Cookies on this website
We use cookies to ensure that we give you the best experience on our website. If you click 'Continue' we'll assume that you are happy to receive all cookies and you won't see this message again. Click 'Find out more' for information on how to change your cookie settings.

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.

Original publication

DOI

10.1074/jbc.M116.773333

Type

Journal article

Journal

J Biol Chem

Publication Date

14/07/2017

Volume

292

Pages

11631 - 11640

Keywords

Calcium, Rab, calcium, endothelial cell, endothelial cells, membrane traffic, membrane trafficking, vascular biology, von-Willebrand factor, ADP-Ribosylation Factors, Amino Acid Substitution, Calcium Signaling, Cells, Cultured, Down-Regulation, Endothelium, Vascular, Exocytosis, GTPase-Activating Proteins, Green Fluorescent Proteins, Histamine, Human Umbilical Vein Endothelial Cells, Humans, Immunoprecipitation, Membrane Proteins, Mutagenesis, Site-Directed, Point Mutation, RNA Interference, Recombinant Fusion Proteins, Weibel-Palade Bodies, rab GTP-Binding Proteins