Adenosine A_2A-dopamine D₂ receptor-receptor heteromerization: Qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer

There is evidence for strong functional antagonistic interactions between adenosine A_2A receptors (A_2ARs) and dopamine D ₂ receptors (D₂Rs). Although a close physical interaction between both receptors has recently been shown using co-immunoprecipitation and co-localization assays, the existence of a A_2AR-D₂R protein-protein interaction still had to be demonstrated in intact living cells. In the present work, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques were used to confirm the occurrence of A_2AR-D₂R interactions in cotransfected cells. The degree of A_2AR-D₂R heteromerization, measured by BRET, did not vary after receptor activation with selective agonists, alone or in combination. BRET competition experiments were performed using a chimeric D₂R-D₁R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D₂R were replaced by those of the dopamine D₁ receptor (D₁R). Although the wild type D₂R was able to decrease the BRET signal, the chimera failed to achieve any effect. This suggests that the helix 5-I3-helix 6-E3 portion of D₂R holds the site(s) for interaction with A_2AR. Modeling of A_2AR and D₂R using a modified rhodopsin template followed by molecular dynamics and docking simulations gave essentially two different possible modes of interaction between D₂R and A_2AR. In the most probable one, helix 5 and/or helix 6 and the N-terminal portion of I3 from D ₂R approached helix 4 and the C-terminal portion of the C-tail from the A_2AR, respectively.