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.

G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo

“…), we also found (Janovick et al, 2007b) two amino acid substitutions at positions 54 and 300 (in the rhesus macaque and human, respectively) that affect the affinity of ligand binding without a substantial effect on the B max of the protein expressed. Because of the position of the amino acid 300 in the receptor molecule, it is easier to imagine that a change at this position (ECL3) might alter ligand affinity by direct interaction with the ligand, although this appears close to a binding pocket (Cui et al, 2000). This case is harder to make with 236 position 54 located near the cytoplasmic side of the first TM helix (Fig.…”

“…Nevertheless, the fact that agonists, antagonists, and inverse agonists may exhibit distinct selectivities toward the active and the inactive conformation of the receptor suggests that competitive antagonism may occur without any overlap with agonist binding sites (Samama et al, 1993). Binding of nonpeptide antagonists is less known; nevertheless, studies on a few nonpeptide small, quinolone-and thienopyridine-based GnRH antagonists indicate that their binding sites partially overlap GnRHR residues important for GnRH binding (Cho et al, 1998;Cui et al, 2000); in addition, Phe 313 has been proposed as a site critical for the binding of this class of antagonists to the human GnRHR (Cui et al, 2000).…”

Specializations of a G‐protein‐coupled receptor that appear to aid with detection of frequency‐modulated signals from its ligand

“…We also describe amino acid substitutions (positions 54 and 300 in the rhesus and human, respectively) that affect the binding affinity without a substantial effect on the B max of the protein expressed. Because of the position of the amino acid 300 in the molecule, it is easier to imagine that a change at this position (ECL3) alters affinity by direct interaction with the ligand, although this appears close to a binding pocket (21). This case is harder to make with position 54 located near the cytoplasmic side of the first TMS (Fig.…”

Trafficking and quality control of the gonadotropin releasing hormone receptor in health and disease

“…Glu 90 is conserved in mammals, but Val 90 is present in eels, reptiles, avians and flies and perciform fish and the residue is Met 90 in trout and catfish. Another apparent point of contact for quinolone pharmacoperones is the highly conserved Phe 313 (Leu 313 in canines and equines) which has already been reported to be the basis of the inability of these species to recognize these drugs (Cui et al, 2000). It is certainly possible that this could result in further structural stability to the receptor.…”