Essential role of extracellular charged residues of the human CCK1 receptor for interactions with SR 146131, SR 27897 and CCK-8S

We hypothesized that charge-charge interactions may be important for the bi nding of the human cholecystokinin type 1 (CCK1) receptor-specific non-pept ide full agonist SR 146131, (2-[4-(4-chloro-2,5-dimethoxyphenyl)-5-(2-cyclo hexyl-ethyl)-thiazol-2-ylcarbamoyl]-5,7-dimethyl-indol-1-yl-1-acetic acid), the competitive antagonist SR 27897, (1-[2-(4-(2-chlorophenyl)thiazol-2-yl ) aminocarbonyl indoyl] acetic acid) and the natural octapeptide CCK-8S to the CCK1 receptor. Alanine replacement studies of positively charged residu es in the extracellular domains of the receptor showed that only the R336A mutation affected SR 146131 potency of mutated receptors transiently expres sed in monkey kidney epithelial COS-7 cells. Two residues, Lys(115) and Lys (187), were implicated in SR 27897 binding. Only the replacement of Lys(115 ), Arg(197) and Arg(336) significantly affected CCK-8S binding or activity. These results clearly indicated the importance of certain charged residues , but not others, in SR 146131, SR 27897 and CCK-8S binding. Furthermore, a lthough these molecules probably occupy different binding sites on the CCK1 receptor, we show that a small non-peptide agonist, SR 146131, can stimula te the dual signaling pathways mediated by the CCK1 receptor. (C) 2000 Else vier Science B.V. All rights reserved.