DIRECT IDENTIFICATION OF A 2ND-DISTINCT SITE OF CONTACT BETWEEN CHOLECYSTOKININ AND ITS RECEPTOR

We have developed a biologically active analogue of cholecystokinin (C CK) that incorporates a photolabile benzoylphenylalanine (Bpa) moiety in the middle of its pharmacophoric domain, which efficiently establis hes a covalent bond with an interacting domain of the CCK receptor, Th is probe incorporated L-Bpa in the position of Gly(29) of the well cha racterized, radioiodinatable CCK analogue, D-Tyr-Gly-[(Nle(28,31))CCK- 26-33]. It was a potent pancreatic secretagogue (EC50 = 28 +/- 6 nM) t hat was equally efficacious with natural CCK, and bound to the CCK rec eptor with moderate affinity (IC50 = 450 +/- 126 nM). This was adequat e to allow specific covalent labeling of the receptor. The labeled dom ain was within the cyanogen bromide fragment of the receptor including the top of TM6 (the sixth transmembrane domain), the third extracellu lar loop, and TM7 (the seventh transmembrane domain), as proven by dir ect Edman degradation sequencing, When this fragment was modified by t he replacement of Val(342) with Met to generate an additional site of cyanogen bromide cleavage, the labeled fragment was reduced in apparen t size consistent with its representing the carboxyl-terminal portion of this fragment. Radiochemical sequencing of that fragment demonstrat ed covalent attachment of the probe to His(347) and Leu(348) in this d omain, This represents the second experimentally demonstrated contact between a CCH analogue and this receptor, complementing the labeling o f the domain just above TM1 (the first transmembrane domain) by a phot olabile residue at the carboxyl terminus of CCK (Ji, Z. S., Hadac, E. M., Henne, R. M., Patel, S. A., Lybrand, T. P., and Miller, L. J. (199 7) J. Biol. Chem, 272, 24393-24401). Both contacts are consistent with the conformational model of CCB. binding proposed on the basis of the initial contact.