Determination of ligand binding affinities at the human adenosine A1-receptor using the fluorescent ligand xanthine amine congener-bodipy ® 630/650

Alison Carter1 , Eleanor Henshaw2, Kirsty Rich2, Stephen Hill1&3. 1CellAura Technologies Ltd, Nottingham, United Kingdom, 2Lead Generation, Molecular Biology, AstraZeneca R&D Charnwood, Loughborough, United Kingdom, 3Institute of Cell Signalling, Medical School, Queen’s Medical Centre, Nottingham, United Kingdom.


Ligand binding affinities to G protein-coupled receptors have traditionally been determined using radioligand binding studies. However, safety and disposal issues associated with the use of radioligands suggest the need for a safer alternative, which does not compromise the quality of data obtained. Here, we have developed a cell-based fluorescent ligand binding assay which uses the adenosine antagonist xanthine amine congener (XAC) conjugated to the fluorophore BODIPY® 630/650 (BY630; Briddon et al., 2004), to measure binding affinities at the human adenosine A1-receptor.

A CHO cell line stably expressing the human adenosine A1-receptor (CHO-A1) was used in the present study (Briddon et al., 2004). Cells were grown to semi-confluence in 384 well black view plates (Greiner) in DMEM/F12 medium supplemented with 5% foetal calf serum at 37oC in 5% CO2/humidified air. Experiments were performed in 384 well plates in HEPES buffered saline (HBS) supplemented with 0.1% BSA and 5mM HEPES. In saturation experiments, non-specific binding was determined with 1μM unlabelled XAC. Unlabelled competitors were added 30 min prior to the addition of XAC-BY630 (40nM for competition experiments) at room temperature. Incubations were continued for 20 min at room temperature with fluorescent antagonist. Prior to fixing all buffer was removed and the cells fixed in 4% paraformaldehyde for 10 min at room temperature. Once fixed the paraformaldehyde was removed and the cells washed once in buffer and resuspended in a final volume of 40 μl/well buffer. Cellular binding of the fluorescent antagonist XAC-BY630 was then determined using the 8200 Cellular Detection System (Applied Biosystems; Mellentin-Michelotti et al., 1999 ). Whole cell saturation binding studies in CHO-A1 cells using the fluorescent XAC-BY630 ligand gave a KD value of 50.95 + 0.81nM (n = 27) which is similar to that obtained previously in membrane-based radioligand binding studies (Briddon et al., 2004). Native CHO cells (not expressing the human A1-receptor) showed no specific binding of XAC-BY630. Specific binding of XAC-BY630 to the adenosine A1-receptor in CHO-A1 cells was displaced using a range of A1-receptor ligands. The A1-selective antagonist DPCPX and the selective agonist CPA inhibited XAC-BY630 binding to give a logKi of –9.74 + 0.06 (n = 3) and –7.72 + 0.14 (n = 7) respectively. The adenosine A2a selective antagonist ZM 241385 and agonist CGS 21680 had a lower affinity for the A1-receptor with logKi values of –7.46 + 0.06 (n = 7) and –7.36 + 0.31 (n = 3) respectively. Other ligands tested gave logKi values of -7.65 + 0.09 (NECA, n =4), -8.54 + 0.02 (XAC, n = 5) and -9.49 + 0.11 (CGS 15943, n = 3). These data indicate that fluorescence technology can provide an equally sensitive approach to the determination of ligand binding affinities in intact cells.


Briddon, S.J., et al. (2004) Proc. Natl. Acad. Sci. U. S. A. 101: 4673-4678.
Mellentin-Michelotti, J., et al. (1999) Anal. Biochem. 272: 182-190.