Guanine exchange factor RalGDS mediates exocytosis of Weibel-Palade bodies from endothelial cells

Abstract

IntroductionWeibel-Palade bodies (WPBs) are endothelial cell-specific storage organelles 1 that contain a number of hormones, chemokines, enzymes, and adhesive molecules that are rapidly released or recruited to the cell surface upon stimulation with specific agonists. Its main constituent, von Willebrand factor (VWF), 2 is also the driving force for the biogenesis of WPBs from the trans-Golgi network. [3][4][5] Current evidence suggests that VWF provides a platform for the cosegregation of a number of other bioactive compounds that include P-selectin, angiopoietin-2, and the chemokines interleukin-8 (IL-8) and eotaxin-3 into this intracellular storage compartment. 6 Release or surface presentation of WPB constituents enables the endothelium to control vascular homeostasis, by participating in diverse processes such as the arrest of bleeding, inflammatory responses, and angiogenesis.Regulation of WPB exocytosis by both Ca 2ϩ -and cAMPraising agonists involves signaling pathways initiated by agonist binding to G protein-coupled receptors (GPCRs) that ultimately result in fusion of WPBs with the plasma membrane. 7 Real-time imaging of the secretory behavior has shown that rapid release of WPBs is observed after stimulation of endothelial cells with Ca 2ϩ -raising agonists such as thrombin or histamine. 8,9 Stimulation of endothelial cells with agents that raise intracellular cAMP, such as epinephrine or vasopressin, promote a relatively slow release of WPBs, whereas a subpopulation of WPBs escapes cAMP-mediated exocytosis by clustering at the microtubule organizing center (MTOC). 8,10,11 We previously reported that the small GTP-binding protein RalA cosediments with WPBs on density gradients and multiple lines of evidence suggest that activation of Ral is a crucial step in both thrombin-and epinephrine-induced exocytosis of WPBs. [12][13][14] Ral has been implicated in regulated release of secretory granules of various origins, 15-18 playing a dual role in the process of exocytosis. Ral is involved in tethering secretory vesicles to specific sites on the plasma membrane through its GTP-dependent interaction with components of the exocyst complex. 16 In addition, Ral modulates exocytosis by enhancing ADP-ribosylation factor 6 (ARF6)-dependent phospholipase D1 activity, 19 resulting in the formation of fusogenic lipids that promote membrane fusion.The guanine exchange factor (GEF) involved in the activation of RalA in response to GPCR-mediated signaling in endothelial cells has not been identified. Recently, the activity of the RalGEF Ral-GDP dissociation stimulator (RalGDS) was found to be regulated by GPCR activation via receptor activation-mediated dissociation of RalGDS/␤-arrestin complexes. 20 In this study, we investigated whether RalGDS is involved in Ral-mediated exocytosis of WPBs. We report here that siRNA-mediated knockdown of RalGDS markedly inhibited thrombin-and epinephrine-induced WPB exocytosis. Furthermore, a RalGDS⌬382-597 mutant lacking The online version of this article contains a data supplement...

Ral GTPases Regulate Cell-Mediated Cytotoxicity in NK Cells

“…Ral is linked to various polarized secretion events that require vesicle targeting to specific membrane domains; these include basolateral secretion in epithelia (16), synaptic vesicle release in neurons (29), and secretion of Weibel-Palade bodies in endothelial cells (30). Ral might thus be involved in lytic granule polarization during cell-mediated cytotoxicity.…”

Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells

“…9). Likewise, RalA activation followed by PLD2-dependent PA formation is also required for caveolae fusion with the basal membrane during exocytosis, a process known to be dependent on yet another downstream RalA effector, the exocyst complex (23,51,52). …”

Molecular Machinery Regulating Exocytosis

“…Active GTP-bound Ras facilitates activation by binding to RalGEFs (Spaargaren & Bischoff, 1994), and delivering them to Ral in the plasma membrane. RalGDS has been shown to be required for the activation of RalA 70 dependent exocytosis from Weibel-Palade bodies in endothelial cells and the secretion of insulin in β-pancreatic cells (Kim, et al, 2010;Ljubicic, et al, 2009;Rondaij, et al, 2008), while calcium induced exocytosis from endosomes is predicted to be regulated by Rgl2/Rlfmediated activation of Ral (Takaya, et al, 2007). Thus, the regulation of Ral dependent exocytosis may be at the nexus of a number of upstream signaling pathways.…”

“…Subsequently, Ral small GTPases were linked to exocytosis in neural, epithelial, endothelial endocrinal, and pancreatic tissues (Hazelett, et al, 2011;Lopez, et al, 2008;Moskalenko, et al, 2003;Polzin, et al, 2002;Rondaij, et al, 2004;Rondaij, et al, 2008;Takaya, et al, 2007). The two mammalian Ral homologues RalA and RalB share 85% protein sequence identity, and are well conserved in evolution (van Dam & Robinson, 2006).…”

“…In the epithelium, RalB is required for delivery of membrane to the dynamic leading edge of migrating cells (Rosse, et al, 2006); while RalA is involved in polarised delivery of the membrane protein, ECadherin, to the basolateral surface of epithelial cells (Shipitsin & Feig, 2004). Exocytosis of vesicular content, such as hormones, chemokines, enzymes, and adhesion molecules from Weibel-Palade bodies (endothelial cell-specific storage organelles), occurs in response to a specific agonist that requires Ral regulation (Kim, et al, 2010;Rondaij, et al, 2008). RalA is required in glucose regulation where it mediates insulin secretion from pancreatic cells (Ljubicic, et al, 2009;Lopez, et al, 2008), and translocation of the glucose transporter GLUT4 in adipocytes (Chen, et al, 2007).…”