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J. Exp. Med. 206 (12): 2761-2777

Copyright © 2009 by the Rockefeller University Press.

Article

The G protein β{gamma} subunit mediates reannealing of adherens junctions to reverse endothelial permeability increase by thrombin

Nebojsa Knezevic, Mohammad Tauseef, Tracy Thennes, , and Dolly Mehta

Center for Lung and Vascular Biology, Department of Pharmacology, University of Illinois, Chicago, IL 60612

CORRESPONDENCE Dolly Mehta: dmehta{at}uic.edu

Abstract: The inflammatory mediator thrombin proteolytically activates protease-activated receptor (PAR1) eliciting a transient, but reversible increase in vascular permeability. PAR1-induced dissociation of G{alpha} subunit from heterotrimeric Gq and G12/G13 proteins is known to signal the increase in endothelial permeability. However, the role of released Gβ{gamma} is unknown. We now show that impairment of Gβ{gamma} function does not affect the permeability increase induced by PAR1, but prevents reannealing of adherens junctions (AJ), thereby persistently elevating endothelial permeability. We observed that in the naive endothelium Gβ1, the predominant Gβ isoform is sequestered by receptor for activated C kinase 1 (RACK1). Thrombin induced dissociation of Gβ1 from RACK1, resulting in Gβ1 interaction with Fyn and focal adhesion kinase (FAK) required for FAK activation. RACK1 depletion triggered Gβ1 activation of FAK and endothelial barrier recovery, whereas Fyn knockdown interrupted with Gβ1-induced barrier recovery indicating RACK1 negatively regulates Gβ1-Fyn signaling. Activated FAK associated with AJ and stimulated AJ reassembly in a Fyn-dependent manner. Fyn deletion prevented FAK activation and augmented lung vascular permeability increase induced by PAR1 agonist. Rescuing FAK activation in fyn–/– mice attenuated the rise in lung vascular permeability. Our results demonstrate that Gβ1-mediated Fyn activation integrates FAK with AJ, preventing persistent endothelial barrier leakiness.

Abbreviations: Ab, antibody • AJ, adherens junctions • CT-βARK-1, C terminus of β-adrenergic receptor kinase 1 • EBA, Evans blue–labeled albumin • EBAE, Evans blue albumin extravasation • ECM, extracellular matrix • FAK, focal adhesion kinase • HPAE, human pulmonary arterial endothelial • PAR1, protease-activating receptor 1 • RACK1, receptor for activated C kinase 1 • siRNA, small interfering RNA • TER, transendothelial electrical resistance

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