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Biochem Pharmacol. 2013 Apr 15;85(8):1171-81. doi: 10.1016/j.bcp.2013.01.021. Epub 2013 Jan 31.

Characterization by flow cytometry of fluorescent, selective agonist probes of the A(3) adenosine receptor.

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  • 1Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0810, USA.


Various fluorescent nucleoside agonists of the A3 adenosine receptor (AR) were compared as high affinity probes using radioligands and flow cytometry (FCM). They contained a fluorophore linked through the C2 or N(6) position and rigid A3AR-enhancing (N)-methanocarba modification. A hydrophobic C2-(1-pyrenyl) derivative MRS5704 bound nonselectively. C2-Tethered cyanine5-dye labeled MRS5218 bound selectively to hA3AR expressed in whole CHO cells and membranes. By FCM, binding was A3AR-mediated (blocked by A3AR antagonist, at least half through internalization), with t1/2 for association 38min in mA3AR-HEK293 cells; 26.4min in sucrose-treated hA3AR-CHO cells (Kd 31nM). Membrane binding indicated moderate mA3AR affinity, but not selectivity. Specific accumulation of fluorescence (50nM MRS5218) occurred in cells expressing mA3AR, but not other mouse ARs. Evidence was provided suggesting that MRS5218 detects endogenous expression of the A3AR in the human promyelocytic leukemic HL-60 cell line. Therefore, MRS5218 promises to be a useful tool for characterizing the A3AR.

Published by Elsevier Inc.

[PubMed - indexed for MEDLINE]
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