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CELLULAR AND MOLECULAR

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

Departments of Biochemistry (R.M.-D., N.C., Z.X., N.W., M.S.) and Medicine (M.T.), Case Western Reserve University, Cleveland, Ohio

Whereas arginine vasopressin binds to its receptor subtypes V₁R and V₂R with equal affinity of approximately 2 nM, nonpeptide antagonists interact differently with vasopressin receptor subtypes. The V₂R antagonist binding site was mapped by site-directed mutagenesis at six selected amino acid positions, K100D, A110W, M120V, L175Y, R202S, and F307I, predicted to be involved in antagonist binding differences between V₂ R and V₁R. These mutations did not alter the affinity for arginine vasopressin. However, the affinity for six nonpeptide receptor antagonists SR121463B [1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzenesulfonyl]-5-ethoxy-3-spiro-[4[(2 morpholinoethoxy)cy-clohexane]indoline-2-one, phosphate monohydrate cis-isomer], SR49059 [(2S)1-[(2R3S)-(5-chloro-3-(2 chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide], SSR149415 [(2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxyphenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2pyrrolidine carboxamide, isomer(-)], OPC21268 [1-[1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl]-3,4-dihydro-2(1H)-quinolinone], OPC41061 [(±)-4'-[(7-chloro-2,3,4,5-tetrahydro-5-hydroxy-1H-1-benzazepin-1-yl)carbonyl]-o-tolu-m-toluidide], and OPC31260, [(±)-5-dimethylamino-1-[4-(2-methylbenzoylamino)benzoyl]-1,2, 3,4,5-tetrahydro-1H-benzazepine monohydrochloride], was altered to varying degrees, resulting in differences up to 6000-fold. Replacement of the small alanine for the bulky tryptophan in position 110 resulted in a reduced affinity for all six antagonists. In contrast, replacement of the large methionine for the smaller valine in position 120 caused a dramatic increase in affinity, up to a K_i of 7 fM for OPC31260. Molecular modeling revealed that the binding sites for arginine vasopressin and the nonpeptide antagonists are partially overlapping. Whereas arginine vasopressin binds on the extracellular surface of V₂ R, the nonpeptide antagonists penetrate deeper into the transmembrane region of the receptor, in particular OPC21268. The mutagenesis data point to significant differences in the shape of the V₁R and V₂R antagonist binding pockets. The most important factor determining the specificity of nonpeptide antagonists seems to be the shape of the binding pocket on the receptor.

Address correspondence to: Menachem Shoham, Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4935. E-mail: mxs10{at}case.edu

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