The Guanine Exchange Factor RalGDS Is Involved in Regulated Exocytosis of Weibel-Palade Bodies from Endothelial Cells.
Authors: Voorberg Jan , Rondaij Mariska G. , Gijzen Karina A. , Bierings Ruben , Sellink Erica , Fernandez-Borja Mar , van Mourik Jan A. 
Published: October 2019

Abstract
Endothelial cells contribute to vascular homeostasis and mediate pathophysiological responses to hypoxia-induced injury and inflammatory events. To ensure rapid responses to vascular perturbation endothelial cells contain intracellular storage pools for inflammatory mediators and pro-thrombotic compounds. One of the best characterized storage granules within endothelial cells are the Weibel-Palade bodies (WPB), rod-shaped organelles that contains P-selectin, Von Willebrand Factor (VWF), interleukin-8 (IL-8) and a number of other proteins with diverse biological activities. Agonist-induced triggering of heterotrimeric G protein coupled receptors (GPCR) present on endothelial cells promote exocytosis of WPB. We have previously shown that the small GTP binding protein RalA is involved in thrombin induced exocytosis of Weibel-Palade bodies. Exocytosis of Weibel-Palade bodies was found to coincide with the activation of RalA in response to thrombin. More recently, we have shown that cAMP-raising stimuli such as epinephrine also coincide with the activation of RalA. Consistent with these findings constitutively active RalG23V was capable of inducing release of WPB from endothelial cells. Small GTPases are activated by guanine exchange factors (GEFs) that induce GDP release thereby enhancing GTP binding to the small GTPase. RalGDS is a widely expressed GEF for Ral that has recently been implicated in cytoskeletal rearrangements that result from activation of GPCRs. Here, we investigated whether RalGDS is involved in thrombin- and/or epinephrine-induced exocytosis of WPB from endothelial cells. First, we showed by RT-PCR that human endothelial cells express RalGDS. Overexpression of a GFP-tagged variant of RalGDS in endothelial cells reduces the number of WPB in endothelial cells suggesting that RalGDS can promote exocytosis of these subcellular organelles. To investigate whether endogenously synthesized RalGDS plays a role in exocytosis of WPBs we designed short hairpin RNAs that acts as small interfering RNA (siRNA). Co-expression of siRalGDS and GFP-RalGDS in heterologously transfected 293 cells markedly reduced expression levels of GFP-RalGDS. Subsequently, we addressed the effect of siRalGDS on exocytosis of WPB in endothelial cells. Knockdown of endogenous RalGDS using siRNA prevented thrombin-induced release of WPBs. Expression of siRalGDS also interfered with WPB release in response to epinephrine. These results show that knock down of RalGDS interferes with exocytosis of WPB in endothelial cells. A dominant negative RalGDS variant, RalGDSΔGEF, lacking its catalytic exchange domain, was subsequently introduced in endothelial cells. As expected, no release of WPB was observed in endothelial cells expressing RalGDSΔGEF. Remarkably, both thrombin- and epinephrine-induced exocytosis were impaired in cells expressing RalGDSΔGEF. Together, these findings indicate that the Ral-specific guanine exchange factor RalGDS is involved in exocytosis of WPB from endothelial cells.