G PROTEINS AND RAT PAROTID SECRETORY GRANULE

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The overall goal of the proposed research is to understand the function/role of GTP-binding proteins in salivary exocrine cells. Evidence obtained from Our laboratory suggests that both heterotrimeric and monomeric G proteins are located on the rat parotid acinar cell secretory granule as well as on the plasma membranes. The hypothesis to be tested in this proposal is that G proteins, located on secretory granule membranes function to modulate the gating of ion channels and exocytosis in the rat parotid gland. Specific aims are: l) Identification and localization of heterotrimeric and monomeric G proteins. G proteins will be identified in various cellular fractions including the plasma membrane, cytosol and secretory granule membranes by two-dimensional gel electrophoresis using [32P] ADP-ribosylation, radiolabeled GTP binding and immunoblot analysis with specific G protein antisera. Localization of G proteins in parotid tissue sections and in acinar cells will be by immunohistochemistry/immunocytochemistry (light and electron microscopy). 2) Determination of a role for G proteins on the Cl- conductance of isolated secretory granules. For these studies GTP, GTP analogs, A1F4, bacterial toxins, ADP-ribosylating factor (ARF), specific G protein antisera, and agents that affect monomeric G protein release/activation will be utilized. Modulation of G protein-regulated Cl- conductance by phosphorylation/dephosphorylation mechanisms will also be examined in isolated granules and in permeabilized cells by using radiolabeled [32P]ATP and [32P]orthophosphate, respectively. 3) Examination of the role of G proteins in regulating secretion (exocytosis) from parotid acinar cells. The effects of GTP and GTP analogs on the late stage of secretion, i.e. amylase release will be determined in permeabilized cells. The translocation of G proteins from secretory granules to other cellular compartments will be determined in acinar cells following cell stimulation; translocation will be examined by immunofluorescence/immunoelectron microscopy, immunoblot analysis and radiolabeled GTP binding. Results obtained will be correlated with amylase release. The proposed research should provide significant information on the identity of G proteins as well as significant insight as to their function in salivary exocrine cells. These studies will also provide a framework for examining cellular biochemical defects in diseases relating to salivary dysfunction.