Galas M C, Helms J B, Vitale N, Thiersé D, Aunis D, Bader M F
Institut National de la Santé et de la Recherche Médicale, U-338 Biologie de la Communication Cellulaire, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France.
J Biol Chem. 1997 Jan 31;272(5):2788-93. doi: 10.1074/jbc.272.5.2788.
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 betagamma 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.
ADP核糖基化因子（ARF）GTP结合蛋白被认为在分泌途径中作为囊泡出芽和融合的调节因子发挥作用。为了研究ARF在调节性胞吐作用中的作用，我们通过亚细胞分级分离和免疫复制品分析，研究了其在培养的嗜铬细胞中的细胞内分布。我们发现ARF6与纯化的分泌性嗜铬颗粒膜特异性相关。化学交联和免疫沉淀实验表明，ARF6可能是三聚体G蛋白βγ亚基复合物的一部分。刺激完整的嗜铬细胞或在通透细胞中直接升高胞质钙会引发ARF6从分泌颗粒的快速解离。这种效应可被选择性激活三聚体G蛋白的AlF4-抑制。此外, 一种与ARF6 N端结构域对应的合成肉豆蔻酰化肽强烈抑制链球菌溶血素-O通透的嗜铬细胞中钙诱发的分泌。本文讨论了ARF6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。
