G protein-coupled receptor kinase-5 regulates thrombin-activated signalingin endothelial cells

We studied the function of G protein-coupled receptor kinases (GRKs) in the regulation of thrombin-activated signaling in endothelial cells. GRK2, GRK 5, and GRK6 isoforms were expressed predominantly in endothelial cells. The function of these isoforms was studied by expressing wild-type and dominan t negative (dn) mutants in endothelial cells. We determined the responses t o thrombin, which activates intracellular signaling in endothelial cells by cleaving the NH2 terminus of the G protein-coupled proteinase-activated re ceptor-1 (PAR-1). We measured changes in phosphoinositide hydrolysis and in tracellular Ca2+ concentration ([Ca2+](i)) in response to thrombin as well as the state of endothelial activation. In the latter studies, the transend othelial monolayer electrical resistance, a measure of the loss of endothel ial barrier function, was measured in real time. Of the three isoforms, GRK 5 overexpression was selective in markedly reducing the thrombin-activated phosphoinositide hydrolysis and increased [Ca2+](i). GRK5 overexpression al so inhibited the thrombin-induced decrease in endothelial monolayer resista nce by 75%, These effects of GRK5 overexpression occurred in association wi th 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 [Ca2+](i) in response to thr ombin, whereas dn-GRK2 had no effect. These results indicate the crucial ro le of the GRK5 isoform in the mechanism of thrombin-induced desensitization of PAR-1 in endothelial cells.