Biochemical and Biophysical Research Communications
Regulation of pharmacology by hetero-oligomerization between A1 adenosine receptor and P2Y2 receptor☆,☆☆
Section snippets
Experimental procedures
Immunoprecipitation and Western blot analysis. HEK293T cells were transfected using Effectene transfection reagent (Qiagen, Hilden, Germany) with plasmids for expressing rat A1R with an N-terminal HA epitope tag (YPYDVPDYA) and rat P2Y2R with an N-terminal Myc epitope tag (EQKLISEEDL) as described previously [5]. Transfected cells (approximately 107 cells) were collected and washed twice with DPBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4). Cells were then disrupted by sonication
Myc-tagged P2Y2R coimmunoprecipitates with HA-tagged A1R
To explore the formation of hetero-oligomers by A1R and P2Y2R, we performed immunoprecipitation of HA-A1R and Myc-P2Y2R using solubilized membranes from HA-A1R/Myc-P2Y2R-cotransfected HEK293T cells. We detected both Myc-P2Y2R and HA-A1R in the complex precipitated with anti-Myc (Fig. 1, upper right and left panels, respectively). In addition, as shown in the lower panel of Fig. 1, we found that Myc-P2Y2R in the complex precipitated with anti-HA antibody (right). These results indicate the
Discussion
In the present study, a direct association between A1R and P2Y2R was shown by coimmunoprecipitation in A1R/P2Y2R-cotransfected HEK293T cells. Confocal immunofluorescence and immunoelectron microscopy also showed that these receptors colocalize at the cell membrane (data not shown). Although similar hetero-oligomer formation between A1R and P2Y1R was previously reported [5], differences in the pharmacological properties of A1R/P2Y1R and A1R/P2Y2R hetero-oligomers were revealed in this study.
Acknowledgment
We thank Prof. Tatsuya Haga of Gakushuin University for helping with the Ca2+ measurements.
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This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Abbreviations: GPCR, G protein-coupled receptor; A1R, A1 adenosine receptor; P2Y1R, P2Y1 receptor; P2Y2R, P2Y2 receptor; PLCβ, phospholipase C-β; HEK, human embryonic kidney; ADPβS, adenosine 5′-O-(2-thiodiphosphate); DPCPX, 8-cyclopentyl-1, 3-dipropylxanthine; MAPK, mitogen-activated protein kinase; DPBS, Dulbecco’s phosphate-buffered saline; CPA, N6-cyclopentyladenosine; HA, hemagglutinin; NECA, 5′-N-ethylcarboxamidoadenosine; CCPA, 2-chloro-N6-cyclopentyladenosine; ADA, adenosine deaminase; FSK, forskolin; CPA, N6-cyclopentyladenosine; PTX, pertussis toxin.