There is evidence for strong functional antagonistic interactions between adenosine A_2A receptors (A_2ARs) and dopamine D₂ 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 co-transfected 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 D₂R approached helix 4 and the C-terminal portion of the C-tail from the A_2AR, respectively.

The function of dopamine D₃ receptors present in the striatum has remained elusive. In the present study evidence is provided for the existence of dopamine D₁–D₃ receptor heteromers and for an intramembrane D₁–D₃ receptor cross-talk in living cells and in the striatum. The formation of D₁–D₃ receptor heteromers was demonstrated by fluorescence resonance energy transfer and bioluminescence resonance energy transfer techniques in transfected mammalian cells. In membrane preparations from these cells, a synergistic D₁–D₃ intramembrane receptor-receptor interaction was observed, by which D₃ receptor stimulation enhances D₁ receptor agonist affinity, indicating that the D₁–D₃ intramembrane receptor-receptor interaction is a biochemical characteristic of the D₁–D₃ receptor heteromer. The same biochemical characteristic was also observed in membrane preparations from brain striatum, demonstrating the striatal co-localization and heteromerization of D₁ and D₃ receptors. According to the synergistic D₁–D₃ intramembrane receptor-receptor interaction, experiments in reserpinized mice showed that D₃ receptor stimulation potentiates D₁ receptor-mediated behavioral effects by a different mechanism than D₂ receptor stimulation. The present study shows that a main functional significance of the D₃ receptor is to obtain a stronger dopaminergic response in the striatal neurons that co-express the two receptors.