The long-term goal of the research proposed in this application is to understand mechanisms controlling exocytosis in acinar cells of parotid and other salivary glands. The main focus is on a new family of proteins called SCAMPs (for Secretory Carrier Membrane Proteins). SCAMPs are present in parotid secretion granule membranes but are also found in other membranes (e.g., synaptic vesicles, endosomes) that function as recycling carriers between intracellular compartments and the cell surface. They are widely distributed among different cell types and are highly conserved among mammalian species. Intracellular administration of an anti-SCAMP monoclonal antibody inhibits exocytosis of secretion granules, and the structure and properties of one SCAMP, SCAMP37, are consistent with a potential role in promoting membrane fusion. A combination of molecular biological and biophysical approaches will be used to map the epitope of the inhibitory monoclonal antibody and to evaluate how intermolecular and membrane associations of specific domains of SCAMPs might relate to their hypothesized role in fusion during exocytosis. As a part of these studies, new anti-SCAMP antibodies and recombinant peptides encoding domains of SCAMP37 will be evaluated as perturbants of exocytosis in permeabilized neuroendocrine and parotid acinar cell models. Overexpression of exogenous SCAMP constructs and antisense expression in mammalian cells will address whether exocytosis and constitutive membrane recycling are perturbed in situ. Molecular cloning approaches will be used to deduce the structures of new SCAMP family members including prospective homologs from lower in the phylogenetic tree. Finally, an existing panel of monoclonal antibodies raised against parotid secretion granule membranes will be screened to search for new reagents that perturb exocytosis in permeabilized parotid acinar cells. Secretion of salivary proteins is essential to maintaining oral physiology and host defense and to initiating digestive processes. These studies originated in response to an NIDR program announcement requesting applications that address basic mechanisms of salivary secretion. Thus this continuation proposal reflects an ongoing commitment to address issues at the interface of molecular cell biology and oral physiology.
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