Modulation of A₂a receptor antagonist on D₂ receptor internalization and ERK phosphorylation

Acta Pharmacol Sin. 2013 Oct;34(10):1292-300. doi: 10.1038/aps.2013.87. Epub 2013 Aug 12.

Abstract

Aim: To explore the effects of heterodimerization of D2 receptor/A2a receptor (D2R/A2aR) on D2R internalization and D2R downstream signaling in primary cultured striatal neurons and HEK293 cells co-expressing A2aR and D2R in vitro.

Methods: Primary cultured rat striatal neurons and HEK293 cells co-expressing A2aR and D2R were treated with A2aR- or D2R-specific agonists. D2R internalization was detected using a biotinylation assay and confocal microscopy. ERK, Src kinase and β-arrestin were measured using Western blotting. The interaction between A2aR and D2R was detected using bioluminescence resonance energy transfer (BRET) and immunoprecipitation.

Results: D2R and A2aR were co-localized and formed complexes in striatal neurons, while both the receptors formed heterodimers in the HEK293 cells. In striatal neurons and the HEK293 cells, the D2R agonist quinpirole (1 μmol/L) marked increased Src phosphorylation and β-arrestin recruitment, thereby D2R internalization. Co-treatment with the A2aR antagonist ZM241385 (100 nmol/L) significantly attenuated these D2R-mediated changes. Furthermore, both ZM241385 (100 nmol/L) and the specific Src kinase inhibitor PP2 (5 μmol/L) blocked D2R-mediated ERK phosphorylation. Moreover, expression of the mutant β-arrestin (319-418) significantly attenuated D2R-mediated ERK phosphorylation in HEK293 cells expressing both D2R and A2aR, but not in those expressing D2R alone.

Conclusion: A2aR antagonist ZM241385 significantly attenuates D2R internalization and D2R-mediated ERK phosphorylation in striatal neurons, involving Src kinase and β-arrestin. Thus, A2aR/D2R heterodimerization plays important roles in D2R downstream signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine A2 Receptor Antagonists / pharmacology*
  • Animals
  • Arrestins / metabolism
  • Bioluminescence Resonance Energy Transfer Techniques
  • Cells, Cultured
  • Corpus Striatum / drug effects
  • Corpus Striatum / metabolism
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • HEK293 Cells
  • Humans
  • Neurons / drug effects
  • Neurons / metabolism
  • Phosphorylation / drug effects
  • Protein Multimerization
  • Quinpirole / pharmacology
  • Rats
  • Rats, Sprague-Dawley
  • Receptor, Adenosine A2A / drug effects*
  • Receptor, Adenosine A2A / metabolism
  • Receptors, Dopamine D2 / drug effects*
  • Receptors, Dopamine D2 / metabolism
  • Triazines / pharmacology*
  • Triazoles / pharmacology*
  • beta-Arrestins
  • src-Family Kinases / metabolism

Substances

  • Adenosine A2 Receptor Antagonists
  • Arrestins
  • Receptor, Adenosine A2A
  • Receptors, Dopamine D2
  • Triazines
  • Triazoles
  • ZM 241385
  • beta-Arrestins
  • Quinpirole
  • src-Family Kinases
  • Extracellular Signal-Regulated MAP Kinases