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

Abstract

Whereas arginine vasopressin binds to its receptor subtypes V 1 R and V 2 R with equal affinity of approximately 2 nM, nonpeptide antagonists interact differently with vasopressin receptor subtypes. The V 2 R 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 V 2 R and V 1 R. These mutations did not alter the affinity for arginine vasopressin. However, the affinity for six nonpeptide receptor,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 V 2 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 V 1 R and V 2 R 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.Arginine vasopressin (AVP), the antidiuretic hormone, is a cyclic nonapeptide that modulates various physiological functions, such as water reabsorption, blood volume, blood pressure, cellular proliferation, and adrenocorticotropic hormone secretion (Thibonnier et al., 1998(Thibonnier et al., , 2001). The antidiuretic effect of AVP is mediated by the vasopressin V 2 receptor (V 2 R), a member of the large family of G protein-coupled receptors (Robben et al., 2004). V 2 R is a 41-kDa seven-transmembrane protein of 371 residues (Birnbaumer et al., 1992). V 2 R is expressed in the basolateral membrane of epithelial cells of the renal distal tubule and the collecting ducts (Hermosilla et al., 2004;Robben et al., 2004). In the collecting duct of the kidney, AVP binds to the V 2 R, thereby activating

Vasopressin antagonists

“…Mutagenesis data point to significant differences in the shape of the V1 and V2-receptor antagonistbinding pockets. The most important factor determining the specificity of non-peptide antagonists seems to be the shape of the binding pocket on the receptor [34].…”

Hyponatremia Treatment Guidelines 2007: Expert Panel Recommendations

“…Molecular modeling of binding sites indicates that the nonpeptide antagonists penetrate deeper into the transmembrane region of the V 2 R than does native AVP, thereby preventing binding of native hormone without themselves interacting with the H1 helix site that is critical for V 2 R-mediated G-protein activation. 107 Consequently, binding of the antagonists to V 2 R blocks activation of the receptor by endogenous AVP. The increased urine output produced by the V 2 R antagonists is quantitatively equivalent to diuretics such as furosemide, but qualitatively it is different in that only water excretion is produced without significant increases in urine solute excretion, including sodium and potassium.…”

A case of nephrogenic diabetes insipidus with a novel missense mutation in the AVPR2 gene

“…Therefore, to investigate the structural effects on the AVPR2 molecule resulting from the Y205F substitution, we constructed a molecular model of AVPR2 and analyzed the intermolecular interaction of the Tyr-205 hydroxy group. The model structure of bacteriorhodopsin [21] had been used in many structural analyses of vasopressin receptors [22][23][24]. However, the X-ray structure of rhodopsin was reported recently [25], and it has been shown to have higher homology with vasopressin receptors in comparison with bacteriorhodopsin.…”