Agonist-promoted heteromeric oligomerization between adenosine A 1 and P2Y 1 receptors in living cells

Abstract

We have explored the process of oligomerization of G protein-coupled purinergic receptors, adenosine A 1 receptor (A 1R) and P2Y 1 receptor (P2Y 1R), in intact HEK293T cells by means of modified bioluminescence resonance energy transfer technology (BRET 2) 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 P2Y 1R fused to a donor, Renilla luciferase (Myc-P2Y 1R-Rluc) and HA-tagged A 1R fused to an acceptor, a different form of green fluorescent protein (HA-A 1R-GFP 2). The BRET 2 signal increased in a time-dependent manner in the cells expressing HA-A 1R-GFP 2/Myc-P2Y 1R-Rluc upon addition of agonists for both receptors, which could be inhibited by pretreatment with the P2Y 1R antagonist MRS2179. A high degree of HA-A 1R-GFP 2 and Myc-P2Y 1R-Rluc co-localization in the co-transfected HEK293T cells was also observed by confocal laser microscopy. These results indicate that A 1R and P2Y 1R can form constitutive hetero-oligomers in living cells and this process is promoted by the simultaneous activation of both receptors.