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(Circulation Research. 2004;94:1041.)
© 2004 American Heart Association, Inc.

Molecular Medicine

GIT1 Mediates Thrombin Signaling in Endothelial Cells

Role in Turnover of RhoA-Type Focal Adhesions

Geerten P. van Nieuw Amerongen, Kanchana Natarajan, Guoyong Yin, Ryan J. Hoefen, M. Osawa, Judith Haendeler, A.J. Ridley, K. Fujiwara, Victor W.M. van Hinsbergh, Bradford C. Berk

From the Center for Cardiovascular Research and Department of Medicine (G.P.v.N.A., K.N., G.Y., R.J.H., M.O., J.H., K.F., B.C.B.), Aab Institute for Biomedical Sciences, University of Rochester, Rochester, NY; Department of Physiology (G.P.v.N.A., V.W.M.v.H.), Institute for Cardiovascular Research, Vrije Universiteit Medical Center, Amsterdam, The Netherlands; Gaubius Laboratory TNO-Prevention and Health (V.W.M.v.H.), Leiden, The Netherlands; Ludwig Institute for Cancer Research (A.J.R.), Royal Free and University College School of Medicine, London, UK; Department of Biochemistry and Molecular Biology (A.J.R.), University College London, London, UK.

Correspondence to Bradford C. Berk, MD, PhD, University of Rochester, Box MED, 601 Elmwood Ave, Rochester, NY 14642. E-mail Bradford_Berk{at}URMC.Rochester.edu

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.

Key Words: contractility • endothelium • focal adhesion kinase • thrombin

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