Rab35 protein regulates evoked exocytosis of endothelial Weibel-Palade bodies.
Rab35 protein regulates evoked exocytosis of endothelial Weibel-Palade bodies.
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DOI：
10.1074/jbc.m116.773333
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发表时间：
2017-07-14
期刊：
影响因子：
--
通讯作者：
Gerke V
中科院分区：
文献类型：
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作者：
Biesemann A;Gorontzi A;Barr F;Gerke V
Webel-Palade小体(WPB)是内皮细胞的分泌细胞器，在细胞内钙离子或cAMP升高后发生诱发性胞吐，从而为血管系统提供控制止血的因子。一些胞浆和膜相关蛋白，包括Rab家族成员Rab3、Rab15和Rab27a，参与调节WPB的急性胞吐。在这里，我们进行了全基因组筛选，以确定影响WPB胞吐的RAB途径。过表达特定的Rab GTP酶激活蛋白(RabGAP)抑制组胺诱导的、钙依赖的WPB胞吐，可能是通过灭活靶标Rab GTP酶来实现的。在这些RabGAP中，我们集中在TBC1D10A上，表明其抑制作用依赖于其GAP活性。我们证实Rab35是人内皮细胞中TBC1D10A的靶向Rab，Rab35与TBC1D10A相互作用，GAP不敏感的Rab35(Q67A)突变体的表达挽救了TBC1D10A过表达对WPB胞吐的抑制作用。此外，Rab35基因敲除和显性负性Rab35突变体的表达都抑制了组胺诱导的WPB货物von Willebrand因子和P-选择素的分泌。下拉和免疫共沉淀实验证明，含有螺旋卷曲、Ank重复和Pleckstrin同源结构域的蛋白ACAP2的ArfGAP是内皮细胞中的Rab35效应器，耗竭和过表达方法表明ACAP2作为WPB胞吐的负调节因子。有趣的是，一个已知的ACAP2靶标，小GTPase Arf6，支持组胺引起的WPB胞吐，正如一个显性负Arf6突变体的击倒和过表达所显示的那样。我们的数据确定Rab35是一种新的WPB胞吐调节因子，很可能通过下游效应器ACAP2和Arf6发挥作用。
Weibel–Palade bodies (WPB) are secretory organelles of endothelial cells that undergo evoked exocytosis following intracellular Ca2+ or cAMP elevation, thereby supplying the vasculature with factors controlling hemostasis. Several cytosolic and membrane-associated proteins, including the Rab family members Rab3, Rab15, and Rab27a, have been implicated in regulating the acute exocytosis of WPB. Here, we carried out a genome-wide screen to identify Rab pathways affecting WPB exocytosis. Overexpression of a specific subset of Rab GTPase–activating proteins (RabGAPs) inhibited histamine-evoked, Ca2+-dependent WPB exocytosis, presumably by inactivating the target Rab GTPases. Among these RabGAPs, we concentrated on TBC1D10A and showed that the inhibitory effect depends on its GAP activity. We confirmed that Rab35 was a target Rab of TBC1D10A in human endothelial cells; Rab35 interacted with TBC1D10A, and expression of the GAP-insensitive Rab35(Q67A) mutant rescued the inhibitory effect of TBC1D10A overexpression on WPB exocytosis. Furthermore, knockdown of Rab35 and expression of a dominant-negative Rab35 mutant both inhibited histamine-evoked secretion of the WPB cargos von Willebrand factor and P-selectin. Pulldown and co-immunoprecipitation experiments identified the ArfGAP with coiled-coil, Ank repeat, and pleckstrin homology domain–containing protein ACAP2 as an Rab35 effector in endothelial cells, and depletion as well as overexpression approaches revealed that ACAP2 acts as a negative regulator of WPB exocytosis. Interestingly, a known ACAP2 target, the small GTPase Arf6, supported histamine-evoked WPB exocytosis, as shown by knockdown and overexpression of a dominant-negative Arf6 mutant. Our data identify Rab35 as a novel regulator of WPB exocytosis, most likely acting through the downstream effectors ACAP2 and Arf6.