TY - JOUR
T1 - Adenosine A2A-dopamine D2 receptor-receptor heteromerization
T2 - Qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer
AU - Canals, Meritxell
AU - Marcellino, Daniel
AU - Fanelli, Francesca
AU - Ciruela, Francisco
AU - De Benedetti, Piero
AU - Goldberg, Steven R.
AU - Neve, Kim
AU - Fuxe, Kjell
AU - Agnati, Luigi F.
AU - Woods, Amina S.
AU - Ferré, Sergi
AU - Lluis, Carme
AU - Bouvier, Michel
AU - Franco, Rafael
PY - 2003/11/21
Y1 - 2003/11/21
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0344875553&partnerID=8YFLogxK
U2 - 10.1074/jbc.M306451200
DO - 10.1074/jbc.M306451200
M3 - Article
C2 - 12933819
AN - SCOPUS:0344875553
SN - 0021-9258
VL - 278
SP - 46741
EP - 46749
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 47
ER -