MEMBRANE FUNCTION IN PAROTID SECRETORY MECHANISMS

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The long-term objective of the research proposed in this application is to provide new insights into the mechanisms of salivary protein secretion. The intracellular events that couple the input of secretory stimuli and generation of second messengers to the discharge of salivary proteins are largely unknown but are likely to be mediated by proteins of the secretion granule and plasma membranes. The focus of these studies is on the structure and function of certain polypeptides that are components of the membranes of the secretion granules of parotid acinar cells. These polypeptides have been identified using monoclonal antibodies and are among the first to be reported as common components of storage granule membranes in exocrine, endocrine and neural cells. Their broad distribution suggests that they may be common denominators in the distal steps of protein export and thus may be essential to the secretory process. The monoclonal antibodies will be used to characterize the distribution and membrane organization of the antigens by immunocytochemical and immunochemical techniques and to immunopurify the polypeptides from parotid membranes. The primary structures of the polypeptides will be deduced by peptide sequencing and cloning from a parotid cDNA library. Three kinds of studies are planned toward the major goal of establishing the function of these granule membrane proteins. First, the identification of polypeptides having related sequences and known functions will be sought by computer- searching of centralized sequence data banks. Second, the effects on secretion will be examined in cultured cells in which full-length cDNAs encoding the proteins have been expressed. Finally, the monoclonal antibodies, as well as polyclonal antibodies generated to immunopurified antigens, will be examined as prospective perturbants of protein discharge using a cell-free analog of parotid secretion granule exocytosis. These studies may define some of the final effectors of salivary protein secretion. As such, they will be of substantial interest to other investigators who are attempting to clarify the integration and transduction of secretory stimuli, and they may lead to new insight concerning the regulation of the macromolecular composition of the fluid phase of the oral cavity.