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


CONCLUSIONS
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.
CAS Key Laboratory of Receptor Research and Department of Neuropharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Li
L
Huang
Dong-Dong
DD
Wu
Lei
L
Zhang
Lin-Yin
LY
Feng
D058917:Adenosine A2 Receptor Antagonists / Q000494:pharmacology*; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D060807:Bioluminescence Resonance Energy Transfer Techniques; D002478:Cells, Cultured; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D055503:Protein Multimerization; D019257:Quinpirole / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D043705:Receptor, Adenosine A2A / Q000187:drug effects* / Q000378:metabolism; D017448:Receptors, Dopamine D2 / Q000187:drug effects* / Q000378:metabolism; D014227:Triazines / Q000494:pharmacology*; D014230:Triazoles / Q000494:pharmacology*; D000071557:beta-Arrestins; D019061:src-Family Kinases / Q000378:metabolism
D058917:Adenosine A2 Receptor Antagonists; D019390:Arrestins; D043705:Receptor, Adenosine A2A; D017448:Receptors, Dopamine D2; D014227:Triazines; D014230:Triazoles; C097270:ZM 241385; D000071557:beta-Arrestins; D019257:Quinpirole; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases