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

H Zhou1, F Yan, H H Tai

  • 1Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082, USA.

The Journal of Pharmacology and Experimental Therapeutics
|August 16, 2001
PubMed

Insights

G protein-coupled receptor kinases (GRKs), specifically GRK5 and GRK6, may phosphorylate the thromboxane A(2) receptor alpha (TP alpha) following agonist stimulation. This phosphorylation by GRKs can lead to TP alpha desensitization, impacting its signaling pathways.

Area of Science:

  • Biochemistry
  • Molecular Pharmacology
  • Cell Signaling

Background:

  • The thromboxane A(2) receptor (TP) mediates crucial physiological processes including vasoconstriction and platelet aggregation.
  • TP undergoes agonist-induced desensitization, a process critical for regulating cellular responses.
  • Two TP isoforms, alpha and beta, have been identified, with TP alpha's desensitization mechanism under investigation.

Purpose of the Study:

  • To investigate the role of G protein-coupled receptor kinases (GRKs) in the phosphorylation and desensitization of the thromboxane A(2) receptor alpha (TP alpha).
  • To elucidate the specific GRKs involved in TP alpha phosphorylation and their impact on receptor function.

Main Methods:

  • Utilized human embryonic kidney (HEK) 293 cells stably expressing His-tagged TP alpha.
  • Employed (32)P(i) labeling and Ni(2+)-nitrilotriacetic acid agarose for rapid isolation of phosphorylated TP alpha after agonist stimulation with I-BOP.
  • Investigated the effects of GRK5 and GRK6 overexpression and a protein kinase C (PKC) inhibitor (GF 109203X) on TP alpha phosphorylation and calcium (Ca(2+)) release.

Main Results:

  • Agonist I-BOP induced time- and dose-dependent phosphorylation of TP alpha and subsequent Ca(2+) release, with prior I-BOP treatment desensitizing the receptor.
  • Overexpression of GRK5 or GRK6 enhanced I-BOP-induced TP alpha phosphorylation but inhibited I-BOP-stimulated Ca(2+) release.
  • The PKC inhibitor GF 109203X blocked both agonist- and GRK-mediated TP alpha phosphorylation, indicating GF 109203X also inhibits GRKs, necessitating caution when interpreting PKC inhibitor effects on agonist-induced receptor phosphorylation.

Conclusions:

  • GRK5 and GRK6 may phosphorylate TP alpha in an agonist-dependent manner, contributing to receptor desensitization.
  • The findings highlight a potential role for GRKs in regulating TP alpha signaling.
  • Results suggest that the interpretation of studies using PKC inhibitors to assess PKC's role in agonist-induced receptor phosphorylation should be approached with caution due to potential off-target effects on GRKs.

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