The long-range objective of this proposal is to characterize the structural and functional properties of the cholecystokinin receptor on pancreatic acinar cells and retinal neuronal cells. Comparative studies will be directed at examining the affinities and molecular weights of the CCK receptors on rat pancreas and toad retina by competition binding analyses and affinity labeling techniques. Functional comparisons of these receptors will be obtained by monitoring the relative effectiveness of CCK-8 to stimulate 45Ca2+ efflux and polyphosphoinositide turnover in intact pancreatic lobules and retinal monolayers, respectively. We will also compare the pancreatic and retinal CCK receptors using specific antibody probes. To this end, a major emphasis has been placed on obtaining specific polyclonal antibodies to the Mr 81 K CCK recognition subunit of the receptor isolated from rat pancreatic plasma membranes. Rat pancreatic plasma membranes will be solubilized with Nonidet P-40 and the Mr 81 K protein will be purified by a number of sequential chromatographic steps followed by SDS gel electrophoresis. It will then be electroeluted from gel slices and either immobilized onto nitrocellulose paper and used to immunize rabbits or electroblotted onto activated glass for NH2-terminal microsequence analysis on a gas-phase sequenator. In the latter case, we will obtain the sequence of the first 10-20 NH2- terminal amino acid residues of this protein and with this data, have mg quantities of the corresponding peptide synthesized. Synthetic peptide will then be used to immunize rabbits and obtain site-specific antibodies to the CCK receptor. Using the specific antibody probes generated, we will complement our comparisons of the pancreatic and retinal CCK receptors by Western blotting analysis and immunoprecipitation of biosynthetically labeled receptor. We will also determine whether the CCK receptor in pancreas undergoes phosphorylation on tyrosyl residues in response to CCK binding or whether it undergoes regulatory phosphorylation by agents such as protein kinase C. These studies will provide considerable information, at the molecular level, of CCK receptor structure and function and will greatly enhance our understanding of the role CCK play in the regulation of pancreatic and retinal function.
