With the long-term goal of better understanding the role of the gastrointestinal peptide hormone cholecystokinin (CCK) in health and disease, the current proposal focuses on the structure, function and regulation of the type of CCK receptor found on classical gastrointestinal targets such as pancreas and gallbladder, the Type A CCK receptor. This is known to be important physiologically and is potentially important in the pathogenesis of digestive dysfunctional states. Our general hypothesis is that the structure of this receptor will provide insight into the molecular basis for its initiation of signalling cascades within the cell and for receptor regulation. We will test the specific hypothesis that the CCK-binding subunit follows the structural motif of other guanine nucleotide-binding protein (G-p)- associated receptors, with seven transmembrane segments, and domains which are glycosylated, cysteine-rich, phosphorylated, and fatty acid- acrylated. This will be accomplished using approaches of affinity labeling, enzymatic digestion, affinity chromatography, precursor incorporation in biosynthesis, and purification with chemical characterization. Unlike most G-p-coupled receptors cloned, CCK has trophic effects on the acinar cell, suggesting the possibility of its receptor having additional novel domains. Particular attention will be directed toward the processes of receptor phosphorylation and fatty acid acylation, since these have potentially profound functional and regulatory roles. Affinity labeling probes with photolabile and chemically-reactive moieties within the receptor binding domain will be used to identify domains, and possibly residues, within the active site of this receptor. Partial amino acid sequencing of some of these domains will be performed, and used as a basis for cDNA cloning strategies and for generation of site-specific receptor antibodies. While this structural motif also carries predictions for receptor function and regulation, because of the growth factor activity and unusual biphasic dose-response curve of CCK, new regulatory themes may exist as well. Novel analogues of CCK exist, which will be used to dissect the intracellular events involved in these processes, and to help test a new model for the function and regulation of this receptor. This model predicts the sequential existence of two affinity states of one receptor molecule, two independently initiated intracellular activation cascades, and the presence and sites of regulation of this receptor. This model predicts the sequential existence of two affinity states of one receptor molecule, two independently initiated intracellular activation cascades, and the presence and sites of regulation of a CCK receptor kinase and phosphatase. Focus will be directed toward the molecular basis of receptor affinity state, the role of G-p-association in biological responses, and mechanisms of receptor desensitization, including uncoupling, sequestration, and down-regulation. This should establish a paradigm for future investigation of other types of CCK/gastrin receptors, and to thereby gain insights into the molecular basis of ligand-receptor interaction and cell activation.
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