Abstract

The thromboxane A₂ receptor (TP), which mediates vasoconstriction, mitogenesis, and platelet aggregation, has been shown to undergo rapid agonist-induced desensitization. Two isoforms (α and β) of TP have been recognized. The potential role of the G protein-coupled receptor kinases (GRKs) in the phosphorylation and desensitization of TPα was investigated. Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged TPα was used to study the phosphorylation and desensitization of the receptor. Rapid isolation of the ³²P-labeled receptor was achieved by Ni²⁺-nitrilotriacetic acid agarose after agonist stimulation of HEK293 cells prelabeled with³²P_i. [1S-[1α,2α(Z),3β(1E,3S*),4α]]-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 Ca²⁺release in a time- and dose-dependent manner. Pretreatment of cells with I-BOP abolished subsequent induction of Ca²⁺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 Ca²⁺ 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 TPα 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.