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

Blood. 2008 Jul 1;112(1):56-63. doi: 10.1182/blood-2007-07-099309. Epub 2008 Apr 16.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites / genetics
  • Calmodulin / metabolism
  • Cell Line
  • Endothelial Cells / drug effects
  • Endothelial Cells / physiology
  • Endothelial Cells / ultrastructure
  • Epinephrine / pharmacology
  • Exocytosis / drug effects
  • Exocytosis / physiology*
  • Genetic Variation
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • RNA Interference
  • RNA, Small Interfering / genetics
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction
  • Thrombin / pharmacology
  • Transfection
  • Weibel-Palade Bodies / physiology*
  • ral GTP-Binding Proteins / metabolism
  • ral Guanine Nucleotide Exchange Factor / antagonists & inhibitors
  • ral Guanine Nucleotide Exchange Factor / chemistry
  • ral Guanine Nucleotide Exchange Factor / genetics
  • ral Guanine Nucleotide Exchange Factor / physiology*

Substances

  • Calmodulin
  • RNA, Small Interfering
  • Recombinant Fusion Proteins
  • ral Guanine Nucleotide Exchange Factor
  • Green Fluorescent Proteins
  • Thrombin
  • RALA protein, human
  • ral GTP-Binding Proteins
  • Epinephrine