Identification of a plasma membrane-associated guanine nucleotide exchangefactor for ARF6 in chromaffin cells - Possible role in the regulated exocytotic pathway

ADP-ribosylation factors (ARFs) constitute a family of structurally related proteins that forms a subset of the Ras superfamily of regulatory GTP-bind ing proteins. Like other GTPases, activation of ARFs is facilitated by spec ific guanine nucleotide exchange factors (GEFs), In chromaffin cells, ARF6 is associated with the membrane of secretory granules. Stimulation of intac t cells or direct elevation of cytosolic calcium in permeabilized cells tri ggers the rapid translocation of ARF6 to the plasma membrane and the concom itant activation of phospholipase D (PLD) in the plasma membrane. Both calc ium-evoked PLD activation and catecholamine secretion in permeabilized cell s are strongly inhibited by a synthetic peptide corresponding to the N-term inal domain of ARF6, suggesting that the ARF6-dependent PLD activation near the exocytotic sites represents a key event in the exocytotic reaction in chromaffin cells. In the present study, we demonstrate the occurrence of a brefeldin A-insensitive ARF6-GEF activity in the plasma membrane and in the cytosol of chromaffin cells, Furthermore, reverse transcriptase-polymerase chain reaction and immunoreplica analysis indicate that ARNO, a member of the brefeldin A-insensitive ARF-GEF family, is expressed and predominantly localized in the cytosol and in the plasma membrane of chromaffin cells. Us ing permeabilized chromaffin cells, we found that the introduction of anti- ARNO antibodies into the cytosol inhibits, in a dose-dependent manner, both PLD activation and catecholamine secretion in calcium-stimulated cells. Fu rthermore, co-expression in PC12 cells of a catalytically inactive ARNO mut ant with human growth hormone as a marker of secretory granules in transfec ted cells resulted in a 50% inhibition of growth hormone secretion evoked b y depolarization with high K+. The possibility that the plasma membrane-ass ociated ARNO participates in the exocytotic pathway by activating ARF6 and downstream PLD is discussed.