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