Regulated exocytosis in chromaffin cells - A potential role for a secretory granule-associated ARF6 protein
Regulated exocytosis in chromaffin cells - A potential role for a secretory granule-associated ARF6 protein
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DOI：
10.1074/jbc.272.5.2788
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发表时间：
1997-01-31
影响因子：
4.8
通讯作者：
Bader, MF
中科院分区：
文献类型：
--
作者：
Galas, MC;Helms, JB;Bader, MF
ADP-核糖基化因子（ARF）GTP结合蛋白被认为是沿着分泌途径的囊泡出芽和融合的调节剂。为了研究ARF在调节胞吐中的作用，我们用亚细胞分离和免疫组化分析方法研究了ARF在培养的嗜铬细胞中的胞内分布。我们发现，ARF 6是专门与纯化的分泌嗜铬颗粒的膜。化学交联和免疫沉淀实验表明，ARF 6可能是与三聚体G蛋白的β-γ亚基复合的一部分。刺激完整的嗜铬细胞或直接升高通透细胞中的胞浆钙离子，触发了分泌颗粒中ARF 6的快速解离。这种作用可以被选择性激活三聚体G蛋白的AlF 4-抑制。此外，一个合成的豆蔻酰化肽对应的N-末端结构域的ARF 6强烈抑制钙诱导的分泌链球菌溶血素-O-透性嗜铬细胞。ARF 6在三聚体G蛋白控制嗜铬细胞胞吐作用的效应途径中发挥作用的可能性进行了讨论。
The ADP-ribosylation factor (ARF) GTP-binding proteins are believed to function as regulators of vesicular budding and fusion along the secretory pathway. To investigate the role of ARF in regulated exocytosis, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation and immunoreplica analysis. We found that ARF6 is specifically associated with the membrane of purified secretory chromaffin granules. Chemical cross-linking and immunoprecipitation experiments suggested that ARF6 may be part of a complex with beta gamma subunits of trimeric G proteins. Stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid dissociation of ARF6 from secretory granules. This effect could be inhibited by AlF4- which selectively activates trimeric G proteins. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 strongly inhibited calcium-evoked secretion in streptolysin-O-permeabilized chromaffin cells. The possibility that ARF6 plays a role in the effector pathway by which trimeric G proteins control exocytosis in chromaffin cells is discussed.