Conserved aromatic residues in the transmembrane region VI of the V1a vasopressin receptor differentiate agonist vs. antagonist ligand binding

Despite their opposite effects on signal transduction, the nonapeptide hormone arginine-vasopressin (AVP) and its V-1a receptor-selective cyclic peptide antagonist d(CH2)(5)[Tyr(Me)2]AVP display homologous primary structures, differing only at residues 1 and 2. These structural similarities led us to hypothesize that both ligands could interact with the same binding pocket in the V-1a receptor. To determine receptor residues responsible for discriminating binding of agonist and antagonist ligands, we performed site-directed mutagenesis of conserved aromatic and hydrophilic residues as well as nonconserved residues, all located in the transmembrane binding pocket of the V-1a receptor. Mutation of aromatic residues of transmembrane region VI (W304, F307, F308) reduced affinity for the d(CH2)(5)[Tyr(Me)2]AVP and markedly decreased affinity for the unrelated strongly hydrophobic V-1a-selective nonpeptide antagonist SR 49059. Replacement of these aromatic residues had no effect on AVP binding, but increased AVP-induced coupling efficacy of the receptor for its G protein. Mutating hydrophilic residues Q108, K128 and Q185 in transmembrane regions II, III and IV, respectively, led to a decrease in affinity for both agonists and antagonists. Finally, the nonconserved residues T333 and A334 in transmembrane region VII, controlled the V-1a/V-2 binding selectivity for both nonpeptide and cyclic peptide antagonists. Thus, because conserved aromatic residues of the V-1a receptor binding pocket seem essential for antagonists and do not contribute at all to the binding of agonists, we propose that these residues differentiate agonist vs. antagonist ligand binding.