Agonist‐promoted heteromeric oligomerization between adenosine A1 and P2Y1 receptors in living cells

FEBS Letters

Nakata, Hiroyasu¹  Yoshioka, Kazuaki¹  Saitoh, Osamu¹ 

[1] Department of Molecular Cell Signaling, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan

关键词: Purinergic receptor;    Adenosine A1 receptor;    P2Y1 receptor;    Hetero-oligomerization;    Bioluminescence resonance energy transfer;    HEK293T cell;    GPCR;    G protein-coupled receptor;    BRET;    bioluminescence resonance energy transfer;    FRET;    fluorescence resonance energy transfer;    BRET2;    improved BRET system offered by Perkin-Elmer Life Sciences;    GFP2;    modified green fluorescent protein;    Rluc;    Renilla luciferase;    A1R;    adenosine A1 receptor;    P2Y1R;    P2Y1 receptor;    HA;    hemagglutinin;    HA-A1R;    HA-tagged A1R;    Myc-P2Y1R;    Myc-tagged P2Y1R;    HA-A1R-GFP2;    HA-A1R fused to GFP2;    HA-A1R-Rluc;    HA-A1R fused to Rluc;    Myc-P2Y1R-Rluc;    Myc-P2Y1R fused to Rluc;    CPA;    N 6-cyclopentyladenosine;    ADPβS;    adenosine 5′-O-(2-thiotriphosphate);    MRS2179;    N 6-methyl-2′-deoxyadenosine-3′;    5′-bisphosphate;   

DOI  :  10.1016/S0014-5793(02)02965-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown