We have explored the process of oligomerization of G protein-coupled purinergic receptors, adenosine A1 receptor (A1R) and P2Y1 receptor (P2Y1R), in intact HEK293T cells by means of modified bioluminescence resonance energy transfer technology (BRET2) that offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared to traditional BRET. This approach identified both constitutive and agonist-promoted heteromeric oligomerization between Myc-tagged P2Y1R fused to a donor, Renilla luciferase (Myc-P2Y1R-Rluc) and HA-tagged A1R fused to an acceptor, a different form of green fluorescent protein (HA-A1R-GFP2). The BRET2 signal increased in a time-dependent manner in the cells expressing HA-A1R-GFP2/Myc-P2Y1R-Rluc upon addition of agonists for both receptors, which could be inhibited by pretreatment with the P2Y1R antagonist MRS2179. A high degree of HA-A1R-GFP2 and Myc-P2Y1R-Rluc co-localization in the co-transfected HEK293T cells was also observed by confocal laser microscopy. These results indicate that A1R and P2Y1R can form constitutive hetero-oligomers in living cells and this process is promoted by the simultaneous activation of both receptors.
