GIT1 Mediates Thrombin Signaling in Endothelial Cells: Role in Turnover of RhoA-Type Focal Adhesions
article
Thrombin mediates changes in endothelial barrier function and increases endothelial permeability. A feature of thrombin-enhanced endothelial hyperpermeability is contraction of endothelial cells (ECs), accompanied by formation of focal adhesions (FAs). Recently, a G protein-coupled receptor kinase-interacting protein, GIT1, was shown to regulate FA disassembly. We hypothesized that GIT1 modulates thrombin-induced changes in FAs. In human umbilical vein ECs (HUVECs), thrombin recruited GIT1 to FAs, where GIT1 colocalized with FAK and vinculin. Recruitment of GIT1 to FAs was dependent on activation of the small GTPase RhoA, and Rho kinase, as demonstrated by adenoviral transfection of dominant-negative RhoA and treatment with Y-27632. Thrombin stimulated GIT1 tyrosine phosphorylation with a time course similar to FAK phosphorylation in a Rho kinase- and Src-dependent manner. Depletion of GIT1 with antisense GIT1 oligonucleotides had no effect on basal cell morphology, but increased cell rounding and contraction of HUVECs, increased FA formation, and increased FAK tyrosine phosphorylation in response to thrombin, concomitant with increased endothelial hyperpermeability. These data identify GIT1 as a novel mediator in agonist-dependent signaling in ECs, demonstrate that GIT1 is involved in cell shape changes, and suggest a role for GIT1 as a negative feedback regulator that augments recovery of cell contraction. Chemicals / CAS: 4 (1 aminoethyl) n (4 pyridyl)cyclohexanecarboxamide, 146986-50-7; guanosine triphosphatase, 9059-32-9; protein tyrosine kinase, 80449-02-1; thrombin, 9002-04-4; Adaptor Proteins, Signal Transducing; Amides; Cell Cycle Proteins; Enzyme Inhibitors; Focal Adhesion Kinase 1, EC 2.7.1.112; Focal Adhesion Protein-Tyrosine Kinases, EC 2.7.1.112; GIT1 protein, human; GTPase-Activating Proteins; Oligodeoxyribonucleotides, Antisense; Phosphoproteins; Protein-Tyrosine Kinases, EC 2.7.1.112; Proto-Oncogene Proteins pp60(c-src), EC 2.7.1.112; PTK2 protein, human, EC 2.7.1.112; Pyridines; rac GTP-Binding Proteins, EC 3.6.5.2; rhoA GTP-Binding Protein, EC 3.6.5.2; RNA, Small Interfering; Thrombin, EC 3.4.21.5; Vinculin, 125361-02-6; Y 27632, 138381-45-0
Topics
Biomedical ResearchContractilityEndotheliumFocal adhesion kinaseThrombin4 (1 aminoethyl) n (4 pyridyl)cyclohexanecarboxamideantisense oligonucleotidefocal adhesion kinaseG protein coupled receptor kinaseG protein coupled receptor kinase interacting proteinGIT1 proteinguanosine triphosphataseprotein tyrosine kinaseRho kinaseRhoA guanine nucleotide binding proteinthrombinunclassified drugvinculinAdenovirusanimal cellarticlecell migrationcell shapecell structurecellular distributioncontrolled studydepletionendothelium cellenzyme activationenzyme metabolismenzyme phosphorylationenzyme substratefocal adhesiongenetic transfectionhumanhuman cellmembrane permeabilitynegative feedbacknonhumanpriority journalprotein functionprotein localizationprotein phosphorylationprotein protein interactionprotein transportregulatory mechanismsignal transductionumbilical veinAdaptor Proteins, Signal TransducingAmidesAnimalsAortaCattleCell Cycle ProteinsCell SizeCells, CulturedEndothelial CellsEndothelium, VascularEnzyme InhibitorsFeedback, BiochemicalFocal Adhesion Kinase 1Focal Adhesion Protein-Tyrosine KinasesFocal AdhesionsGTPase-Activating ProteinsHumansOligodeoxyribonucleotides, AntisensePhosphoproteinsPhosphorylationProtein Processing, Post-TranslationalProtein TransportProtein-Tyrosine KinasesProto-Oncogene Proteins pp60(c-src)Pyridinesrac GTP-Binding ProteinsrhoA GTP-Binding ProteinRNA, Small InterferingSignal TransductionThrombinTransduction, GeneticTransfectionUmbilical VeinsVinculin
TNO Identifier
237720
ISSN
00097330
Source
Circulation Research, 94(8), pp. 1041-1049.
Pages
1041-1049
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