On the Existence of a Possible A2A–D2–β-Arrestin2 Complex: A2A Agonist Modulation of D2 Agonist-Induced β-Arrestin2 Recruitment

Given that coactivation of adenosine A2A (A2AR) and dopamine D2 (D2R) receptors results in the coaggregation, cointernalization, and codesensitization of the A2AR and D2R and the role of scaffolding protein β-arrestin2 in the desensitization, internalization, and signaling of G-protein-coupled receptors, in this study we explored the ability of the A2AR agonist CGS21680 in A2AR–D2R-coexpressing cells to modulate the D2R agonist-induced recruitment of β-arrestin2 to the D2R by means of proximity-based bioluminescence resonance energy transfer (BRET2) and co-trafficking analysis. We found evidence that CGS21680 can increase the maximal BRET2 signal between β-arrestin2RLuc and D2LRGFP2 upon D2R activation, by increasing the potency of the D2R agonist to exert this action. In addition, this change was associated with an increased formation of cytoplasmic clusters containing β-arrestin2GFP2 and D2LRYFP as seen from the co-trafficking analysis. Furthermore, the A2AR agonist advanced the time for the increase in Akt phosphorylation obtained with the D2R agonist. Finally, using a novel bioinformatics approach to predict the protein–protein interface, we have also found that amino acid pro-triplets TNY, LLS, RAF, and VSR may be crucial for the -induced β-arrestin2 recruitment by A2AR–D2R heteromers. Taken together, the results indicate that the antagonistic A2AR–D2R allosteric receptor–receptor interaction in A2AR–D2R heteromers favors β-arrestin2 recruitment to the D2LR protomer with subsequent cointernalization associated with a reduced time onset of Akt phosphorylation followed by a rapid dephosphorylation. Thus, β-arrestin2 action becomes more rapid and short-lasting and, in this way, mimics G-protein-mediated signaling.

Research Highlights
► A2A agonist can increase the maximal BRET2 signal between β-arrestin2 and the D2R.
► BRET2 change is associated with an increased formation of cytoplasmic clusters.
► A2A agonist advances the time for the increase of Akt phosphorylation upon D2R activation.
► A set of amino acid pro-triplets may be crucial for β-arrestin2 recruitment.