Regulated exocytosis in chromaffin cells - Translocation of ARF6 stlmulates a plasma membrane-associated phospholipase D
Regulated exocytosis in chromaffin cells - Translocation of ARF6 stlmulates a plasma membrane-associated phospholipase D
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DOI：
10.1074/jbc.273.3.1373
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发表时间：
1998-01-16
影响因子：
4.8
通讯作者：
Bader, MF
中科院分区：
文献类型：
--
作者：
Caumont, AS;Galas, MC;Bader, MF
ADP-核糖基化因子 (ARF) GTP 结合蛋白与分泌途径中的多种囊泡转运和融合步骤有关。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) 的既定调节剂，因此我们检查了静息和刺激嗜铬细胞中 ARF6 和 PLD 活性的细胞内分布。我们发现，刺激完整的嗜铬细胞或直接升高透化细胞中的胞浆钙会触发ARF6从分泌颗粒快速易位到质膜，并同时激活质膜中的PLD。为了探究嗜铬细胞中 ARF6 依赖性 PLD 的存在，我们测量了纯化质膜中的 PLD 活性。 PLD 可被不可水解的 GTP 类似物和重组肉豆蔻酰化 ARF6 激活，并被特异性抗 ARF6 抗体抑制。此外，与 ARF6 N 末端结构域相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。讨论了 ARF6 通过控制质膜结合的 PLD 参与胞吐反应，从而在胞吐位点产生融合脂质的可能性。
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.