In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 97, No. 13 ( 2000-06-20), p. 7440-7445
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.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.97.13.7440
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2000
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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