This is a revised application to investigate the function of the Rab3 GTPase in the regulation of exocytosis from neuroendocrine cells. Rab3A is unusual among members of the Rab GTPases in the sense that it is expressed exclusively in neuroendocrine cells, where it is localized predominantly on synaptic granules in neurons and dense secretory granules in PC12 and adrenal chromaffin cells. The subcellular location of Rab3A together with two other observations suggest a role for the protein in exocytosis. The first observation is that overexpression of wild-type Rab3A, or the activated Q81 mutant, potently inhibits the initial rate of agonist-stimulated secretion. The second observation is that removal of a domain from a putative effector of Rab3A, Rabphilin, also inhibits secretion. Dr. Macara proposes that Rab3A targets Rabphilin to vesicle membrane, at which point cytosolic calcium can promote an association of Rabphilin with the cytoskeleton as a prequel to vesicle docking. Another protein relevant to the actions of Rab3A is Rabin3, which is implicated in the association of Rab3-guanine nucleotide exchange activity with Rab3. With that as the background, the specific aims of the proposal are: 1) to determine whether Rabin3 interacts with a Rab3-specific nucleotide exchange factor, and whether such a factor on membranes preferentially acts on a Rab3:GDI complex; 2) to determine whether Rab3A targets Rabphilin to secretory vesicle membrane, or vice versa; 3) to identify the functions of the Rabphilin domains, particularly that of the C2 domain, and to search for Rabphilin-binding proteins; 4) to investigate the cellular function of the Rab3A:Rabphilin complex, that is, effects of mutants on the distribution of secretory vesicles and the effects of Rab3A binding on the interaction of Rabphilin with beta-adducin and other proteins; and 5) to search for alternate downstream effectors in tissues that express Rab3 isoforms other than Rab3A.
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