Agonist-promoted heteromeric oligomerization between adenosine A(1) and P2Y(1) receptors in living cells.
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.
Yoshioka
Kazuaki
K
Department of Molecular Cell Signaling, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, 183-8526, Tokyo, Japan.
Saitoh
Osamu
O
Nakata
Hiroyasu
H
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D001704:Biopolymers; D002460:Cell Line; D004735:Energy Transfer; D006801:Humans; D049449:Luminescence; D018613:Microscopy, Confocal; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D058906:Purinergic P1 Receptor Agonists; D058910:Purinergic P2 Receptor Agonists; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000378:metabolism
D001704:Biopolymers; C546382:P2RY1 protein, human; D058906:Purinergic P1 Receptor Agonists; D058910:Purinergic P2 Receptor Agonists; D018047:Receptors, Purinergic P1; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D011993:Recombinant Fusion Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); C048599:N(6)-cyclopentyladenosine; D000255:Adenosine Triphosphate; D000241:Adenosine