EVIDENCE FOR THE INVOLVEMENT OF RAB3A IN CA2-DEPENDENT EXOCYTOSIS FROM ADRENAL CHROMAFFIN CELLS()

Bovine chromaffin cells are nondividing primary secretory cells that s tore and secrete catecholamine and a variety of proteins including chr omogranins, opiate peptides, and opiate precursors. A transient transf ection technique based upon the expression of human growth hormone as a reporter for the regulated secretory pathway was used to study the r ole of a Ras-like, GTP-binding protein, Rab3a, in Ca2+-dependent exocy tosis. Immunocytochemistry and flow cytometry revealed that growth hor mone and Rab proteins were coexpressed in the same cells. Overexpressi on of the wild type protein and expression of a mutant protein Rab3aQ8 1L both inhibited nicotinic agonist-stimulated exocytosis in intact ce lls. Expression of Rab3aQ81L also inhibited Ca2+-dependent secretion f rom permeabilized cells. Two other mutants, Rab3aN135I and Rab3aT36N, which correspond to dominant acting mutants of Ras, caused limited and no inhibition, respectively, of agonist-stimulated exocytosis. These data provide direct evidence that Rab3a plays an important role in Ca( 2+-)triggered exocytosis. We suggest that Rab3a is an inhibitor of sec retion, perhaps as part of a pre-fusion complex with secretory vesicle s. Elevated Ca2+ may trigger exocytosis by overcoming the inhibition b y Rab3a.