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Molecular Mechanism of Action of Pharmacoperone Rescue of Misrouted GPCR Mutants: The GnRH Receptor

Oregon Health & Science University (J.A. J., A.C., P.M.C.), Oregon National Primate Research Center, Beaverton, Oregon 97006; Merck Research Laboratories (A.P., R.M.), Department of Molecular Systems, Rahway, New Jersey 07065; and Merck Research Laboratories (M.T.G., M.D.A., T.S.R.), Department of Drug Design and Optimization, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: P. Michael Conn, Oregon National Primate Research Center/Oregon Health Sciences University, 505 North West 185th Avenue, Beaverton, Oregon 97006. E-mail: connm{at}ohsu.edu.

The human GnRH receptor (hGnRHR), a G protein-coupled receptor, is a useful model for studying pharmacological chaperones (pharmacoperones), drugs that rescue misfolded and misrouted protein mutants and restore them to function. This technique forms the basis of a therapeutic approach of rescuing mutants associated with human disease and restoring them to function. The present study relies on computational modeling, followed by site-directed mutagenesis, assessment of ligand binding, effector activation, and confocal microscopy. Our results show that two different chemical classes of pharmacoperones act to stabilize hGnRHR mutants by bridging residues D⁹⁸ and K¹²¹. This ligand-mediated bridge serves as a surrogate for a naturally occurring and highly conserved salt bridge (E⁹⁰–K¹²¹) that stabilizes the relation between transmembranes 2 and 3, which is required for passage of the receptor through the cellular quality control system and to the plasma membrane. Our model was used to reveal important pharmacophoric features, and then identify a novel chemical ligand, which was able to rescue a D⁹⁸ mutant of the hGnRHR that could not be rescued as effectively by previously known pharmacoperones.