title: The G protein betagamma subunit mediates reannealing of adherens junctions to reverse endothelial permeability increase by thrombin.
abstract: The inflammatory mediator thrombin proteolytically activates protease-activated receptor (PAR1) eliciting a transient, but reversible increase in vascular permeability. PAR1-induced dissociation of Galpha subunit from heterotrimeric Gq and G12/G13 proteins is known to signal the increase in endothelial permeability. However, the role of released Gbetagamma is unknown. We now show that impairment of Gbetagamma 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 Gbeta1, the predominant Gbeta isoform is sequestered by receptor for activated C kinase 1 (RACK1). Thrombin induced dissociation of Gbeta1 from RACK1, resulting in Gbeta1 interaction with Fyn and focal adhesion kinase (FAK) required for FAK activation. RACK1 depletion triggered Gbeta1 activation of FAK and endothelial barrier recovery, whereas Fyn knockdown interrupted with Gbeta1-induced barrier recovery indicating RACK1 negatively regulates Gbeta1-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 Gbeta1-mediated Fyn activation integrates FAK with AJ, preventing persistent endothelial barrier leakiness.
authors.affiliation: Center for Lung and Vascular Biology, Department of Pharmacology, University of Illinois, Chicago, IL 60612, USA.
authors.forename: Nebojsa
authors.initials: N
authors.lastname: Knezevic
authors.forename: Mohammad
authors.initials: M
authors.lastname: Tauseef
authors.forename: Tracy
authors.initials: T
authors.lastname: Thennes
authors.forename: Dolly
authors.initials: D
authors.lastname: Mehta
mesh_terms: D051702:Activating Transcription Factor 6; D022005:Adherens Junctions / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism; D002199:Capillary Permeability / Q000502:physiology*; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006490:Hemostatics / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008168:Lung / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*
chemical_list: C487807:ATF6B protein, human; D051702:Activating Transcription Factor 6; D050976:Basic-Leucine Zipper Transcription Factors; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D006490:Hemostatics; D009363:Neoplasm Proteins; D009479:Neuropeptides; C419368:RACK1 protein, human; C090294:RACK1 protein, mouse; D044463:Receptor, PAR-1; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; C494957:FYN protein, human; D051417:Focal Adhesion Kinase 1; C494960:Fyn protein, mouse; C496050:PTK2 protein, human; D051076:Proto-Oncogene Proteins c-fyn; C496051:Ptk2 protein, mouse; D013917:Thrombin; D019204:GTP-Binding Proteins; D044365:GTP-Binding Protein alpha Subunits, G12-G13