Species Selectivity of Nonpeptide Antagonists of the Gonadotropinreleasing Hormone Receptor Is Determined by Residues in Extracellular Loops II and III and the Amino Terminus

Abstract

Efforts to develop orally available gonadotropin-releasing hormone (GnRH) receptor antagonists have led to the discovery of several classes of potent nonpeptide antagonists. Here we investigated molecular interactions of three classes of nonpeptide antagonists with human, rat, and macaque GnRH receptors. Although all are high affinity ligands of the human receptor (K i <5 nM), these compounds show reduced affinity for the macaque receptor and bind only weakly (K i >1 M) to the rat receptor. To identify residues responsible for this selectivity, a series of chimeric receptors and mutant receptors was constructed and evaluated for nonpeptide binding. Surprisingly, 4 key residues located in the amino terminus (Met-24) and extracellular loops II (Ser-203, Gln-208) and III (Leu-300) of the GnRH receptor appear to be primarily responsible for species-selective binding. Comparisons of reciprocal mutations suggest that these may not be direct contacts but rather may be involved in organizing extracellular portions of the receptor. These data are novel because most previous reports of residues involved in binding of nonpeptide ligands to peptide-activated G protein-coupled receptors, including the GnRH receptor as well as mono-amine receptors, have identified binding sites in the transmembrane regions. Gonadotropin-releasing hormone (GnRH)1 is a linear decapeptide amide, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 , that regulates the reproductive endocrine axis in both male and female vertebrates (1, 2). The GnRH receptor (Gn-RHR) is a member of the G protein-coupled receptor superfamily (3). Its activation stimulates phospholipase C and phosphatidyl inositol turnover by coupling to the GTP-binding proteins G q and G 11 (4). Stimulation of pituitary GnRH receptors by hypothalamic GnRH released into the pituitary portal circulation results in secretion of the gonadotropins, luteinizing hormone, and follicle-stimulating hormone into the peripheral circulation. The gonadotropins in turn regulate the production of steroids and gametes by the gonads. Receptors for GnRH have been cloned from a wide range of species (5), and numerous studies have been reported elucidating key residues in the receptor responsible for peptide binding and signal transduction (5).Peptide drugs that inhibit the action of GnRH have proven useful in regulating the hypothalamic-pituitary-gonadal axis and are now routinely used for ablation of gonadal steroids. This approach has proven effective in the management of endometriosis, prostate cancer, uterine fibroids, breast cancer, and other conditions, including infertility (6). However, the requirement for daily injection or implantation of long acting depots has prompted a number of groups to attempt to develop orally active, nonpeptide antagonists (for a review, see Ref. 7). These efforts have been hindered in part by the species selectivity that is observed for most nonpeptide GnRH antagonists. Cho et al. (8) reported synthesis of T-98475, a nonpeptide GnRH antagonist with high affinity to the hu...

Expression and distribution of GnRH, LH, and FSH and their receptors in gastrointestinal tract of man and rat

“…The GnRH-R and LH-R both have more than 80% amino acid homology between rat and man [24,25]. LH-R has been demonstrated in submucous and myenteric neurons in small and large intestine, glia cells and endothelial cells in man [17], while in rat LH-R-IR was exclusively confined to enteric neurons [15].…”

Identification of a Novel Allosteric Binding Site in the CXCR2 Chemokine Receptor

“…Translating the results of animal studies to humans, however, can be hampered by differences between receptor orthologs. Species-related differences in the sequences of protein targets ("natural mutants") have previously been used to identify the molecular determinants of ligand binding to GPCRs (Reinhart et al, 2004;Lim et al, 2010;Milligan, 2011). Species differences have also been observed for CXCR2.…”

Molecular Determinants of Ligand Binding to H4R Species Variants

“…Therefore, to study therapeutic effects of H 4 R ligands in animal models of disease, it is important to characterize the H 4 R of the corresponding species. For several GPCRs, including the H 3 R, significant species differences are known and have seriously hampered drug discovery efforts (Oksenberg et al, 1992;Maconi et al, 2002;Reinhart et al, 2004;Hancock, 2006). Various species orthologs of H 4 R were promptly cloned based on their homology to the human H 4 R sequence (Oda et al, 2000), including those of mouse, rat, guinea pig, pig, monkey (Macaca fascilularis), and dog (Liu et al, 2001b;Oda et al, 2002Oda et al, , 2005Jiang et al, 2008).…”