function Gonadotropin-Releasing Hormone Receptor Mutants Retain Wild-Type Receptors in the Endoplasmic Reticulum: Molecular Basis of the Dominant-Negative Effect Mol. Endocrinol., July 1, 2004; 18(7): 1787 - 1797. [Abstract] [Full Text] [PDF]

				A. Ulloa-Aguirre, J. A. Janovick, A. Leanos-Miranda, and P. M. Conn Misrouted cell surface GnRH receptors as a disease aetiology for congenital isolated hypogonadotrophic hypogonadism Hum. Reprod. Update, March 1, 2004; 10(2): 177 - 192. [Abstract] [Full Text] [PDF]

				S. P. Brothers, J. A. Janovick, and P. M. Conn Unexpected Effects of Epitope and Chimeric Tags on Gonadotropin-Releasing Hormone Receptors: Implications for Understanding the Molecular Etiology of Hypogonadotropic Hypogonadism J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 6107 - 6112. [Abstract] [Full Text] [PDF]

				A. Leanos-Miranda, A. Ulloa-Aguirre, T. H. Ji, J. A. Janovick, and P. M. Conn Dominant-Negative Action of Disease-Causing Gonadotropin-Releasing Hormone Receptor (GnRHR) Mutants: A Trait That Potentially Coevolved with Decreased Plasma Membrane Expression of GnRHR in Humans J. Clin. Endocrinol. Metab., July 1, 2003; 88(7): 3360 - 3367. [Abstract] [Full Text] [PDF]

				J. A. Janovick, M. Goulet, E. Bush, J. Greer, D. G. Wettlaufer, and P. M. Conn Structure-Activity Relations of Successful Pharmacologic Chaperones for Rescue of Naturally Occurring and Manufactured Mutants of the Gonadotropin-Releasing Hormone Receptor J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 608 - 614. [Abstract] [Full Text] [PDF]

				G. Y. Bedecarrats, K. D. Linher, and U. B. Kaiser Two Common Naturally Occurring Mutations in the Human Gonadotropin-Releasing Hormone (GnRH) Receptor Have Differential Effects on Gonadotropin Gene Expression and on GnRH-Mediated Signal Transduction J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 834 - 843. [Abstract] [Full Text] [PDF]

				J. A. Janovick, G. Maya-Nunez, and P. M. Conn Rescue of Hypogonadotropic Hypogonadism-Causing and Manufactured GnRH Receptor Mutants by a Specific Protein-Folding Template: Misrouted Proteins as a Novel Disease Etiology and Therapeutic Target J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3255 - 3262. [Abstract] [Full Text] [PDF]