Contrasting roles of Leu(356) in the human CCK1 receptor for antagonist SR27897 and agonist SR 146131 binding

A new highly specific, potent non-peptide agonist for the cholecystokinin s ubtype 1 receptor (CCK1), SR 146131 (2-[4-(4-chloro-2,5-dimethoxyphenyl)-5- (2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl]-5,7-dimethyl-indol-1-yl-1-acet ic acid) was recently described [Bignon, E., Bachy, A., Boigegrain, R., Bro din, R., Cottineau, M., Gully, D., Herbert, J.-M., Keane, P., Labie, C., Mo limard, J.-C., Olliero, D., Oury-Donat, F., Petereau, C., Prabonneaud, V., Rockstroh, M.-P., Schaeffer, P., Servant, O. Thurneyssen, O., Soubrie, P., Pascal, M., Maffrand, J.-P., Le Fur, G., 1999. SR 146131: a new, potent, or ally active and selective non-peptide cholecystokinin subtype I receptor ag onist: I. In vitro studies. J. Pharmacol. Exp. Ther. 289, 742-751]. From bi nding and activity assays with chimeric constructs of human CCK1 and the ch olecystokinin subtype 2 receptor (CCK2) and receptors carrying point mutati ons, we show that Leu(356), situated in transmembrane domain seven in the C CK1 receptor, is a putative contact point for SR 146131. In contrast, Leu(3 56) is probably not in contact with the CCK1 receptor specific antagonist S R 27897 (1-[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl indoyl]acetic a cid), a compound structurally related to SR 146131, since its replacement b y alanine, histidine or asparagine gave receptors having wild-type CCK1 rec eptor SR 27897 binding affinity. Previous mutational analysis of His(381), the cognate position in the rat CCK2 receptor, had implicated it as being i nvolved in subtype specificity for SR 27897, results which we confirm with corresponding mutations in the human CCK2 receptor. Moreover, binding and a ctivity assays with the natural CCK receptor agonist, CCK-8S, show that CCK -8S is more susceptible to the mutations in that position in the CCK1 recep tor than in the CCK2 receptor. The results suggest different binding modes for SR 27897, SR 146131 and CCK-8S in each CCK receptor subtype. O 1999 Els evier Science B.V. All rights reserved.