Evidence for the involvement of Rab3A in Ca(2+)-dependent exocytosis from adrenal chromaffin cells.
Bovine chromaffin cells are nondividing primary secretory cells that store and secrete catecholamine and a variety of proteins including chromogranins, opiate peptides, and opiate precursors. A transient transfection technique based upon the expression of human growth hormone as a reporter for the regulated secretory pathway was used to study the role of a Ras-like, GTP-binding protein, Rab3a, in Ca(2+)-dependent exocytosis. Immunocytochemistry and flow cytometry revealed that growth hormone and Rab proteins were coexpressed in the same cells. Overexpression of the wild type protein and expression of a mutant protein Rab3aQ81L both inhibited nicotinic agonist-stimulated exocytosis in intact cells. Expression of Rab3aQ81L also inhibited Ca(2+)-dependent secretion from 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 secretion, perhaps as part of a pre-fusion complex with secretory vesicles. Elevated Ca2+ may trigger exocytosis by overcoming the inhibition by Rab3a.
Holz
R W
RW
Department of Pharmacology, University of Michigan, Ann Arbor 48109-0626.
Brondyk
W H
WH
Senter
R A
RA
Kuizon
L
L
Macara
I G
IG
D000311:Adrenal Glands / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D004072:Digitonin / Q000494:pharmacology; D005089:Exocytosis; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D020692:rab3 GTP-Binding Proteins
D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D004072:Digitonin; D002118:Calcium