Involvement of amino acids flanking Glu^7.32 of the Gonadotropin-releasing hormone receptor in the selectivity of antagonists

The Glu/Asp^7.32 residue in extracellular loop 3 of the mammalian type-I gonadotropin-releasing hormone receptor (GnRHR) interacts with Arg⁸ of GnRH-I, conferring preferential ligand selectivity for GnRH-I over GnRH-II. Previously, we demonstrated that the residues (Ser and Pro) flanking Glu/Asp^7.32 also play a role in the differential agonist selectivity of mammalian and non-mammalian GnRHRs. In this study, we examined the differential antagonist selectivity of wild type and mutant GnRHRs in which the Ser and Pro residues were changed. Cetrorelix, a GnRH-I antagonist, and Trptorelix-2, a GnRH-II antagonist, exhibited high selectivity for mammalian type-I and non-mammalian GnRHRs, respectively. The inhibitory activities of the antagonists were dependent on agonist concentration and subtype. Rat GnRHR in which the Ser-Glu-Pro (SEP) motif was changed to Pro-Glu-Val (PEV) or Pro-Glu-Ser (PES) had increased sensitivity to Trptorelix-2 but decreased sensitivity to Cetrorelix. Mutant bullfrog GnRHR-1 with the SEP motif had the reverse antagonist selectivity, with redueed sensitivity to Trptorelix-2 but increased sensitivity to Cetrorelix. These findings indicate that the residues flanking Glu^7.32 are important for antagonist as well as agonist selectivity.