Docking of Linear Peptide Antagonists into the Human V1a Vasopressin Receptor

Abstract

Phpa-LVA (covalent attachment to transmembrane domain VII), threedimensional models of the antagonist-bound receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V 1a receptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone vasopressin.Over the past few years, interest in locating ligand-binding sites in G protein-coupled receptors has increased exponentially. Indeed, identification of these binding sites is of prime importance both for a better understanding of the structure and the function of the G protein-coupled receptor superfamily and for facilitating rational design of potential therapeutic agents. Extensive mutational analysis and receptor three-dimensional molecular modeling have led to valuable information concerning "small ligand" and peptide/protein ligand receptor-binding sites (for review, see Refs. 1-6).In 1995, we published the mapping of arginine-vasopressin (AVP) 1 -binding site in the V 1a receptor subtype and described a major localization within transmembrane regions (TMR) in a position equivalent to that defined for the cationic neurotransmitters (7). Because all receptor residues potentially interacting with AVP are conserved in the different members of the AVP/oxytocin (OT) receptor family, we proposed that the binding pocket identified in the V 1a might be common to V 2 , V 1b , and OT receptor subtypes. Extracellular residues responsible for receptor-selective and species-selective binding have also been identified (8 -10). Unfortunately, these first analyses of AVP receptor structure/function relationships did not provide much information on AVP receptor antagonist-binding domains (Refs. 11 and 12, and for review see Ref. 13). The photoaffinity labeling technique is an essential complement to modeling and mutagenesis approaches and allows direct unambiguous identification of the contact regions between a receptor and its specific photoactivatable ligands (for review see Ref. 14). At the present time, very few photoaffinity labeling studies have led to the direct determination of labeled amino acid residues in peptide G protein-coupled receptor; remarkable results with bovine V 2 receptor (15), human NK1 tachykinin receptor (16), and rat type A cholecystokinin receptor (17) allowed identification of covalently labeled residues with photoactivatable agonist analogues of AVP, substance P, and cholecystokinin, respectively.Very recently, a first radioiodinated photoreactive linear peptide antagonist has been used in our laboratory to photolabel the human and rat V 1a receptors (12,18,19). Our results have clearly indicated that covalent attachment of the [ 125 I]3N 3 Phpa-LVA occurs in a restricted domain of the human receptor including TMR VII. Based both on this photolabeling result and on th...

Molecular basis of ligand binding and receptor activation in the oxytocin and vasopressin receptor family

“…This model has been extensively tested by means of site-directed mutagenesis (Mouillac et al 1995) and successfully used to identify the receptor residues responsible for selective agonist binding affinity (Chini et al 1995) and efficacy (Chini et al 1996). It has also been recently exploited to explain the pharmacological behaviour of the vasopressin 2 (V,) receptor mutants responsible for clinical forms of congenital nephrogenic diabetes insipidus (Ala et al Albertazzi et al 2000), and to identify the putative docking site for peptidergic linear antagonists in the VIa receptor subtype (Phalipou et al 1999). …”

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

“…1) in the human V 1a receptor, we used 3D models of the rat and human AVP receptors [4,16,17,21]. These models suggest that hydrophobic residues potentially involved in the ligand binding are aromatic and localized at the bottom of the receptor binding pocket.…”

“…Studies of chimeric OT/V 2 receptors have shown that TM VII contributes to the binding of an OT antagonist [15]. To identify the receptor domains that contribute to the binding of V 1a antagonists, we developed two radioiodinated photosensitive ligands used to covalently label the human V 1a receptor [16,17]. Combining photoaffinity labeling and receptor fragmentation, we clearly demonstrated that covalent attachment of the antagonists occurs in TM VII and in the first extracellular loop of the receptor, respectively.…”

“…A rapid interconversion between different b-turn geometries and multiple slowly exchanging conformations were observed for AVP and for the antagonists, respectively. Taking into account these structural data and our previous findings describing that the binding sites for peptide agonists/linear peptide antagonists partially overlap in the receptor TM core [4,16,17], we hypothesized that AVP and its cyclic peptide antagonist d(CH 2 ) 5 [Tyr(Me)2]AVP could occupy the same transmembrane binding pocket in the V 1a receptor.…”

“…In the present study, based on the 3D models of the rat and human AVP receptors [4,16,17,21], and in order to determine residues of the human V 1a receptor able to differentiate binding of AVP and d(CH 2 ) 5 [Tyr(Me)2]AVP antagonist, conserved aromatic or hydrophilic, as well as nonconserved residues, were mutated. The consequences on the ligand affinity were determined.…”