Evidence for a direct interaction between the penultimate aspartic acid of cholecystokinin and histidine 207, located in the second extracellular loop of the cholecystokinin B receptor

Abstract

Recently, we reported that the mutation of His207 to Phe located in the second extracellular loop of the cholecystokinin B receptor strongly affected cholecystokinin (CCK) binding (Silvente-Poirot, S., Escrieut, C., and Wank, S. A. (1998) Mol. Pharmacol. 54, 364-371). To characterize the functional group in CCK that interacts with His207, we first substituted His207 to Ala. This mutation decreased the affinity and the potency of CCK to produce total inositol phosphates 302-fold and 456-fold without affecting the expression of the mutant receptor. The screening of L-alanine-modified CCK peptides to bind and activate the wild type and mutant receptors allowed the identification of the interaction of the C-terminal Asp8 of CCK with His207. The H207A-CCKBR mutant, unlike the wild type receptor, was insensitive to substitution of Asp8 of CCK to other amino acid residues. This interaction was further confirmed by mutating His207 to Asp. The affinity of CCK for the H207DCCKBR mutant was 100-fold lower than for the H207ACCKBR mutant, consistent with an electrostatic repulsion between the negative charges of the two interacting aspartic acids. Peptides with neutral amino acids in position eight of CCK reversed this effect and displayed a gain of affinity for the H207D mutant compared with CCK. To date, this is the first report concerning the identification of a direct contact point between the CCKB receptor and CCK.