Vitale Nicolas, Chasserot-Golaz Sylvette, Bader Marie-France
CNRS UPR-2356 Neurotransmission et Sécrétion Neuroendocrine, 67084 Strasbourg cedex, France.
Ann N Y Acad Sci. 2002 Oct;971:193-200. doi: 10.1111/j.1749-6632.2002.tb04463.x.
ARFs constitute a family of structurally related proteins that forms a subset of the ras GTPases. In chromaffin cells, secretagogue-evoked stimulation triggers the rapid translocation of ARF6 from secretory granules to the plasma membrane and the concomitant activation of PLD in the plasma membrane. Both PLD activation and catecholamine secretion are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6. ARNO, a potential guanine nucleotide exchange factor for ARF6, is expressed and localized in the plasma membrane of chromaffin cells. Using permeabilized cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits both PLD activation and catecholamine secretion. Chromaffin cells express PLD1 at the plasma membrane. We found that microinjection of the catalytically inactive PLD1(K898R) dramatically reduces catecholamine secretion monitored by amperometry, most likely by interfering with a late postdocking step of calcium-regulated exocytosis. We propose that ARNO-ARF6 participate in the exocytotic reaction by controlling the plasma membrane-bound PLD1. By generating fusogenic lipids at the exocytotic sites, PLD1 may represent an essential component of the fusion machinery in neuroendocrine cells.
ARF蛋白构成了一个结构相关蛋白家族，它是ras GTP酶的一个子集。在嗜铬细胞中，促分泌剂诱发的刺激会触发ARF6从分泌颗粒快速转运至质膜，并伴随质膜中PLD的激活。PLD的激活和儿茶酚胺分泌均受到一种与ARF6 N端结构域对应的合成肽的强烈抑制。ARNO是ARF6潜在的鸟嘌呤核苷酸交换因子，在嗜铬细胞质膜中表达并定位。利用透化细胞，我们发现将抗ARNO抗体导入胞质溶胶会抑制PLD激活和儿茶酚胺分泌。嗜铬细胞在质膜表达PLD1。我们发现显微注射催化失活的PLD1(K898R)会显著降低通过安培法监测的儿茶酚胺分泌，这很可能是通过干扰钙调节性胞吐作用的对接后晚期步骤实现的。我们提出ARNO-ARF6通过控制质膜结合的PLD1参与胞吐反应。通过在胞吐位点产生促融合脂质，PLD1可能是神经内分泌细胞融合机制的一个重要组成部分。
