title: G protein-coupled receptor kinase-5 regulates thrombin-activated signaling in endothelial cells.
abstract: We studied the function of G protein-coupled receptor kinases (GRKs) in the regulation of thrombin-activated signaling in endothelial cells. GRK2, GRK5, and GRK6 isoforms were expressed predominantly in endothelial cells. The function of these isoforms was studied by expressing wild-type and dominant negative (dn) mutants in endothelial cells. We determined the responses to thrombin, which activates intracellular signaling in endothelial cells by cleaving the NH(2) terminus of the G protein-coupled proteinase-activated receptor-1 (PAR-1). We measured changes in phosphoinositide hydrolysis and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to thrombin as well as the state of endothelial activation. In the latter studies, the transendothelial monolayer electrical resistance, a measure of the loss of endothelial barrier function, was measured in real time. Of the three isoforms, GRK5 overexpression was selective in markedly reducing the thrombin-activated phosphoinositide hydrolysis and increased [Ca(2+)](i). GRK5 overexpression also inhibited the thrombin-induced decrease in endothelial monolayer resistance by 75%. These effects of GRK5 overexpression occurred in association with the specific increase in the thrombin-induced phosphorylation of PAR-1. In contrast to the effects of GRK5 overexpression, the expression of the dn-GRK5 mutant produced a long-lived increase in [Ca(2+)](i) in response to thrombin, whereas dn-GRK2 had no effect. These results indicate the crucial role of the GRK5 isoform in the mechanism of thrombin-induced desensitization of PAR-1 in endothelial cells.
authors.affiliation: Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL 60612, USA. tiruc@uic.edu
authors.forename: C
authors.initials: C
authors.lastname: Tiruppathi
authors.forename: W
authors.initials: W
authors.lastname: Yan
authors.forename: R
authors.initials: R
authors.lastname: Sandoval
authors.forename: T
authors.initials: T
authors.lastname: Naqvi
authors.forename: A N
authors.initials: AN
authors.lastname: Pronin
authors.forename: J L
authors.initials: JL
authors.lastname: Benovic
authors.forename: A B
authors.initials: AB
authors.lastname: Malik
mesh_terms: D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D054769:G-Protein-Coupled Receptor Kinase 2; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; C517898:GRK5 protein, human; D013917:Thrombin