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Identification of Phe³¹³ of the Gonadotropin-Releasing Hormone (GnRH) Receptor as a Site Critical for the Binding of Nonpeptide GnRH Antagonists

Department of Endocrinology and Chemical Biology (J.C., J-L.L., J.Y., J.M.S., K.C.) Department of Medicinal Chemistry (T.F.W., R.J.D., M.T.G.) Department of Molecular Systems (S.B.S.) Merck Research Laboratories Rahway, New Jersey 07065
Huffington Center of Aging (R.G.S.) Houston, Texas 77030
Temple University (G.M.) School of Medicine Philadelphia, Pennsylvania 19140

The dog GnRH receptor was cloned to facilitate the identification and characterization of selective nonpeptide GnRH antagonists. The dog receptor is 92% identical to the human GnRH receptor. Despite such high conservation, the quinolone-based nonpeptide GnRH antagonists were clearly differentiated by each receptor species. By contrast, peptide antagonist binding and functional activity were not differentiated by the two receptors. The basis of the differences was investigated by preparing chimeric receptors followed by site-directed mutagenesis. Remarkably, a single substitution of Phe³¹³ to Leu³¹³ in the dog receptor explained the major differences in binding affinities and functional activities. The single amino acid replacement of Phe³¹³ of the human receptor with Leu³¹³ resulted in a 160-fold decrease of binding affinity of the nonpeptide antagonist compound 1. Conversely, the replacement of Leu³¹³ of the dog receptor with Phe³¹³ resulted in a 360-fold increase of affinity for this compound. These results show that Phe³¹³ of the GnRH receptor is critical for the binding of this structural class of GnRH antagonists and that the dog receptor can be "humanized" by substituting Leu for Phe. This study provides the first identification of a critical residue in the binding pocket occupied by nonpeptide GnRH antagonists and reinforces cautious extrapolation of ligand activity across highly conserved receptors

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