Abstract

Mammalian gonadotropin-releasing hormone (GnRH) receptors preferentially bind mammalian GnRH, which has Arg in position eight. The Glu^7.32(301) residue, which determines selectivity of the mouse GnRH receptor for Arg⁸-containing GnRH, is Asp^7.32(302) in the human GnRH receptor. We have confirmed that Asp^7.32(302) confers selectivity of the human GnRH receptor for Arg⁸ of GnRH and investigated the mechanism of this specificity using site-directed mutagenesis and ligand modification. We find that although Arg⁸ and Asp^7.32(302) are required for high-affinity binding of GnRH, conformationally constrained peptides, with d-amino acid substitutions in position six or with a 6,7 γ-lactam, bind the human GnRH receptor with high affinity, which is independent of the presence of Asp^7.32(302) in the receptor or Arg⁸ in the ligand. The ability of the ligand constraints to compensate for the absence of both Arg⁸ and Asp^7.32(302) indicates that these residues both have roles in stabilizing a high affinity ligand conformation and that their roles are complementary. This suggests that the Arg⁸ and Asp^7.32(302) side chains interact to induce a high affinity conformation of native GnRH. Thus, Asp^7.32(302) of the human GnRH receptor determines selectivity for mammalian GnRH by its ability to induce a high affinity conformation of its native ligand. However, this initial interaction seems not to contribute to the final ligand-receptor complex. We propose that Arg⁸ interacts transiently with Asp^7.32(302) to induce a high-affinity ligand conformation of GnRH, which then interacts with a binding pocket that is common for both constrained and unconstrained analogs of GnRH.