Regulated exocytosis in chromaffin cells. A potential role for a secretory granule-associated ARF6 protein.
The ADP-ribosylation factor (ARF) GTP-binding proteins are believed to function as regulators of vesicular budding and fusion along the secretory pathway. To investigate the role of ARF in regulated exocytosis, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation and immunoreplica analysis. We found that ARF6 is specifically associated with the membrane of purified secretory chromaffin granules. Chemical cross-linking and immunoprecipitation experiments suggested that ARF6 may be part of a complex with betagamma subunits of trimeric G proteins. Stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid dissociation of ARF6 from secretory granules. This effect could be inhibited by AlF4- which selectively activates trimeric G proteins. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 strongly inhibited calcium-evoked secretion in streptolysin-O-permeabilized chromaffin cells. The possibility that ARF6 plays a role in the effector pathway by which trimeric G proteins control exocytosis in chromaffin cells is discussed.
Galas
M C
MC
Institut National de la Santé et de la Recherche Médicale, U-338 Biologie de la Communication Cellulaire, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France.
Helms
J B
JB
Vitale
N
N
Thiersé
D
D
Aunis
D
D
Bader
M F
MF
D020727:ADP-Ribosylation Factors; D000313:Adrenal Medulla / Q000502:physiology*; D017607:Aluminum Compounds / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002458:Cell Fractionation; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000502:physiology*; D002837:Chromaffin Granules / Q000378:metabolism*; D005089:Exocytosis; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology
D017607:Aluminum Compounds; D000906:Antibodies; D002352:Carrier Proteins; D009227:Myristic Acids; D010446:Peptide Fragments; D019814:Myristic Acid; C050992:tetrafluoroaluminate; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D005459:Fluorides