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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 ...

Using a three-dimensional model of G protein-coupled receptors (GPCR), we have previously succeeded in docking the neurohypophysial hormone argininevasopressin (AVP) into the Vla receptor. According to this model, the hormone is completely embedded in the transmembrane part of the receptor. Only the side chain of the Arg residue at position 8 projects outside the transmembrane core of the receptor and possibly interacts with a Tyr residue located in the first extracellular loop at position 115. Residue 8 varies in the two natural neurohypophysial hormones, AVP and oxytocin (OT); similarly, different residues are present at position 115 in the different members of the AVP/ OT receptor family. Here we show that Arg8 is crucial for high affinity binding of AVP to the rat Vla receptor. Moreover, when Tyrl15 is replaced by an Asp and a Phe, the amino acids naturally occurring in the V2 and in the OT receptor subtypes, the agonist selectivity of the Vla receptor switches accordingly. Our results indicate that the interaction between peptide residue 8 and the receptor residue at position 115 is not only crucial for agonist high affinity binding but also for receptor selectivity.

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