DIRECT IDENTIFICATION OF A DISTINCT SITE OF INTERACTION BETWEEN THE CARBOXYL-TERMINAL RESIDUE OF CHOLECYSTOKININ AND THE TYPE-A CHOLECYSTOKININ RECEPTOR USING PHOTOAFFINITY-LABELING

Mechanisms of ligand binding and activation of G protein-coupled recep tors are particularly important, due to their ubiquitous expression an d potential as drug targets, Molecular interactions between ligands an d these receptors are best defined for small molecule ligands that bin d within the transmembrane helices, Extracellular domains seem to be m ore important for peptide ligands, based largely on effects of recepto r mutagenesis, where interference with binding or activity can reflect allosteric as well as direct effects, We now take the more direct app roach of photoaffinity labeling the active site of the cholecystokinin (CCK) receptor, using a photolabile analogue of CCK having a blocked amino terminus, This probe, yrosyl-Gly-[Nle(28,31),pNO(2)-Phe(33)]CCK- (26-33), binds specifically, saturably, and with high affinity (K-i = 3.3 nM) and has full agonist activity, This makes likely its being sit ed in a natural position within the receptor, As substrate, we used CH O-CCK receptor cells overexpressing functional recombinant rat type A CCK receptor, Covalent labeling of the appropriate M-r = 85,000-95,000 plasma membrane glycoprotein with core of M-r = 42,000 was establishe d by SDS-polyacrylamide gel electrophoresis and autoradiography, A sin gle domain adjacent to transmembrane 1 was labeled, as established by cyanogen bromide cleavage and separation by gel and/or high pressure l iquid chromatography, The site of interaction was further defined by a dditional proteolysis with trypsin, with purification of the labeled f ragment, followed by manual Edman degradation and radiochemical sequen cing, This demonstrated that Trp(39) was specifically labeled and like ly resides proximate to the carboxyl-terminal pNO(2)-Phe(33) residue o f the probe. A model of this ligand-bound receptor has been constructe d and will be used to plan future experiments to refine our understand ing of this interaction.