Rab35 protein regulates evoked exocytosis of endothelial Weibel–Palade bodies

Abstract

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.

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“…. Rab35 has many functions in various cellular processes including membrane trafficking (see below), autophagy, cell polarity, cytokinesis, membrane lipid homeostasis, neurite outgrowth, axonal elongation, immunity, phagocytosis, pathogen infection, myoblast fusion, exosome secretion and Weibel‐Palade bodies secretion . Moreover, the mechanisms controlling Rab35 activation in endocytosis is well understood with the identification of Rab35 GEFs (DENND1A‐C/Connecdenn1‐3) and GAPs (TBC1D10A‐C/EPI64A‐C, TBC1D13, TBC1D24), and reviewed in detail in and …”

Neutrophil recruitment and intracellular vesicle transport: A short overview

“…Recently, GAP activity of overexpressed TBC domain family member 10A (TBC1D10A) on Rab35 was shown to impair WPB exocytosis, while this could be abolished when TBC1D10A‐insensitive Rab35 was used. In addition, knockdown of Rab35 inhibited histamine‐evoked release of VWF as well as P‐selectin 35 . This was the first report that directly related the function of a Rab protein to the regulation of selectin surface expression.…”

RAB-35 aids apoptotic cell clearance by regulating cell corpse recognition and phagosome maturation

“…Rab35 is a multifunctional GTPase that plays important roles in a wide variety of biological processes. Mammalian Rab35 has been implicated in events including, but not limited to: exocytosis (49,50), endocytic recycling (51–54), cytokinesis (55,56), cytoskeleton rearrangement (56–61), and autophagy (62). Recently, Rab35 was found to be an oncogene that promotes proliferation by activating the PI3K/AKT signaling pathway (63).…”