Caumont A S, Vitale N, Gensse M, Galas M C, Casanova J E, Bader M F
INSERM, U-338 Biologie de la Communication Cellulaire, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France.
J Biol Chem. 2000 May 26;275(21):15637-44. doi: 10.1074/jbc.M908347199.
ADP-ribosylation factors (ARFs) constitute a family of structurally related proteins that forms a subset of the Ras superfamily of regulatory GTP-binding proteins. Like other GTPases, activation of ARFs is facilitated by specific guanine nucleotide exchange factors (GEFs). In chromaffin cells, ARF6 is associated with the membrane of secretory granules. Stimulation of intact cells or direct elevation of cytosolic calcium in permeabilized cells triggers the rapid translocation of ARF6 to the plasma membrane and the concomitant activation of phospholipase D (PLD) in the plasma membrane. Both calcium-evoked PLD activation and catecholamine secretion in permeabilized cells are strongly inhibited by a synthetic peptide corresponding to the N-terminal 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. Using 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. Furthermore, co-expression in PC12 cells of a catalytically inactive ARNO mutant with human growth hormone as a marker of secretory granules in transfected cells resulted in a 50% inhibition of growth hormone secretion evoked by depolarization with high K(+). The possibility that the plasma membrane-associated ARNO participates in the exocytotic pathway by activating ARF6 and downstream PLD is discussed.
ADP-核糖基化因子（ARFs）构成了一类结构相关的蛋白质家族，它们是调节性GTP结合蛋白的Ras超家族的一个子集。与其他GTP酶一样，特定的鸟嘌呤核苷酸交换因子（GEFs）促进ARFs的激活。在嗜铬细胞中，ARF6与分泌颗粒的膜相关联。刺激完整细胞或在通透细胞中直接升高胞质钙会触发ARF6迅速转位至质膜，并伴随质膜中磷脂酶D（PLD）的激活。通透细胞中钙诱导的PLD激活和儿茶酚胺分泌均被对应于ARF6 N端结构域的合成肽强烈抑制，这表明在胞吐位点附近依赖ARF6的PLD激活是嗜铬细胞胞吐反应中的关键事件。在本研究中，我们证明了嗜铬细胞质膜和胞质溶胶中存在布雷菲德菌素A不敏感的ARF6-GEF活性。此外，逆转录酶-聚合酶链反应和免疫印迹分析表明，布雷菲德菌素A不敏感的ARF-GEF家族成员ARNO在嗜铬细胞中表达且主要定位于胞质溶胶和质膜中。使用通透的嗜铬细胞，我们发现将抗ARNO抗体引入胞质溶胶会以剂量依赖性方式抑制钙刺激细胞中的PLD激活和儿茶酚胺分泌。此外，在PC12细胞中共表达催化失活的ARNO突变体与人生长激素作为转染细胞中分泌颗粒的标志物，导致高钾（K+）去极化诱发的生长激素分泌受到50%的抑制。本文讨论了质膜相关的ARNO通过激活ARF6和下游PLD参与胞吐途径的可能性。
