Biesemann Anja, Gorontzi Alexandra, Barr Francis, Gerke Volker
Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, University of Münster, D-48149 Münster, Germany.
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
J Biol Chem. 2017 Jul 14;292(28):11631-11640. doi: 10.1074/jbc.M116.773333. Epub 2017 May 31.
Weibel-Palade bodies (WPB) are secretory organelles of endothelial cells that undergo evoked exocytosis following intracellular Ca 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, Ca-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.
魏贝尔-帕拉德小体（WPB）是内皮细胞的分泌细胞器，在细胞内钙或环磷酸腺苷（cAMP）升高后会发生诱导性胞吐作用，从而为脉管系统提供控制止血的因子。包括Rab家族成员Rab3、Rab15和Rab27a在内的几种胞质和膜相关蛋白与调节WPB的急性胞吐作用有关。在此，我们进行了全基因组筛选，以确定影响WPB胞吐作用的Rab途径。Rab GTP酶激活蛋白（RabGAP）特定亚群的过表达抑制了组胺诱导的、钙依赖性的WPB胞吐作用，推测是通过使靶Rab GTP酶失活来实现的。在这些RabGAP中，我们重点研究了TBC1D10A，并表明其抑制作用取决于其GAP活性。我们证实Rab35是人类内皮细胞中TBC1D10A的靶Rab；Rab35与TBC1D10A相互作用，且GAP不敏感的Rab35（Q67A）突变体的表达挽救了TBC1D10A过表达对WPB胞吐作用的抑制效果。此外，Rab35的敲低以及显性负性Rab35突变体的表达均抑制了组胺诱导的WPB货物血管性血友病因子和P-选择素的分泌。下拉实验和免疫共沉淀实验确定了具有卷曲螺旋、锚蛋白重复序列和含pleckstrin同源结构域的蛋白ACAP2（含卷曲螺旋、锚蛋白重复序列和pleckstrin同源结构域的ArfGAP）作为内皮细胞中的Rab35效应器，且通过消耗以及过表达方法表明ACAP2作为WPB胞吐作用的负调节因子。有趣的是，如显性负性Arf6突变体的敲低和过表达所示，一个已知的ACAP2靶标——小GTP酶Arf6支持组胺诱导的WPB胞吐作用。我们的数据确定Rab35是WPB胞吐作用的一种新型调节因子，最有可能通过下游效应器ACAP2和Arf6发挥作用。
