MEMBRANE FUNCTION IN PAROTID SECRETORY MECHANISMS

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DESCRIPTION: (verbatim from application) The long term goal of the proposed research is to understand the mechanisms mediating exocytosis in salivary acinar cells. Exocytosis controls the export of macromolecules to the oral cavity and thus represents a basic mechanism for supplying proteins and peptides that are essential for maintaining oral physiology and host defense and for initiating digestion. The focus of investigations is on a family of highly conserved and broadly distributed proteins known as SCAMPs (for Secretory Carrier Membrane Proteins), which were discovered by the Principal Investigator. SCAMPs reside in secretory granules and related recycling membrane carriers that undergo exocytosis, and at least four different SCAMPs have been identified in acinar cells with distinct distributions. Functional studies have led to the hypothesis that SCAMPs are part of the molecular machinery required for exocytosis and that their most highly conserved domain acts to organize phosphoinositide-rich microdomains in membranes. Five specific aims have been developed to address this hypothesis, initially focusing on the granule-associated SCAMP2. First, mutations in the highly conserved domain of full-length SCAMP will be examined for dominant inhibitory effects on exocytosis using conditional expression in a regulated secretory cell line. Second, complementary studies involving analogous conditional expression will test whether regulatory segments of SCAMP and antisense constructs similarly interfere with exocytosis. Third, recombinant SCAMP will be purified, reconstituted in phospholipid vesicles and tested by spectroscopic techniques for interaction with phosphoinositides and regulation of lipid microdomains. Fourth, the binding partners of SCAMP will be sought using a battery of procedures to detect and analyze protein-protein interactions. Fifth, exocytosis will be analyzed in parotid salivary glands of mice in which the SCAMP1 gene has been ablated (obtained from another laboratory) and in mice lacking SCAMP2, a gene knockout that will be made collaboratively. Taken together, these studies, which originated in response to an NIDCR program announcement, reflect part of an ongoing commitment to advance basic understanding at the interface of molecular cell biology and oral physiology.