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