Dr. Séverine Morisset-Lopez utilizes Bioluminescence Resonance Energy Transfer (BRET)—a powerful technique in neuropharmacology for identifying and characterizing new ligands of G protein-coupled receptors (GPCRs). This method enables real-time, live-cell monitoring of protein-protein interactions and receptor activity, providing valuable insights into the dynamics of GPCR signaling and facilitating the discovery of novel therapeutic compounds.

This video is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives (CC BY-NC-ND) license. You are free to share it for non-commercial purposes with proper attribution, but no modifications or adaptations are allowed.

1.   Why use this method?

The Bioluminescence Resonance Energy Transfer (BRET) is a proximity-based assay used to study protein-protein interactions in living cells. The principle behind BRET involves resonance transfer of energy between bioluminescent enzymes and a fluorescent acceptors, resulting in measurable signals that indicate specific molecular interactions. BRET has been widely adopted in drug discovery as a high-throughput screening method to investigate the behavior of signaling proteins, such as G protein-coupled receptors (GPCRs). Due to its ability to operate under physiological conditions, BRET is a highly sensitive and well-suited tool for real-time, dynamic studies regarding diverse signaling pathways, their downstream messengers, receptor internalization and more.

2.  What you’ll need

Materials:

• Plasmids: e.g., GPCR-NanoLuc and β-arrestin-YFP constructs – for NanoBRET
• Cell line: e.g., HEK293 or primary neuronal culture
• Substrate of choice: e.g., NanoBRET Nano-Glo substrate (furimazine)
• GPCR agonist (ligand)

Reagents:

• Culture medium: Dulbecco’s Modified Eagle Medium (DMEM) + 10%  fetal bovine serum (FBS)
• Phosphate-Buffered Saline (PBS) 
• Hank’s Buffer Saline Solution (HBSS)
• Transfection reagent: e.g., Lipofectamine 3000

Equipment:

• Plate reader equipped with emission filters for donor and acceptor (depending on the BRET type, different filters will be needed) and high sensitivity for low-light detection: e.g., Berthold LB 940 or LB943
• Cell culture hood – in order to handle cells in sterile conditions
• CO₂ incubator – to maintain optimal conditions for cell growth
• Centrifuge – for pelleting cells
• Micropipettes and multichannel pipettes – for liquid handling
• 6-well plates – for cell transfection
• White 96-well  or 384-well plates – for luminescence measurements
• Fluorescence microscope – for verification of transfection efficiency

3. Step-by-step instructions

1. Seed cells and transfect them with plasmids:
   1. Seed cells in 6-well plates and incubate them overnight at 37°C and 5% CO2
   1. Transfect cells with a 1:1 ratio with your plasmids of choice (e.g., GPCR-NanoLuc (donor) and β-arrestin-YFP (acceptor)) using transfection reagent and incubate for 24-48 hours
2. Add cells in microplates:
   1. Wash cells 2 times with PBS
   1. Detach the cells by pipetting or using EDTA
   1. Resuspend the cells in HBSS and distribute in a 96-well plate
3. Stimulate ligands and add substrate:
   1. Prepare serial dilutions of the GPCR agonist in PBS and add to the wells (do not forget about the unstimulated control)
   1. Incubate the cells for 5-30 min depending on the kinetics
   1. Add substrate and read luminescence immediately after

• Read BRET signal

Immediately measure the signal using on a microplate reader capable of sequentially detecting luminescence with two filter settings (e.g., Nluc filter, 480 ± 10 nm; YFP filter, 540 ± 20 nm).

• Analyze the data:
  • Calculate BRET ratio (acceptor/ donor)
  • Subtract background (the unstimulated control)
  • Plot BRET ratio vs agonist concentration to determine EC₅₀
  •  

Alternative protocol : BRET can be  performed on adherent cells

For certain specific interactions, BRET can be performed on adherent cells. In this case, transfection is first carried out in suspension, after which the cells along with the plasmids and transfection reagents are distributed in microplates.

1. Add transfected cells in microplates:
   1. Seed cells in 10cm plates and incubate them overnight at 37°C and 5% CO2
   1. Detach and resuspend the cells in culture medium
   1. Mix cells in suspension with your plasmids of choice (e.g., GPCR-NanoLuc (donor) and β-arrestin-YFP (acceptor)) at a 1:1 ratio (which may need to be optimized depending on the interaction being studied) using a transfection reagent.
   1. Distribute the cells in 96 well-pates (around 10⁵ cells/well)
   1. Incubate for 24 hours
   1. Rinse the cells and add fresh medium
   1. Incubate for 24 hours

Then proceed with the same protocol used for suspension cells (step 3)

4. Practical tips

• Ensure that donor fusion does not affect pharmacology of the receptor/ transfection does not affect the cells
• Maintain consistent expression levels to avoid saturation or insufficient signal
• Use white plates to reduce signal cross-talk and improve detection sensitivity
• Protect the plate from light during detection to minimize signal degradation
• For accurate interpretation always include negative controls (e.g., no ligand, donor-only, or acceptor-only)
• Carefully calibrate your donor/ acceptor ratio to avoid misleading data

5.  Critical appraisal & implications for future research

BRET has been established as a sensitive and reliable technique for investigating protein–protein interactions particularly in the context of GPCR-mediated signaling. The method allows real-time monitoring of dynamic molecular interactions without the interference introduced by external light sources. This is especially critical in live-cell assays, where photobleaching and phototoxicity can significantly compromise data quality.

Despite these advantages, BRET also presents several limitations. The need for specialized equipment and the high costs of the substrates can limit its routine application. Additionally, the genetic fusion of donor and acceptor tags to proteins of interest may alter the structure and function of studied biomolecules, potentially leading to unreliable results.

Nevertheless, BRET has become a powerful tool in drug discovery, particularly for high-throughput screening of novel GPCR ligands. The method is uniquely suited for the identification and characterization of biased ligands and allosteric modulators, where subtle changes in interaction dynamics must be accurately detected. Future research may focus on developing more cost-effective and stable alternatives to current substrates, reducing the financial barrier. Additionally, engineering donor and acceptor systems with adjustable affinities or self-optimizing ratios could minimize the need for extensive preliminary optimization, further increasing the accessibility of BRET in neuropharmacological research.

References

1. El Khamlichi, C., F. Reverchon, N. Hervouet-Coste, E. Robin, N. Chopin, E. Deau, F. Madouri, C. Guimpied, C. Colas, A. Menuet, A. Inoue, A. J. Bojarski, G. Guillaumet, F. Suzenet, E. Reiter and S. Morisset-Lopez (2022). “Serodolin, a beta-arrestin-biased ligand of 5-HT7 receptor, attenuates pain-related behaviors.” Proc Natl Acad Sci U S A 119(21): e2118847119. https://doi.org/10.1073/pnas.2118847119

• El Khamlichi, C., F. Reverchon-Assadi, N. Hervouet-Coste, L. Blot, E. Reiter and S. Morisset-Lopez (2019). “Bioluminescence Resonance Energy Transfer as a Method to Study Protein-Protein Interactions: Application to G Protein Coupled Receptor Biology.” Molecules 24(3). https://doi.org/10.3390/molecules24030537Ph

This protocol is licensed under a Creative Commons Attribution-NonCommercial (CC BY-NC) license, allowing sharing and adaptation for non-commercial purposes with proper attribution.