All eukaryotic cells secrete proteins into their extracellular milieu. Besides the common constitutive pathway, certain more specialized cells package their secretory products into dense-core granules awaiting cell stimulation for release. This has been termed the regulated pathway of secretion. In contrast to the granule but the molecular mechanisms responsible for this sorting remain poorly understood. Investigations to date have suggested a similar mechanism of sorting across different cell types, such as endocrine and isolated the cDNA encoding the major pancreatic zymogen granule membrane protein, GP-2. This protein, expressed only in exocrine pancreas, is specifically targeted to the granules. GP-2, when expressed in an exocrine pancreas-derived tissue culture cell line, can be immunohistochemically localized to the secretory granules. By contrast, when expressed in cell lines derived from endocrine tissues, the protein was excluded from the endogenous granules and accumulated intracellularly in large multivesicular structures also labeling with an endosomal/lysosomal membrane marker. This localization did not appear to be due to endocytosis of plasma membrane GP-2 responsible for its sorting into secretory vesicles. Chimeras of GP-2 and THP and deletion mutations in GP-2 will be constructed and expressed the exocrine AR42J cells to investigate their sorting and detect regions of the protein necessary for its correct trafficking within the cell. 2. To characterize responsible. The same mutated cDNA's will be studied in the endocrine Rin5F and AtT-20 cells in order to determine whether these regions interfere with the incorporation of the protein into the endogenous secretory granules. 3. To generate transgenic mice expressing GP-2 and characterize its localization and sorting in various tissues. The experiments proposed should enable the determination of the mechanisms for the sorting to the regulated secretory pathway and should advance our knowledge of the basic cell biology of this pathway which is important to cells in critical for the homeostasis and nutrition of all animals. In addition, understanding of the normal physiology of regulated secretion should provide a basis for the study of the pathogenesis of some diseases such as acute pancreatitis in which abnormal intracellular organelle trafficking has implicated.
