CHARACTERIZATION AND SORTING OF ZYMOGEN GRANULE PROTEINS

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DESCRIPTION (adapted from investigator's abstract and/or aims): The applicant notes that the establishment of epithelial polarity is essential to the function of the exocrine pancreas. Although the apical plasma membrane of the pancreas represents approximately 5 percent of the total surface area of the plasma membrane, zymogen granules are targeted specifically to this area resulting in the release of digestive enzymes. The biogenesis of epithelial polarity in the exocrine pancreas will be examined by asking whether the zymogen granule represents the sole pathway to the apical membrane. To achieve this goal, the exocrine cell line, AR42J, will be stably transfected with cDNA encoding the hemagglutinin protein of influenza, an apical membrane protein normally not found in secretory granules. Utilizing radioactive pulse-chase techniques and subcellular fractionation, the intracellular pathway of hemagglutinin to the plasma membrane will be compared to that of endogenous membrane proteins present in zymogen granules. Endogenous membrane proteins will be identified and characterized by the use of monoclonal antibodies generated against purified zymogen granules. The applicant hopes that these experiments will offer insight into mechanisms of protein sorting in polarized secretory epithelia. In addition to their utility in defining intracellular pathways, the monoclonal antibodies will also be used to identify proteins that are functionally important in regulated secretion. The monoclonal antibodies will be tested for their ability to inhibit regulated secretion as measured by two assays. One assay measures the fusion of zymogen granules with the plasma membrane in-vitro. The second assay utilizes red blood cells for the delivery of antibodies into AR42J cells. Those antibodies that display inhibition will be characterized further. Finally, efforts will also be directed towards the development of a polarized exocrine pancreatic cell line in culture. The applicant believes that such a cell line will offer greater opportunities to study pancreatic function with molecular and cell biological techniques.