Caumont A S, Galas M C, Vitale N, Aunis D, Bader M F
INSERM, U-338 Biologie de la Communication Cellulaire, Strasbourg, France.
J Biol Chem. 1998 Jan 16;273(3):1373-9. doi: 10.1074/jbc.273.3.1373.
The ADP-ribosylation factor (ARF) GTP-binding proteins have been implicated in a wide range of vesicle transport and fusion steps along the secretory pathway. In chromaffin cells, ARF6 is specifically associated with the membrane of secretory chromaffin granules. Since ARF6 is an established regulator of phospholipase D (PLD), we have examined the intracellular distribution of ARF6 and PLD activity in resting and stimulated chromaffin cells. We found that stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid translocation of ARF6 from secretory granules to the plasma membrane and the concomitant activation of PLD in the plasma membrane. To probe the existence of an ARF6-dependent PLD in chromaffin cells, we measured the PLD activity in purified plasma membranes. PLD could be activated by a nonhydrolyzable analogue of GTP and by recombinant myristoylated ARF6 and inhibited by specific anti-ARF6 antibodies. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 inhibited both PLD activity and catecholamine secretion in calcium-stimulated chromaffin cells. The possibility that ARF6 participates in the exocytotic reaction by controlling a plasma membrane-bound PLD and thereby generating fusogenic lipids at the exocytotic sites is discussed.
ADP核糖基化因子（ARF）GTP结合蛋白参与了分泌途径中广泛的囊泡运输和融合步骤。在嗜铬细胞中，ARF6与分泌性嗜铬颗粒的膜特异性相关。由于ARF6是磷脂酶D（PLD）的既定调节因子，我们研究了静息和刺激的嗜铬细胞中ARF6的细胞内分布和PLD活性。我们发现，刺激完整的嗜铬细胞或通透细胞中胞质钙的直接升高会触发ARF6从分泌颗粒快速转运到质膜，并伴随质膜中PLD的激活。为了探究嗜铬细胞中是否存在ARF6依赖性PLD，我们测量了纯化质膜中的PLD活性。PLD可被GTP的非水解类似物和重组肉豆蔻酰化的ARF6激活，并被特异性抗ARF6抗体抑制。此外，一种与ARF6 N端结构域对应的合成肉豆蔻酰化肽可抑制钙刺激的嗜铬细胞中的PLD活性和儿茶酚胺分泌。本文讨论了ARF6通过控制质膜结合的PLD并由此在胞吐位点产生融合脂质来参与胞吐反应的可能性。
