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Agonist‐promoted heteromeric oligomerization between adenosine A 1 and P2Y 1 receptors in living cells
Author(s) -
Yoshioka Kazuaki,
Saitoh Osamu,
Nakata Hiroyasu
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)02965-4
Subject(s) - receptor , purinergic receptor , chemistry , agonist , transfection , hek 293 cells , p2y receptor , g protein coupled receptor , adenosine , microbiology and biotechnology , green fluorescent protein , bioluminescence , luciferase , biophysics , biochemistry , biology , gene
We have explored the process of oligomerization of G protein‐coupled purinergic receptors, adenosine A 1 receptor (A 1 R) and P2Y 1 receptor (P2Y 1 R), 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 1 R fused to a donor, Renilla luciferase (Myc‐P2Y 1 R‐Rluc) and HA‐tagged A 1 R fused to an acceptor, a different form of green fluorescent protein (HA‐A 1 R‐GFP 2 ). The BRET 2 signal increased in a time‐dependent manner in the cells expressing HA‐A 1 R‐GFP 2 /Myc‐P2Y 1 R‐Rluc upon addition of agonists for both receptors, which could be inhibited by pretreatment with the P2Y 1 R antagonist MRS2179. A high degree of HA‐A 1 R‐GFP 2 and Myc‐P2Y 1 R‐Rluc co‐localization in the co‐transfected HEK293T cells was also observed by confocal laser microscopy. These results indicate that A 1 R and P2Y 1 R can form constitutive hetero‐oligomers in living cells and this process is promoted by the simultaneous activation of both receptors.