Rab35 Regulates Evoked Exocytosis of Endothelial Weibel-Palade Bodies [Cell Biology]

Weibel-Palade bodies (WPB) are secretory organelles of endothelial cells that undergo evoked exoyctosis following intracellular Ca2+ or cAMP elevation, thereby supplying the vasculature with factors controlling haemostasis. 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 RabGTPases. Among the inhibitory RabGAPs, we concentrated on TBC1D10A and showed that the inhibitory effect depends on its GAP activity. We confirmed 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 (VWF) and P-selectin. Pulldown and co-immunoprecipitation experiments identified the ArfGAP with coiled-coil, Ank repeat and PH domain-containing protein ACAP2 as 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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