The Binding Site of Neuropeptide Vasopressin V1a Receptor

Abstract

To identify receptor functional domains underlying binding of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT), we have constructed a threedimensional (3D) model of the V1a vasopressin receptor subtype and docked the endogenous ligand AVP. To verify and to refine the 3D model, residues likely to be involved in agonist binding were selected for site-directed mutagenesis. Our experimental results suggest that AVP, which is characterized by a cyclic structure, could be completely buried into a 15-20-Å deep cleft defined by the transmembrane helices of the receptor and interact with amino acids located within this region. Moreover, the AVP-binding site is situated in a position equivalent to that described for the cationic neurotransmitters. Since all mutated residues are highly conserved in AVP and OT receptors, we propose that the same agonist-binding site is shared by all members of this receptor family. In contrast, the affinity for the antagonists tested, including those with a structure closely related to AVP, is not affected by mutations. This indicates a different binding mode for agonists and antagonists in the vasopressin receptor.The neurohypophysial hormones arginine vasopressin (AVP) 1 and oxytocin (OT) are two closely related nonapeptides that exhibit a high degree of functional diversity through interaction with specific receptors. Four different receptor subtypes have been characterized and possess different pharmacological and G protein coupling properties (1). V2 vasopressin receptors are coupled to adenylyl cyclase and are responsible for the antidiuretic effect of AVP. V1a and V1b vasopressin receptors are both coupled to phospholipase C. V1a receptors are involved in blood pressure control and in all other known functions of AVP, except for the stimulation of corticotropin secretion by the adenohypophysis which is mediated via V1b receptor subtype. OT receptors are coupled to phospholipase C and are responsible for the galactobolic and uterotonic effects. Recently, cDNAs for rat, human, and pig V2 receptors (2-4), rat and human V1a receptors (5, 6), pig and human OT receptors (4, 7), fish arginine vasotocin (AVT) receptor (8), and human V1b receptor (9) have been cloned and sequenced. This confirmed that these receptors belong to the G protein-coupled receptor (GPCR) family, characterized by seven hydrophobic putative ␣-helical transmembrane regions. All subtypes cloned so far are similar to each other in both identity and size. The level of conservation is remarkable, not only in the transmembrane domains but also in the first and second extracellular loops. Despite this high degree of receptor identity and a strong structural homology between the two hormones, the receptor subtypes are distinguished on the basis of different pharmacological and functional profiles. Many potent selective agonists as well as peptidic and nonpeptidic antagonists have been developed and used as pharmacological probes for the characterization of AVP and OT receptor subtypes (10).Previous molecular ...

Mapping Peptide-binding Domains of the Human V1a Vasopressin Receptor with a Photoactivatable Linear Peptide Antagonist

“…8 -11) led to the production of a profusion of valuable pharmacological probes for assessing receptor selectivity and activity but failed to provide relevant information on the organization of the receptor-binding sites. Recently, we have tentatively localized and mapped the binding site of vasopressin on the V1a receptor subtype by combining three-dimensional molecular modeling and site-directed mutagenesis approaches (12). Our results suggest that the larger part of the vasopressin molecule is anchored within the transmembrane portion of the receptor in a position equivalent to that described for the cationic neurotransmitters.…”

“…However, although peptide agonists and antagonists for vasopressin receptors have similar amino acid composition, they probably adopt very different conformations and probably fit into different states of the V1a receptor protein. Indeed, our previous mutagenesis study on the V1a receptor-binding sites have shown that mutations affecting agonist binding have little or no effect on antagonist binding (12).…”

“…The pharmacological properties of the receptor were not modified by this treatment (data not shown). Membrane preparations and radioligand binding assays were performed as already described (12). Briefly, CHO cells were harvested, washed two times in PBS without Ca 2ϩ and Mg 2ϩ , Polytron-homogenized in lysis buffer (15 mM Tris-HCl pH 7.4, 2 mM MgCl 2 , 0.3 mM EDTA), and centrifuged at 100 ϫ g for 5 min at 4°C.…”

“…Our results suggest that the larger part of the vasopressin molecule is anchored within the transmembrane portion of the receptor in a position equivalent to that described for the cationic neurotransmitters. Since all amino acids in the receptor and hormone, potentially interacting in hormone binding, are conserved in the different members of the receptor family and natural neurohypophysial peptides, we proposed that the binding pocket identified in the V1a might be common to V2, V1b, and oxytocin subtypes (12). In this model, only the side chain of vasopressin Arg 8 residue is projecting outside the transmembrane core of the receptor in a direction allowing interaction with Tyr 115 in the first extracellular loop.…”

“…So far all point mutations affecting agonist binding to V1a receptors were found to have no or far less pronounced effect on antagonist binding, suggesting that agonist and antagonistbinding sites might be structurally distinct (12). To further investigate structure-function relationships of AVP receptors, the aim of the present study was to localize the peptide antagonist-binding domains of the human V1a vasopressin receptor using a radiolabeled photoactivatable antagonist.…”