Eukaryotic cells show a high degree of macromolecular organization in which specific components are selectively segregated into specialized organelles. Of central interest in the field of cell biology is the elucidation of the molecular mechanisms operating in the cell to appropriately distribute proteins to organelles such as the secretory granules that serve to store hormones, enzymes, neurotransmitters, and other important molecules for release upon appropriate stimulus. Exocrine secretory granules are the product of cells that are epithelial in nature and specifically release their contents at the apical plasma membrane. The principal objective of this project is to study the mechanisms by which exocrine secretory granules are formed. We have chosen as a model GP-2, the major pancreatic acinar granule membrane protein, and have isolated the cDNA encoding it. In vivo, this protein has a life cycle which includes, in addition to its incorporation into the granule membrane, its acquisition of a glycosyl-phosphoinositol (GPT) linkage, its transport to the cell surface and its partial secretion in a sedimentable form. GP-2, when expressed from its cDNA by transfection, is primarily localized on the cell surface in Hela cells, which lack secretory granules. However, in permanent transformants of pituitary AtT-20 cells, a portion of it enters a novel type of storage organelle in AtT-20 pituitary cells. These GP-2-rich granules are distinct from the endogenous ACTH-containing secretory vesicles. By contrast, uromodulin/Tamm Horsfall protein, a renal apical membrane protein that is 88% homologous to GP-2, does not share this ability and is found exclusively on the cell surface in AtT-20 cells transfected with the cDNA encoding it. Taking advantage of the homology between GP-2 and THP, we propose to use recombinant DNA techniques to define the molecular features of GP-2 that are account for its storage in AtT-20 cells. This will serve as a prelude to the isolation of the cellular components responsible for the sorting of GP-2. We will also determine whether secretory and membrane proteins share the same molecular mechanisms for targetting to the regulated pathway and examine the capability of secretory cell lines of diverse tissue origin to support the storage of GP-2 in granules. In addition, we will use molecular biological techniques to identify other members of the putative GP-2/uromodulin family. It is anticipated that this research will provide important insights into the formation of secretory granules not only in exocrine cells but also in other secretory cell types.
