The long-range goal of this proposal is to characterize the structural and functional properties of the cholecystokinin (CCK) receptor in pancreatic acinar cells using biochemical and immunochemical techniques. In biochemical studies, CCK binding proteins will be labeled in intact acinar cells and isolated membranes with a new photoaffinity analog of CCK-8, as well as by chemical cross-linking protocols. The potential multicomponent nature of these labeled proteins will be analyzed by gel filtration chromatography and sucrose density gradient centrifugation. Using the recently obtained information that the affinity labeled CCK binding proteins are glycoproteins, the combined techniques of lectin affinity chromatography, CCK affinity chromatography and HPLC gel filtration will be used to isolate CCK receptors from detergent extracts of rat pancreatic membranes for detailed structural analysis. Nearest neighbor analysis of the CCK binding proteins to one another and to other membrane proteins will be performed as will a structural comparison of the CCK receptor in brain to that in pancreas. The role receptor oligosaccharide side chains may have in CCK binding or action will also be examined. For immunochemical studies on the CCK receptor, polyclonal and monoclonal antibodies will be raised against purified CCK receptors. Structural studies will include analysis of the potential multicomponent nature of the receptor, studies on the biosynthesis of receptor and the application of these antibodies toward purification of receptor by immunoaffinity chromatography. Studies on receptor function will be directed at examining the effects of anti-receptor antibodies on CCK binding and/or action. Whether the anti-receptor antibodies directly mimic or antagonize CCK action on intact pancreatic acini will be examined as will the covalent modification of the CCK receptor via phosphorylation. These studies should greatly enhance our understanding of the CCK receptor at the molecular level and provide insight into the role CCK plays in the regulation of pancreatic function and satiety.
