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

Recently, we reported that the mutation of His(207) to Phe located in the s econd extracellular loop of the cholecystokinin B receptor strongly affecte d cholecystokinin (CCK) binding (Silvente-Poirot, S., Escrieut, C., and Wan k, S. A. (1998) Mol. Pharmacol. 54, 364-371), To characterize the functiona l group in CCK that interacts with His(207), we first substituted His(207) to Ala. This mutation decreased the affinity and the potency of CCK to prod uce total inositol phosphates 302-fold and 456-fold without affecting the e xpression of the mutant receptor. The screening of L-alanine-modified CCK p eptides to bind and activate the wild type and mutant receptors allowed the identification of the interaction of the C-terminal Asp(8) of CCK with His (207). The H207A-CCKBR mutant, unlike the wild type receptor, was insensiti ve to substitution of Asp(8) of CCK to other amino acid residues. This inte raction was further confirmed by mutating His(207) to Asp, The affinity of CCK for the H207D-CCKBR mutant was 100-fold lower than for the H207ACCKBR m utant, consistent with an electrostatic repulsion between the negative char ges of the two interacting aspartic acids. Peptides with neutral amino acid s in position eight of CCK reversed this effect and displayed a gain of aff inity for the H207D mutant compared with CCK. To date, this is the first re port concerning the identification of a direct contact point between the CC KB receptor and CCK.