Identification of Residues Responsible for the Selective Binding of Peptide Antagonists and Agonists in the V2 Vasopressin Receptor

Abstract

To improve our understanding of the functional architecture of G protein-coupled receptors, we have taken advantage of differences among mammalian species in ligand binding to search for the rat versus human selectivity determinants of the V2 vasopressin receptor and of its peptide ligands. Our data indicate that residue 2 of species-selective peptide antagonists such as d(CH 2 ) 5 -[DIle 2 ,Ile 4 ,Tyr-NH 2 9 ]arginine vasopressin controls their rat versus human selectivity. For species-selective agonists such as desmopressin, residues 1 and 8 modulate the binding selectivity. Among residues different between rat and human V2 receptors, those localized in the upper part of the human V2 receptor have been substituted with their rat V2 homologs. Pharmacological analysis of mutant receptors revealed that residues 202 and 304 fully control the species selectivity of the discriminating antagonists in an independent and additive manner. A third residue (position 100) is necessary to observe an equivalent phenomenon for the discriminating agonists. The substitution of these three residues does not modify the affinity of the nonselective agonists and antagonists. In conclusion, extracellular loops and the top of the transmembrane domains of V2 vasopressin receptors may provide the molecular basis for peptide ligand-binding species selectivity. Very few residues in these regions may control the binding mode of both agonists and antagonists. The arginine vasopressin (AVP)1 /oxytocin receptor family represents a suitable system to investigate structure-function relationships among G protein-coupled receptors. Indeed, all four receptor subtypes for these neurohypophyseal hormones, namely the V1a, V1b, and V2 AVP receptors and the unique oxytocin receptor, have been well characterized by pharmacological and molecular cloning studies (see Ref. 1 for review). These peptide receptors share a high primary sequence homology, but display a great diversity in their functional properties. Moreover, these receptors interact with many potent selective agonists and several classes of antagonist ligands, such as cyclic and linear peptides (2) as well as nonpeptides (3,4). By a combination of receptor three-dimensional molecular modeling and site-directed mutagenesis approaches, our preceding study has led to the mapping of the AVP agonist-binding domain in the V1a receptor subtype (5). The hormone-binding site is localized in a narrow cleft delimited by most of the transmembrane regions ϳ15 Å away from the extracellular surface. Because of the high conservation of residues involved in the interaction with the hormone, this binding site has been proposed to be common to the V1b, V2, and oxytocin receptors. An extracellular residue responsible for receptor subtype agonist selectivity has also been identified (6 -8).These first analyses did not provide much information on the definition of AVP/oxytocin receptor antagonist-binding domains. To date, only the upper part of transmembrane region VII and a hydrophobic cluster of aromatic residue...

Genetic Variations and Polymorphisms of G Protein-Coupled Receptors: Functional and Therapeutic Implications

“…The binding impairment of the mutant receptor suggests that this residue participates in ligand recognition. Cotte et al (97) showed that a Leu residue at the same position in the rat V 2 receptor facilitates ligand binding, whereas the Arg at position 202 in the human receptor is not favorable. A change to Cys at this position may constitute an even more negative determinant restricting receptor interaction with ligand.…”

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

“…Role of the conserved aromatic residues on antagonist binding properties of the V 1a vasopressin receptor 9]AVP, suggesting that as for agonists, aromatic residues do not contribute to its affinity. It is interesting to point out that this compound is an antagonist with partial agonist activity on the V 2 receptor [25].…”

“…Indeed, measuring a putative gain in affinity is much more significant. The pharmacological properties of the mutant V 2 receptors are presented in 9]AVP. Surprisingly, the L310T mutation led to a decrease in affinity for this compound (K i was 10.2 nm, compared to 0.79 nm).…”

Mapping the Binding Site of Six Nonpeptide Antagonists to the Human V2-Renal Vasopressin Receptor

“…Pharmacological and molecular cloning studies of the V 1 , V 2 , and V 3 AVP receptors, as well as the related oxytocin receptor, have shown that these peptide receptors display a great diversity in their functional properties despite high sequence homology. These receptors can bind not only the native hormone AVP but also potent and selective cyclic and linear peptide analogs, as well as nonpeptide antagonists (Cotte et al, 1998). Mutagenesis studies have indicated that the ligand-binding pocket includes residues located on the extracellular loops as well as in adjoining transmembrane helices of the receptors (Oksche et al, 2002).…”