GIT1 mediates thrombin signaling in endothelial cells: role in turnover of RhoA-type focal adhesions.
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.
van Nieuw Amerongen
Geerten P
GP
Center for Cardiovascular Research and Department of Medicine,Aab Institute for Biomedical Sciences, University of Rochester, Rochester, NY 14642, USA.
Natarajan
Kanchana
K
Yin
Guoyong
G
Hoefen
Ryan J
RJ
Osawa
M
M
Haendeler
Judith
J
Ridley
A J
AJ
Fujiwara
K
K
van Hinsbergh
Victor W M
VW
Berk
Bradford C
BC
D048868:Adaptor Proteins, Signal Transducing; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D002417:Cattle; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D048429:Cell Size / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D025461:Feedback, Physiological; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000502:physiology*; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D006801:Humans; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013917:Thrombin / Q000235:genetics / Q000502:physiology*; D014161:Transduction, Genetic; D014162:Transfection; D014471:Umbilical Veins; D016596:Vinculin / Q000032:analysis; D020744:rac GTP-Binding Proteins / Q000502:physiology; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D000577:Amides; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; C485647:GIT1 protein, human; D020690:GTPase-Activating Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D010750:Phosphoproteins; D011725:Pyridines; D034741:RNA, Small Interfering; D016596:Vinculin; C108830:Y 27632; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D016392:Proto-Oncogene Proteins pp60(c-src); D013917:Thrombin; D020744:rac GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein