Adenosine A2A-dopamine D2 receptor-receptor heteromerization: Qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer

Meritxell Canals, Daniel Marcellino, Francesca Fanelli, Francisco Ciruela, Piero De Benedetti, Steven R. Goldberg, Kim Neve, Kjell Fuxe, Luigi F. Agnati, Amina S. Woods, Sergi Ferré, Carme Lluis, Michel Bouvier, Rafael Franco

Research output: Contribution to journalArticlepeer-review

369 Scopus citations


There is evidence for strong functional antagonistic interactions between adenosine A2A receptors (A2ARs) 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 A2AR-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 A2AR-D2R interactions in cotransfected cells. The degree of A2AR-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 A2AR. Modeling of A2AR 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 A2AR. 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 A2AR, respectively.

Original languageEnglish (US)
Pages (from-to)46741-46749
Number of pages9
JournalJournal of Biological Chemistry
Issue number47
StatePublished - Nov 21 2003
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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