title: Identification of Phe313 of the gonadotropin-releasing hormone (GnRH) receptor as a site critical for the binding of nonpeptide GnRH antagonists.
abstract: 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.
authors.affiliation: Department of Endocrinology and Chemical Biology, Merck Research Laboratories, Rahway, New Jersey 07065, USA. jisong cui@merck.com
authors.forename: J
authors.initials: J
authors.lastname: Cui
authors.forename: R G
authors.initials: RG
authors.lastname: Smith
authors.forename: G R
authors.initials: GR
authors.lastname: Mount
authors.forename: J L
authors.initials: JL
authors.lastname: Lo
authors.forename: J
authors.initials: J
authors.lastname: Yu
authors.forename: T F
authors.initials: TF
authors.lastname: Walsh
authors.forename: S B
authors.initials: SB
authors.lastname: Singh
authors.forename: R J
authors.initials: RJ
authors.lastname: DeVita
authors.forename: M T
authors.initials: MT
authors.lastname: Goulet
authors.forename: J M
authors.initials: JM
authors.lastname: Schaeffer
authors.forename: K
authors.initials: K
authors.lastname: Cheng
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004285:Dogs; D007987:Gonadotropin-Releasing Hormone / Q000037:antagonists & inhibitors*; D006727:Hormone Antagonists / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010649:Phenylalanine / Q000737:chemistry*; D011485:Protein Binding; D015363:Quinolones / Q000737:chemistry; D011966:Receptors, LHRH / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship
chemical_list: D006727:Hormone Antagonists; D009842:Oligopeptides; D015363:Quinolones; D011966:Receptors, LHRH; D011993:Recombinant Fusion Proteins; D007987:Gonadotropin-Releasing Hormone; D010649:Phenylalanine; D007930:Leucine