Cocaine produces D2R-mediated conformational changes in the adenosine A2AR-dopamine D2R heteromer

Adenosine A2A receptors (A2ARs) and dopamine D2 receptors (D2Rs) form constitutive heteromers in living cells and exhibit a strong functional antagonistic interaction. Recent findings give neurochemical evidence that extended cocaine self-administration in the rat give rise to an up-regulation of functional A2ARs in the nucleus accumbens that return to baseline expression levels during cocaine withdrawal. In the present work, the acute in vitro effects of a concentration of cocaine known to fully block the dopamine (DA) transporter without exerting any toxic actions were investigated on A2AR and D2LR formed heteromers in transiently co-transfected HEK-293T cells. In vitro treatment of cocaine was found to produce changes in D2R homodimers and in A2AR–D2R heterodimers detected through bioluminescent energy transfer (BRET). Cocaine was found to produce a time- and concentration-dependent reduction in the BRETmax between A2AR–D2LR heterodimers and D2LR homodimers, but not A2AR homodimers, indicating its effect on D2R. Cocaine was evaluated with regard to D2R binding using a human D2LR stable expressing CHO cell line and was found to produce an increase in the affinity of hD2LR for DA. At the level of G protein-coupling, cocaine produced a small, but significant increase in DA-stimulated binding of GTPγS. However, cocaine failed to modulate D2R agonist-induced inhibition of cAMP in stable hD2LR CHO cells or the gating of GIRK channels in oocytes. Taken together, these results indicate a direct and specific effect of a moderate concentration of cocaine on the DA D2LR, that results in enhanced agonist recognition, G protein-coupling and an altered conformational state of D2R homodimers and A2AR–D2R heterodimers.