title: Guanine exchange factor RalGDS mediates exocytosis of Weibel-Palade bodies from endothelial cells.
abstract: The small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein-coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca(2+)- or cAMP-raising agonists.
authors.affiliation: Department of Plasma Proteins, Sanquin Research and Landsteiner Laboratory Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Mariska G
authors.initials: MG
authors.lastname: Rondaij
authors.forename: Ruben
authors.initials: R
authors.lastname: Bierings
authors.forename: Ellen L
authors.initials: EL
authors.lastname: van Agtmaal
authors.forename: Karina A
authors.initials: KA
authors.lastname: Gijzen
authors.forename: Erica
authors.initials: E
authors.lastname: Sellink
authors.forename: Astrid
authors.initials: A
authors.lastname: Kragt
authors.forename: Stephen S G
authors.initials: SS
authors.lastname: Ferguson
authors.forename: Koen
authors.initials: K
authors.lastname: Mertens
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Hannah
authors.forename: Jan A
authors.initials: JA
authors.lastname: van Mourik
authors.forename: Mar
authors.initials: M
authors.lastname: Fernandez-Borja
authors.forename: Jan
authors.initials: J
authors.lastname: Voorberg
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D004837:Epinephrine / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D014644:Genetic Variation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection; D020302:Weibel-Palade Bodies / Q000502:physiology*; D020725:ral GTP-Binding Proteins / Q000378:metabolism; D020726:ral Guanine Nucleotide Exchange Factor / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
chemical_list: D002147:Calmodulin; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D020726:ral Guanine Nucleotide Exchange Factor; D049452:Green Fluorescent Proteins; D013917:Thrombin; C492198:RALA protein, human; D020725:ral GTP-Binding Proteins; D004837:Epinephrine