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

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

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CAS Key Laboratory of Receptor Research and Department of Neuropharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.



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.


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.


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.


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.

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