Contrasting roles of Leu 356 in the human CCK 1 receptor for antagonist SR 27897 and agonist SR 146131 binding

Abstract

A new highly specific, potent non-peptide agonist for the cholecystokinin subtype 1 receptor (CCK 1), SR 146131 (2-[4-(4-chloro-2,5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl]-5,7-dimethyl-indol-1-yl-1-acetic acid) was recently described [Bignon, E., Bachy, A., Boigegrain, R., Brodin, R., Cottineau, M., Gully, D., Herbert, J.-M., Keane, P., Labie, C., Molimard, J.-C., Olliero, D., Oury-Donat, F., Petereau, C., Prabonneaud, V., Rockstroh, M.-P., Schaeffer, P., Servant, O.Thurneyssen, O., Soubrié, P., Pascal, M., Maffrand, J.-P., Le Fur, G., 1999. SR 146131: a new, potent, orally active and selective non-peptide cholecystokinin subtype I receptor agonist: I. In vitro studies. J. Pharmacol. Exp. Ther. 289, 742–751]. From binding and activity assays with chimeric constructs of human CCK 1 and the cholecystokinin subtype 2 receptor (CCK 2) and receptors carrying point mutations, we show that Leu 356, situated in transmembrane domain seven in the CCK 1 receptor, is a putative contact point for SR 146131. In contrast, Leu 356 is probably not in contact with the CCK 1 receptor specific antagonist SR 27897 (1-[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl indoyl]acetic acid), a compound structurally related to SR 146131, since its replacement by alanine, histidine or asparagine gave receptors having wild-type CCK 1 receptor SR 27897 binding affinity. Previous mutational analysis of His 381, the cognate position in the rat CCK 2 receptor, had implicated it as being involved in subtype specificity for SR 27897, results which we confirm with corresponding mutations in the human CCK 2 receptor. Moreover, binding and activity assays with the natural CCK receptor agonist, CCK-8S, show that CCK-8S is more susceptible to the mutations in that position in the CCK 1 receptor than in the CCK 2 receptor. The results suggest different binding modes for SR 27897, SR 146131 and CCK-8S in each CCK receptor subtype.