Abstract

The affinity of the nonpeptide antagonist OPC-21268 is greater for the rat V₁ arginine vasopressin (AVP) receptor (V₁R) than for the human V₁R. Site-specific mutagenesis was carried out to identify the residues that determine interspecies selectivity for nonpeptide antagonist binding. The introduction of rat amino acids in position 224, 310, 324, or 337 of the human V₁R sequence dramatically altered OPC-21268 affinity for the receptor, whereas binding of AVP, the peptide V₁R antagonist d(CH₂)₅Tyr(Me)AVP, and the nonpeptide V₁R antagonist SR49059 was not altered by these mutations. Computer modeling explained the mutagenesis results. Docking of OPC-21268 onto a homology-built model of the V₁R receptor yielded a model for the bound ligand in which the hydrophobic part is deeply embedded in the transmembrane region, whereas the polar part is located on the surface of the extracellular side. The increased affinity of the G337A mutant is due to two additional van der Waals contacts of the alanine methyl group with carbon atoms on the antagonist. The I310V mutant reduces the hydrophobicity in the vicinity of the polar oxygen atom of the antagonist. The I224V mutant relieves overcrowding in a hydrophobic binding pocket involving the aromatic residues Trp¹⁷⁵, Phe¹⁷⁹, Phe³⁰⁷, and Trp³⁰⁴. Finally, the E324D mutant enables the formation of a hydrogen bond of the carboxylate side chain with the amide side chain of Gln³¹¹, which in turn forms a hydrogen bond with the N57 nitrogen atom of OPC-21268. Thus, a few residues, distinct from those involved in agonist binding, control interspecies selectivity toward OPC-21268 nonpeptide antagonist binding.