Conserved aromatic residues in the transmembrane region VI of the V_1a vasopressin receptor differentiate agonist vs. antagonist ligand binding

Abstract

Despite their opposite effects on signal transduction, the nonapeptide hormone arginine-vasopressin (AVP) and its V 1a receptor-selective cyclic peptide antagonist d(CH 2 ) 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(CH 2 ) 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.Keywords: vasopressin receptors; antagonist-binding sites; signal transduction; site-directed mutagenesis; three-dimensional model. G protein-coupled receptors (GPCR) constitute an extremely important target in medicinal chemistry. Thus, there is considerable interest in the docking of the natural ligands and their analogues to these receptors, both for rational drug design and for a better understanding of their functional architecture. To date, the structures of binding sites of peptide hormones have been elucidated primarily by a combination of molecular modeling hypotheses and validation by site-directed mutagenesis analysis (reviewed in [1±3]). Such an approach was applied to the receptors specific for the mammalian hormones arginine-vasopressin (AVP) and oxytocin (OT), which constitute typical GPCR. Indeed, a detailed 3D model of bound AVP to the rat V 1a receptor has been developed and then experimentally verified [4]. Interestingly, AVP binds to its receptor in a hydrophobic pocket of 15±20 A Ê defined by the transmembrane regions (TMs), in a position similar to that of the cationic neurotransmitters [5]. The residues responsible for the binding of AVP are highly conserved and the agonist-binding site was proposed to be common through the different AVP/OT receptor subtypes. Molecular modeling of the V 2 receptor, docking of the hormone and peptide structu...

Vasopressin V1a and V1b Receptors: From Molecules to Physiological Systems

“…(F225) in the TM5 directly participates in the binding of the V1a receptor antagonist SR49059 (480). In the TM6, the mutation of three aromatic residues of the human V1a receptor, Trp-304 (W304), Phe-307 (F307), and Phe-308 (F308), reduced affinity for d(CH 2 ) 5 [Tyr(Me) 2 ]AVP and SR49059, but not for AVP binding, suggesting that these amino acids are necessary for agonist/antagonist discrimination (99). The hydrophilic residues Gln-108 (Q108), Lys-128 (K128), and Gln-185 (Q185) in the TM2, TM3, and TM4 of the human V1a receptor, respectively, are needed for high-affinity binding of both agonists and antagonists.…”

“…The hydrophilic residues Gln-108 (Q108), Lys-128 (K128), and Gln-185 (Q185) in the TM2, TM3, and TM4 of the human V1a receptor, respectively, are needed for high-affinity binding of both agonists and antagonists. The residues Thr-333 (T333) and Ala-334 (A334) in the ECL3 near the TM7 control the binding selectivity of the V1a/V2 receptors for both nonpeptide and cyclic peptide antagonists (99).…”

Probing the Existence of G Protein-Coupled Receptor Dimers by Positive and Negative Ligand-Dependent Cooperative Binding

“…COS-7 cells were transiently transfected by electroporation as described previously (Cotte et al, 2000). In brief, electroporation was performed with 1 g of a pRK5-containing hemagglutinin (HA)-tagged or 6-histidine (6His)-tagged human V 1a receptor and 9 g of empty vector.…”

“…Third, we observed that the estimation of the affinity of a ligand for a receptor can vary depending on the method of its evaluation. As reported in Table 2, when considering the classic model of ligand binding, vasopressin had a K d value of 0.7 Ϯ 0.2 nM when estimated by saturation and a K i value of 3.4 Ϯ 1.1 nM for the human vasopressin V 1a receptor (Cotte et al, 2000) expressed in COS-7 cells, when measured by competition experiments with 125 I-HO-LVA as radiolabeled ligand (Table 2). This discrepancy can be greater in some cases.…”

“…This discrepancy can be greater in some cases. For instance, when experiments are performed on the Q185A V 1a mutant, which displays a loss of affinity for agonists and antagonists (Cotte et al, 2000), the K d and K i values are 46 Ϯ 7 nM and 810 Ϯ 148 nM, respectively (Table 2). Such discrepancies have also been observed for two other mutations on rat vasopressin V 1a receptor, K128A and Q131A, which have been shown to be involved in the binding site of ligands.…”