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

The thromboxane A(2) receptor (TP), which mediates vasoconstriction, mitoge nesis, and platelet aggregation, has been shown to undergo rapid agonist-in duced desensitization. Two isoforms (alpha and beta) of TP have been recogn ized. The potential role of the G protein-coupled receptor kinases (GRKs) i n the phosphorylation and desensitization of TP alpha was investigated. Hum an embryonic kidney (HEK) 293 cells stably transfected with the His-tagged TP alpha was used to study the phosphorylation and desensitization of the r eceptor. Rapid isolation of the P-32- labeled receptor was achieved by Ni2-nitrilotriacetic acid agarose after agonist stimulation of HEK293 cells pr elabeled with P-32(i). [1S-[1 alpha ,2 alpha (Z),3 beta (1E,3S*),4 alpha]]- 7-[3-[3-Hydroxy-4-(4-iodophenoxy)-1 1-butenyl]-7-oxabicyclo[2,2,1]hept-2-yl ]-5-heptenoic acid (I-BOP) induced receptor phosphorylation and Ca2+ releas e in a time- and dose-dependent manner. Pretreatment of cells with I-BOP ab olished subsequent induction of Ca2+ release through a second dose of I-BOP . Transfection with expression plasmids encoding the cDNA of GRK5 or GRK6 a ugmented I-BOP-induced phosphorylation and inhibited I-BOP-stimulated Ca2release. Both I-BOP-induced and GRK-mediated phosphorylation and phorbol es ter-induced phosphorylation were blocked by the addition of 2-[1-(3-dimethy laminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) (GF 109203X). Thi s indicates that GF 109203X, a known protein kinase C (PKC) inhibitor, also inhibits GRKs. This finding was further supported by in vitro studies in w hich 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 i n an agonist-dependent manner. Furthermore, the results obtained with PKC i nhibitors in assessing the role of PKC in agonist-induced receptor phosphor ylation should be interpreted with caution.