title: Adenosine A2A-dopamine D2 receptor-receptor heteromerization: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer.
abstract: There is evidence for strong functional antagonistic interactions between adenosine A2A receptors (A2ARs) 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 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 co-transfected 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 D2R approached helix 4 and the C-terminal portion of the C-tail from the A2AR, respectively.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Barcelona, Martí y Franquès 1, E-08028 Barcelona, Spain.
authors.forename: Meritxell
authors.initials: M
authors.lastname: Canals
authors.forename: Daniel
authors.initials: D
authors.lastname: Marcellino
authors.forename: Francesca
authors.initials: F
authors.lastname: Fanelli
authors.forename: Francisco
authors.initials: F
authors.lastname: Ciruela
authors.forename: Piero
authors.initials: P
authors.lastname: de Benedetti
authors.forename: Steven R
authors.initials: SR
authors.lastname: Goldberg
authors.forename: Kim
authors.initials: K
authors.lastname: Neve
authors.forename: Kjell
authors.initials: K
authors.lastname: Fuxe
authors.forename: Luigi F
authors.initials: LF
authors.lastname: Agnati
authors.forename: Amina S
authors.initials: AS
authors.lastname: Woods
authors.forename: Sergi
authors.initials: S
authors.lastname: Ferré
authors.forename: Carme
authors.initials: C
authors.lastname: Lluis
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
authors.forename: Rafael
authors.initials: R
authors.lastname: Franco
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D003198:Computer Simulation; D019281:Dimerization; D004735:Energy Transfer; D005453:Fluorescence; D006801:Humans; D008163:Luminescent Measurements; D008958:Models, Molecular; D011485:Protein Binding; D043705:Receptor, Adenosine A2A / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D014162:Transfection
chemical_list: D043705:Receptor, Adenosine A2A; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins