Adenosine A2A Receptor Ligand Binding Experiments by Using Real-time Single-cell FRET

Authors

Víctor Fernández-Dueñas, Kenneth A. Jacobson and Francisco Ciruela

Summary

We designed a fluorescence resonance energy transfer (FRET)-based approach to study the ligand binding constants of the adenosine A2A receptor (A2AR). Our assay is based in the interaction of a fluorescent A2AR agonist ligand (MRS5424) with an A2AR tagged with the cyan fluorescent protein (CFP) at the N-terminus (i.e. A2ARCFP) and expressed in living cells. Thus, upon fast superfusion of the A2ARCFP expressing cells with MRS5424, the ligand-receptor interaction is determined by single-cell FRET in a real-time mode. Accordingly, our approach allowed immediate ‘real-time’ readout of the ligand-receptor interaction, thus allowing kinetic binding experiments, a feature impossible to achieve using conventional radioisotope-labelled ligands. In addition, since our assay permitted the visual confirmation of receptor localization it also allowed localized saturation binding experiments.

Materials

1. Cell line (i.e. HEK-293 cells)
2. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich)
3. Sodium pyruvate
4. L-glutamine
5. Antibiotics: streptomycin and penicillin
6. Fetal bovine serum
7. TransFectinTM Lipid Reagent (Bio-Rad Laboratories)
8. Hank’s balanced salt solution (HBSS) (see Recipes)
9. Cell culture medium (see Recipes)

Equipment

1. 18 mm diameter glass coverslips
2. Attofluor holder
3. Inverted Axio Observer microscope (ZEISS) equipped with a 63x oil immersion objective
4. Polychrome V (TILL Photonics)
5. Avalanche photodiodes (TILL Photonics)
6. Focal drug application system (ALA Scientific Instruments, OCTAFLOWTM)
7. Digidata 1440A analog/digital converter (Molecular Devices)

Software

1. pCLAMP (Molecular Devices)
2. GraphPad Prism (GraphPad Software)

Procedure

1. Two days before the experiment, the cells (i.e. HEK-293 cells) were seeded onto 18 mm diameter glass coverslips and transiently transfected with an A2AR construct tagged with the CFP at its N-terminal tail (A2ARCFP) .
2. The day of the experiment the transiently transfected cells were mounted in an Attofluor holder and placed on an inverted Axio Observer microscope equipped with a 63x oil immersion objective and a dual-emission photometry system.
3. Then, cells were continuously superfused with a FRET-compatible A2AR fluorescent ligand (i.e. MRS5424) (Fernández-Dueñas et al., 2012) dissolved in HBSS and applied with the aid of a focal drug application system.
4. A Polychrome V was used as the light source in our dual-emission photometry system. Upon excitation with the corresponding donor excitation wavelength (i.e. A2ARCFP) the fluorescent signals of the donor and acceptor fluorophores were detected by avalanche photodiodes and digitized using a Digidata 1440A analog/digital converter.
5. pCLAMP and GraphPad Prism softwares were used for data collection and analysis.
6. Accordingly, a FRET signal was measured upon donor (i.e. A2ARCFP) excitation at 430 ± 10 nm [beam splitter dichroic long-pass (DCLP) 460 nm] and an illumination time set to 10 ms at 10 Hz. Then, the emission light intensities were determined at 535 ± 15 nm (F535; MRS5424 emission) and 480 ± 20 nm (F480; A2ARCFP emission) with a beam splitter DCLP of 505 nm. No corrections for spillover between channels or direct MRS5424 excitation were made.
7. The increase in FRET ratio (F535/F480) was fitted to the equation: r(t) = A x (1 - e-t/τ), where τ is the time constant (in seconds) and A is the magnitude of the FRET signal (Figure 2). When necessary for calculating τ, agonist-independent changes in FRET due to photobleaching were subtracted (Fernández-Dueñas et al., 2012, Fernández-Dueñas et al., 2013).

Recipes

1. HBSS

137 mM NaCl
5.4 mM KCl
0.3 mM Na2HPO4
0.4 mM KH2PO4
4.2 mM NaHCO3
1.3 mM CaCl2
0.5 mM MgCl2
0.6 mM MgSO4
5.6 mM glucose
pH 7.4

2. Cell culture medium

Dulbecco’s modified Eagle’s medium (DMEM) supplemented with:
1 mM sodium pyruvate
2 mM L-glutamine
100 U/ml streptomycin
100 mg/ml penicillin
5% (v/v) fetal bovine serum

References

1. Fernandez-Duenas, V., Gomez-Soler, M., Jacobson, K. A., Kumar, S. T., Fuxe, K., Borroto-Escuela, D. O. and Ciruela, F. (2012). Molecular determinants of A2AR-D2R allosterism: role of the intracellular loop 3 of the D2R. J Neurochem 123(3): 373-384.
2. Fernández-Dueñas, V., Gómez-Soler, M., Morato, X., Núñez, F., Das, A., Kumar, T. S., Jaumà, S., Jacobson, K. A. and Ciruela, F. (2013). Dopamine D2 receptor-mediated modulation of adenosine A2A receptor agonist binding within the A2AR/D2R oligomer framework. Neurochem Inter 63(1): 42-46.

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