Detection of receptor heteromers involving dopamine receptors by the sequential BRET-FRET technology

Abstract

Until very recently, dopamine receptors, like other G-protein-coupled receptors, were believed to function as individual units on the cell surface. Now it has been described by several groups including ours that dopamine receptors not only function as homomers but also form heteromers with other receptors at the membrane level. Bioluminescence and fluorescence resonance energy transfer (BRET and FRET) based techniques have been very useful to determine the interaction between two receptors, but to demonstrate the existence of higher-order complexes involving more than two molecules requires more sophisticated techniques. Combining BRET and FRET in the Sequential BRET-FRET (SRET) technique permits heteromers formed by three different proteins to be identified. In SRET experiments, the oxidation of a Renilla Luciferase substrate triggers acceptor excitation by BRET and subsequent energy transfer to a FRET acceptor. Using this methodology here we describe the heteromerization between adenosine A(2A), dopamine D(2), and cannabinoids CB(1) receptors in living cells.

Fig. 1.

Sequential BRET-FRET (SRET). SRET combines BRET and FRET involving two energy donors and two acceptors. BRET and FRET techniques are combined to detect heterotrimers at the membrane level. Signal is initiated by oxidation of DeepBlueC by the Rluc-fused protein (A_2AR-Rluc) that generates light emission at the indicated wavelength (blue). The acceptor in BRET is a GFP²-fused protein (D₂R-GFP²) that, after excitation, results in emission at the indicated wavelength (green) that excites a YFP-fused protein (CB₁R-YFP) by a FRET process with concomitant light emission peaking at the indicated wavelength (yellow). Emission of YFP after addition of the Rluc substrate is only possible if the three fusion proteins are in close proximity (<10 nm) allowing bioluminescent and fluorescent sequential resonance energy transfer (SRET) to occur. A representation of excitation (top) and emission (bottom) spectra of fused proteins is shown in the right.

Fig. 2.

SRET for A_2AR, D₂R, and CB₁R in living cells. SRET assays are performed 48 h post-transfection in cells expressing A_2AR-Rluc (2 μg of cDNA; approximately 100,000 luminescence units), D₂R-GFP² (3 μg of cDNA; approximately 6,000 fluorescence units), and CB₁R-YFP (9 μg of cDNA; approximately 18,000 fluorescence units) or the equivalent amounts of the fluorescence or luminescence proteins or transfected with the positive SRET construct (1 μg of cDNA of Rluc-GFP²-YFP construct). Net SRET was obtained by monitoring the YFP fluorescence emission after DeepBlueC addition, with subtraction of the value obtained with cells expressing the same amount of A_2AR-Rluc and the corresponding BRET acceptor. Significant net SRET was detected for A_2AR-Rluc/D₂R-GFP²/CB₁R-YFP coupling or for the positive SRET control, while negligible net SRET was obtained in cells expressing equivalent amounts of A_2AR-Rluc, GFP², and CB₁R-YFP, or A_2AR-Rluc, D₂R-GFP², and YFP. Data are expressed as the mean net SRET ± S.E.M. of four independent experiments performed in duplicate. One-way ANOVA followed by Newman-Keuls test showed significant differences with respect to negative controls (***: P < 0.001).

Fig. 3.

SRET assays are performed 48 h post-SRET saturation curve for A_2AR-D₂R-CB₁R heteromers in living cells. SRET saturation,curves were obtained using HEK-293T cells transfected with 2 μg of the cDNA for A_2AR-Rluc (approximately 100,000 luminescence units) and 3 μg of the cDNA for D₂R-GFP² (approximately 6,000 fluorescence units) and increasing amounts of the cDNA for CB₁R-YFP (8,000 to 18,000 fl uorescence units). Values, expressed as net SRET, represent the mean ± S.E.M. of two independent experiments performed in triplicate. Negative control is constituted by cells expressing the equivalent amounts of D_4.4R-Rluc/A_2AR-GFP2/CB₁R-YFP giving linear (nonspecific) SRET with similar amounts of fluorescence and luminescence as those giving saturable SRET.