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 secr etory pathway. To investigate the role of ARF in regulated exocytosis, we have examined its intracellular distribution in cultured chromaffi n cells by subcellular fractionation and immunoreplica analysis. We fo und that ARF6 is specifically associated with the membrane of purified secretory chromaffin granules. Chemical cross-linking and immunopreci pitation experiments suggested that ARF6 may be part of a complex with beta gamma subunits of trimeric G proteins. Stimulation of intact chr omaffin cells or direct elevation of cytosolic calcium in permeabilize d cells triggered the rapid dissociation of ARF6 from secretory granul es. This effect could be inhibited by AlF4- which selectively activate s trimeric G proteins. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 strongly inhibited calc ium-evoked secretion in streptolysin-O-permeabilized chromaffin cells. The possibility that ARF6 plays a role in the effector pathway by whi ch trimeric G proteins control exocytosis in chromaffin cells is discu ssed.