Our goal is to better understand the role of cholecystokinin (CCK) in health and disease. This gut and brain hormone exists in multiple molecular forms, ranging in size from 4 to 58 amino acids, with functions including regulation of pancreatic secretion, gallbladder contraction, gastrointestinal motility, trophic effects, and satiety. Our approach is 3 fold: a) characterization and quantification of CCK peptides, b) functional and biochemical characterization of CCK receptors, and c) description of the molecular events associated with CCK-receptor interaction, including receptor activation and initiation of the effector cascade. The peptide-related components include: 1) the chemical and mass spectrometric elucidation of the primary structure of large human CCK peptides, 2) the establishment and validation of complementary approaches to the quantification of human CCK peptides (RIA, radioreceptor assay, and bioassay), and 3) the experimental (fluorescence, UV, and CD spectroscopy and NMR) and theoretical approaches to the determination of the conformation of the receptor-binding region of CCK. The receptor-related components include a major commitment to the development of new methodology, with initial application to the pancreatic acinar cell receptor; subsequent application will be to gastrointestinal smooth muscle, studying preparations of fresh and cultured, healthy and neoplastic cells and their membranes. We plan to develop a series of new probes for the CCK-binding region of the receptor; these will include 1) short CCK analogues with free amino groups available for chemical crosslinking, 2) analogues with incorporated aryl nitrene and carbene precursors for photoaffinity labeling, 3) anti-idiotypic antibodies, and 4) naturally-occurring auto-antibodies. We also plan to generate a series of immunologic probes for this receptor and its associated molecules, and to solubilize, purify, and reconstitute these in a functional state. These studies should further our understanding of the mechanisms whereby this hormone interacts with its receptor to activate it and initiate the effector cascade within target cells. This may help in the understanding of potential aberrations of CCK-receptor interaction which may occur in disease stages, and may have more general basic cellular and molecular implications for the important process of message transduction across a membrane.
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