His381 of the rat CCKB receptor is essential for CCKB versus CCKA receptor antagonist selectivity.

A great interest is devoted to antagonists of the cholecystokinin type B (CCKB) receptor such as L-365,260, which reduces panic attacks in humans and to antagonists of the cholecystokinin type A (CCKA) receptor, such as L-364,718 which might be efficient in mental diseases. The A/B specificity of these antagonists was proposed to be mainly dependent on the amino acid sequence of the seventh transmembrane domain (Mantamadiotis and Baldwin (1994) Biochem. Biophys. Res. Commun. 201,1382). In our study, one of these residues, His381 was replaced in the rat CCKB receptor by leucine (the corresponding residue in the CCKA receptor), phenylalanine or arginine using site-directed mutagenesis. Changing histidine for leucine or phenylalanine did not modify significantly the affinity of the CCKB receptor antagonists, L-365,260 and PD-134,308 although both compounds belong to different chemical classes, but strongly improved the affinity of the CCKA receptor antagonists tested. Interestingly, the A selectivity of these CCKA receptor antagonists was recovered by substituting His381 by arginine. Moreover, these results are discussed on the basis of a three dimentional model of the CCKB receptor. The mutated receptors possessed unchanged binding properties for agonists, suggesting that determinants confering specificity for agonists and antagonists are different.