Conserved aromatic residues in the transmembrane region VI of the V-1a vasopressin receptor differentiate agonist vs. antagonist ligand binding

Despite their opposite effects on signal transduction, the nonapeptide horm one arginine-vasopressin (AVP) and its V-1a receptor-selective cyclic pepti de antagonist d(CH2)(5)[Tyr(Me)2]AVP display homologous primary structures, differing only at residues 1 and 2. These structural similarities led us t o hypothesize that both ligands could interact with the same binding pocket in the V-1a receptor. To determine receptor residues responsible for discr iminating binding of agonist and antagonist ligands, we performed site-dire cted mutagenesis of conserved aromatic and hydrophilic residues as well as nonconserved residues, all located in the transmembrane binding pocket of t he 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 mark edly decreased affinity for the unrelated strongly hydrophobic V-1a-selecti ve nonpeptide antagonist SR 49059. Replacement of these aromatic residues h ad no effect on AVP binding, but increased AVP-induced coupling efficacy of the receptor for its G protein. Mutating hydrophilic residues Q108, K128 a nd Q185 in transmembrane regions II, III and IV, respectively, led to a dec rease in affinity for both agonists and antagonists. Finally, the nonconser ved 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 po cket seem essential for antagonists and do not contribute at all to the bin ding of agonists, we propose that these residues differentiate agonist vs. antagonist ligand binding.