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J Pharmacol Exp Ther. 2001 Sep;298(3):1243-51.

Phosphorylation and desensitization of the human thromboxane receptor-alpha by G protein-coupled receptor kinases.

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Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082, USA.


The thromboxane A(2) receptor (TP), which mediates vasoconstriction, mitogenesis, and platelet aggregation, has been shown to undergo rapid agonist-induced desensitization. Two isoforms (alpha and beta) of TP have been recognized. The potential role of the G protein-coupled receptor kinases (GRKs) in the phosphorylation and desensitization of TP alpha was investigated. Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged TP alpha was used to study the phosphorylation and desensitization of the receptor. Rapid isolation of the (32)P-labeled receptor was achieved by Ni(2+)-nitrilotriacetic acid agarose after agonist stimulation of HEK293 cells prelabeled with (32)P(i). [1S-[1 alpha,2 alpha(Z),3 beta(1E,3S*),4 alpha]]-7-[3-[3-Hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid (I-BOP) induced receptor phosphorylation and Ca(2+) release in a time- and dose-dependent manner. Pretreatment of cells with I-BOP abolished subsequent induction of Ca(2+) release through a second dose of I-BOP. Transfection with expression plasmids encoding the cDNA of GRK5 or GRK6 augmented I-BOP-induced phosphorylation and inhibited I-BOP-stimulated Ca(2+) release. Both I-BOP-induced and GRK-mediated phosphorylation and phorbol ester-induced phosphorylation were blocked by the addition of 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) (GF 109203X). This indicates that GF 109203X, a known protein kinase C (PKC) inhibitor, also inhibits GRKs. This finding was further supported by in vitro studies in which preparations of GRK5 and GRK6 were found to be inhibited by GF 109203X. These results suggest that GRK5 and GRK6 may phosphorylate the TP alpha in an agonist-dependent manner. Furthermore, the results obtained with PKC inhibitors in assessing the role of PKC in agonist-induced receptor phosphorylation should be interpreted with caution.

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