The goals of this proposal are to understand the mechanism and regulation of the posttranslational processing of pro-cholecystokinin (CCK), which will aid in the eventual understanding of the role of CCK in brain function. A large body of evidence suggests that CCK is an important element in the neurochemical balance that is thought to be essential for normal nervous system function. By understanding the mechanism and regulation of CCK biosynthesis and processing, it may be possible to alter the rate of synthesis and release of CCK in specific brain regions and thus treat specific neurological and psychiatric diseases. The proposal is designed to gain new information on the temporal order of cleavages during proCCK processing and the substrate specificity of the cleavage enzymes present in cultured cells. Further experiments are designed to identify regions of proCCK which are involved in its sorting to secretory vesicles and the possible role of sorting receptors or carrier proteins in this process. The following specific aims are addressed: 1) using pulse-chase studies of recombinant CCK-expressing cell lines, immunoprecipitation, and chromatography the temporal order of proCCK processing will be determined. 2) Using site- directed mutagenesis of rat proCCK cDNA and stable expression of these cDNAs in AtT-20 cells in culture, the effect of alterations of the proCCK sequence on its sorting and processing will be determined. 3) Using rat hippocampal and cerebral cortical slices in vitro, the temporal order of proCCK processing established in cell lines will be confirmed.
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