BRET-based G Protein Biosensors for Measuring G Protein-Coupled Receptor Activity in Live Cells

G protein-coupled receptors (GPCRs) represent the largest superfamily of transmembrane receptors, responsible for transducing extracellular stimuli into intracellular signaling cascades resulting in specific biological responses. They are pivotal pharmacological targets, with approximately 30% of marketed drugs acting on GPCRs¹. GPCR ligand binding induces conformational changes in the receptor, facilitating interactions with heterotrimeric G proteins and/or GPCR kinases (GRKs) and β-arrestins, thereby initiating downstream signaling or receptor internalization².

Heterotrimeric G proteins serve as the primary intracellular transducers of GPCR signaling and consist of three subunits: G_α, G_β, and G_γ. These heterotrimers are classified into four families -- G_i/o, G_s, G_q, and G_12/13 -- based on the G_α subtype, each activating distinct signaling pathways: G_i/o subtype inhibits adenylate cyclase whereas G_s stimulates it, G_q activates phospholipase C-β, and G_12/13 regulates Rho family GTPases.

GPCR activation leads to their conformational rearrangements, enabling rapid G protein engagement within subsecond kinetics. This process induces G protein conformational changes, promoting GDP-GTP exchange in the G_α subunit. GTP binding further alters G_α subunit conformation, resulting in its dissociation from the G_βγ dimer, allowing signal propagation. G proteins are thus crucial in modulating the specificity and temporal dynamics of cellular responses by regulating diverse effector proteins such as adenylate cyclase or ion channels^3,4,5.

This protocol outlines the measurement of G protein activation or inactivation using bioluminescence resonance energy transfer (BRET)-based biosensors upon GPCR ligand stimulation in live cells. Inspired by pioneering work on G protein biosensors^6,7,8,9, the G-CASE (G protein tri-cistronic activity sensors) system, introduced by Schihada et al.¹⁰, is based on BRET between labeled G_α and G_βγ subunits and offers a streamlined approach requiring only a single plasmid transfection. This feature enhances sensitivity and mitigates challenges associated with co-transfection of multiple plasmids coding for the three subunits (G_α, G_β, and G_γ) of the heterotrimeric G protein. These sensors have been made available to academic research groups commercially (see Table of Materials).

BRET offers significant advantages over Förster Resonance Energy Transfer (FRET). In BRET, energy transfer occurs between a bioluminescent donor and a fluorescent acceptor without the need for external light sources. This intrinsic bioluminescence reduces issues associated with FRET, such as autofluorescence and light scattering, resulting in a lower background signal and improved assay sensitivity^6,7,11.

This absence of external excitation in BRET minimizes photobleaching and phototoxic effects, leading to lower background signals compared to FRET. Consequently, BRET assays most often exhibit enhanced sensitivity, allowing for the measurement of G protein activation of constitutively active GPCRs. The enhanced sensitivity of BRET assays facilitates the detection of subtle biological interactions, which is particularly valuable in pharmacological studies where precise measurement of receptor activity is crucial.

These biosensors can be translated into high-throughput screening in 96-well or 384-well plates, as recently demonstrated by Scott-Dennis et al., where a method using these biosensors has been developed in a membrane-based 384-well assay to analyze the activity of cannabinoid receptors CB1R and CB2R¹². This article describes the use of these biosensors in 96-well plates in living cells by measuring the activity of the β2-AR as a class-A prototypical GPCR, which binds G_s protein and the CB1R which belongs to class-A GPCRs and activates the G_i/o family protein. The use of two G-CASE biosensors is validated: G_s and G_i3 in HEK 293T cells with the GPCR either transiently (with the β2-AR) or stably expressed (with the CB1R).

It is important to note that this experiment can be performed in various cell lines, demonstrating the versatility and robustness of this technique across various cellular contexts. This ensures that the method can be applied to diverse experimental models, making it a valuable tool for studying GPCR signaling in different physiological and pharmacological settings. Figure 1 illustrates the principle of BRET-based G protein activity sensors.

Figure 1: Principle of BRET-based G protein activity sensors. G protein sensors consist of three subunits: G_α, native G_β, and G_γ. The G_α subunit is fused to the small and bright NanoLuciferase (Nluc), while the G_γ subunit is N-terminally labeled with circularly permuted Venus (cpVenus¹⁷³). The genes encoding these engineered G protein subunits are combined into a single plasmid. Ligand binding to GPCRs induces GPCR conformational changes, promoting G protein recruitment and subsequent dissociation followed by GTP hydrolysis. This dissociation disrupts energy transfer between the partners, resulting in a decrease in the BRET signal. Please click here to view a larger version of this figure.

The reagents and the equipment used in this study are listed in the Table of Materials.

1. Well plate seeding (Day 1)

NOTE: Follow all cell culture protocols in a sterile laminar flow hood to maintain sterile conditions. White well plates with transparent flat bottoms are used to follow cell growth and viability. Use the full white well plate to avoid using a sticker in step 3.2.1.

1. Well plate coating
   NOTE: Pre-coated plates may be used to bypass this step.
   1. Pour into a multichannel reservoir around 6 mL of sterile-filtered Poly-L-lysine (PLL) solution (0.01%). With a multichannel pipette, fill each well of a 96 white-well microplate with 60 µL of PLL.
   2. Place the microplate in a dark place and incubate for 20 min.
   3. During the incubation, proceed with the preparation of the cells as described in steps 1.2.1-1.2.3.
   4. Aspirate the PLL solution with the multichannel pipette and store it at 4 °C in a 15 mL tube.
   5. Wash the plate three times with DPBS in order to remove free PLL that might cause cell degradation.
2. Cell preparation and plating
   1. Culture HEK293 cells in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in 5% CO₂.
      NOTE: FBS is added to the culture medium to provide the necessary nutrients and growth factors required for proper cell viability.
   2. Remove the medium and rinse the cells with 2 mL of DPBS.
   3. Add 0.5 mL of trypsin and incubate for 3-5 min at 37 °C in order to detach the cells.
   4. Add 4.5 mL of DMEM to the flask to neutralize trypsin, then pipette up and down repeatedly to detach the cells and resuspend them.
   5. Transfer the detached cells to a 15 mL tube and centrifuge for 5 min at 1000 x g (at room temperature, RT).
   6. Remove the supernatant and resuspend the pellet in 2 mL of DMEM.
   7. Count the cells with a Malassez counting chamber and prepare 9 mL at 300,000 cells/mL. Put them into a multichannel reservoir and seed the 96-well microplate by pouring 100 µL per well with the multichannel pipette (final density of 30,000 cells/well).
   8. Incubate the microplate à 37 °C, 5% CO₂ for approximately 24 h.
      NOTE: The cells should reach around 70%-80% confluence for an efficient transfection.

2. Plasmid transfection (Day 2)

NOTE: Cell transfection can be performed on day one on suspended cells before plating, during step 1.2.7, and data acquisition on day 3.

1. Dilute 10 µg of the desired plasmids DNA (e.g., 5 µg of β2-adrenergic receptor and 5 µg of G_s(short)-CASE in HEK wt cells or only 10 µg of G_i3-CASE in HEK CB1 cells) and 20 µL Lipofectamine in the tubes containing 500 µL of Opti-MEM medium. Incubate at RT for 5 min.
   1. For a negative control, replace the β2-adrenergic receptor with empty plasmid pcDNA 3.1 at the same concentration. Combine the solutions into one tube and mix to obtain a transfection mixture (1 mL).
2. Incubate the transfection mixture at RT for 20 min.
3. Add 10 µL of transfection mixture from step 2.2 dropwise to each well, and gently rock the plate back and forth to ensure complete mixing.
   NOTE: A final concentration of 100 ng of DNA per well is obtained.
4. Incubate the microplate in a humidified incubator at 37 °C, 5% CO₂ for 24 h.

3. Data acquisition (Day 3)

1. Ligand preparation
   NOTE: Hank's Balanced Salt Solution (HBSS) buffer: NaCl 140 mM, Potassium Chloride 5 mM, Calcium Chloride 1 mM, Magnesium Sulfate Heptahydrate 0.4 mM, Magnesium Chloride Hexahydrate 0.5 mM, Sodium Phosphate Dibasic Dihydrate 0.3 mM, Potassium Phosphate Monobasic 0.4 mM, D-Glucose (Dextrose) 6 mM, Sodium bicarbonate 4 mM, pH 7.4, filtered 0.22 µm.
   1. Prepare a stock solution at the highest concentration possible in the appropriate solubilization solvent for the different ligands (in this protocol, isoproterenol is prepared in H₂0 mQ and WIN-55.212-2 in DMSO).
      NOTE: The concentration of the ligand stock solution needs to be adjusted depending on the solubility and the known dissociation constant (K_d) of the ligand.
   2. From this stock, prepare a ligand serial dilution ranging around the K_d of the ligand, of 10 µL in the appropriate solubilization solvent. Store this serial dilution at -20 °C.
      NOTE: Depending on the ligand, the serial dilution range can be frozen and thawed up to ten times before degradation occurs. Prepare a solution containing only the solvent used for ligand dissolution to include a vehicle condition (in this protocol, H₂0 or DMSO).
   3. For each ligand concentration and vehicle, perform a 1/100 dilution in HBSS by adding 1.3 µL of each concentration to a total volume of 130 µL. This results in a final concentration of 1% vehicle in HBSS.
      NOTE: This final dilution range is the one used for experiments by adding 10 µL to obtain 100 µL total volume per well. This results in a final concentration of 0.1% vehicle in all wells. The 130 µL volume corresponds to the quantity necessary to fill one row of a 96-well plate: (10 x 12) ± 10%. Each row corresponds to a different ligand concentration, with the last row serving as the vehicle control.
   4. Prepare 980 µL of Furimazine 1/100 dilution from stock solution (9.8 µL in 970.2 µL of HBSS to obtain a total volume of 980 µL).
      NOTE: Prepare a fresh solution of furimazine and let it incubate for at least 3 min. Then, add it immediately prior to acquisition. It should not be prepared too early, as the signal has a half-life of approximately 120 min at room temperature. To overcome this limitation, long-acting furimazine derivatives, such as vivazine and endurazine, can be used. These substrates keep up a stable BRET signal for up to 48 h.
2. Plate readings
   NOTE: Use a multimode plate reader. Adjust the measurement to the conditions studied and the number of replicates. Here, the data acquirement is divided into three readings of 4 columns, which corresponds to 32 wells. All readings are done with top optics with a transparent lid on to prevent buffer evaporation. Before all readings, adjust the measurement gain. In this study, the gain was fixed at 3600. It is possible to avoid manual ligand addition by using an automated injector or syringe system, which is available in many plate readers.
   1. Remove the medium from the first 4 columns of the 96-well plate. Rinse each well with 100 µL of HBSS and add 80 µL of HBSS. Stick a white sticker on the underside of the plate. This improves fluorescence and/or bioluminescence measurements.
   2. Recording of G protein luminescence spectra: Insert the 96-well plate into the plate reader. Record cpVenus¹⁷³ emission using a 535/30-nm monochromator and measure the luminescence emission between 500 and 600 nm with 2-nm resolution.
      NOTE: This recording is a control to ensure fluorescence is emitted upon cpVenus¹⁷³ excitation.
   3. Recording of G protein luminescence spectra: Record Nluc emission intensity using a 450/40-nm monochromator and measure the luminescence emission between 400 nm and 600 nm with 5-nm resolution.
      NOTE: This recording is a control to ensure no bioluminescence is emitted before the addition of Nluc substrate, furimazine.
   4. Remove the plate from the plate reader and add 10 µL of the previously prepared furimazine solution (step 3.1.4.) in each well with the help of a multichannel pipette.
   5. Repeat step 3.2.3.
      NOTE: This recording is a control to ensure bioluminescence is emitted upon furimazine addition. Directly proceed with step 3.2.6 after this measurement to prevent signal variation due to furimazine consumption and/or degradation. BRET signal variation can occur due to furimazine consumption and/or degradation and regulatory processes happening to terminate the response induced by agonist stimulation. To overcome this limitation, GRK inhibitors (CMPD101) and internalization inhibitors (dynasore) or gene-edited cells devoid of GRKs or arrestins could be used.
   6. Recording of G protein basal BRET: Before adding GPCRs ligand, record Nluc, and cpVenus¹⁷³ emission intensity using a 450/40-nm and a 535/30-nm monochromator, respectively. Measure 3 cycles of 60 s with a measurement interval time of 0.30 s (total reading time of 3 min).
      NOTE: Increasing the measurement interval time to 0.90 s usually improves the signal-to-noise ratio significantly but results in a much longer read time. The experimenter should decide whether the higher temporal resolution or the improved signal-to-noise ratio is more important for their application.
   7. Remove the plate from the plate reader and add 10 µL of the previously prepared dilution range of the GPCR ligand (step 3.1) respectively in each well of one column with the help of a multichannel pipette. Follow the same procedure for the 3 other columns.
   8. Recording of G protein ligand-induced BRET: Record Nluc and cpVenus¹⁷³ emission intensity using a 450/40-nm and a 535/30-nm monochromator, respectively. Measure 16 cycles of 60 s with a measurement interval time of 0.30 s (total reading time of 16 min).
      NOTE: Set up the measurement parameters to measure the wells in columns. Duplicates are read with a time difference of 2.4 s.
   9. Repeat step 3.2. for the next four columns 2 times.

4. Data analysis

NOTE: Collect the data of each reading in separate data spreadsheet files.

1. Control readings
   1. Plot the emission intensity against the wavelength.
2. Basal BRET readings
   1. For each well, calculate the BRET ratio of the three reads. The BRET ratio is defined as acceptor emission/ donor emission.
      
   2. For each well, calculate the average of the three BRET ratios prior to ligand addition. This value is called the basal BRET ratio before ligand stimulation: Ratio_basal.
   3. To normalize the previous BRET ratios of all wells, for each of the three measurements, respectively subtract the BRET ratio value calculated from the vehicle control wells from the BRET ratio at the corresponding measurement time.
      NOTE: The data analyzed correspond to the time points before ligand addition. Since the wells in which the ligand is added are known, the vehicle control wells can be identified accordingly.
3. G protein ligand-induced BRET readings
   1. For each well, calculate the BRET ratio of each read, as described in section 4.2.1. Those values are called Ratio_stim.
   2. To quantify ligand-induced changes, calculate the ΔBRET for each Ratio_stim of each well as a percent over basal. For that, use the corresponding Ratio_basal previously calculated in section 4.2.2.
      
   3. Calculate the ΔBRET(%) to normalize the ΔBRET values of each well. For that, subtract the respective ΔBRET values of the vehicle.
   4. To visualize the ΔBRET kinetics in a data spreadsheet, add the three basal BRET readings normalized in step 4.2.3. in front of the corresponding ΔBRET(%) values calculated for each well in section 4.3.3.
   5. Plot the previous data against the time of the read to obtain a kinetic graph.
   6. When a plateau is reached, take the ΔBRET(%) values at a chosen time and plot them against the concentration of the ligand used, as each well corresponds to a different ligand concentration. A dose-response curve is obtained.
      ​NOTE: The plateau is usually reached around 5 min after ligand stimulation. In this protocol, the values obtained after 15 min ligand stimulation are plotted. To compare data from different experiments, adjust the time depending on the kinetic of activation.
   7. Plot the previous data in analysis software and fit using a sigmoidal dose-response three-parameter fit based on the following equation:
      
      where, X is the concentration of the ligand used, Y is the ΔBRET(%) values, Top and Bottom represent the plateaus, and EC₅₀ is the ligand concentration required to reach 50% of the maximal effect. This allows one to obtain the E_max and EC₅₀.
   8. Compare data from the control condition using a non-parametric Mann-Whitney test. Results are considered significant at p < 0.05.

Figure 2: Key steps for performing the BRET assay. Overview of the three main experimental steps outlined in this protocol (cell seeding, cell transfection, and BRET signal acquisition) and detailed step-wise guide for data acquisition. Experimental setup: On day 1, cells are seeded in a PLL-precoated white 96-well plate with a flat, clear bottom at a density of 30,000 cells/well. On day 2, cells are transfected with the selected G protein sensor along with either the studied GPCR or pcDNA3.1 plasmids. After 24 h incubation, G protein sensor activity is measured through bioluminescence and fluorescence readings upon ligand addition. Data acquisition: After the HBSS wash of the cells, 80 µL of HBSS is added to the wells, and cpVenus¹⁷³ fluorescence emission is measured between 500 nm and 600 nm using a 535/30-nm monochromator (1). Next, Nluc bioluminescence is measured between 400 nm and 600 nm using a 450/40-nm monochromator, both before (2) and after (3) the addition of furimazine. Finally, the BRET signal for basal and ligand-induced G protein activity is measured using 450/40-nm and 535/30-nm monochromators, respectively, over 3 min (3 cycles of 60 s with a measurement interval of 0.30 s) (4) and 16 min (16 cycles of 60 s with a measurement interval of 0.30 s) (5). Please click here to view a larger version of this figure.

A generalized scheme of the experimental setup and execution is detailed in Figure 2. The activation of Gs or Gi/o family proteins following ligand activation of two GPCRs was evaluated using the G-CASE biosensors. First, a prototypical family A GPCR was studied, the β2-AR transiently transfected in HEK 293T cells. When an agonist (i.e., isoproterenol) was applied to HEK wt cells expressing the β2-AR along with the Gs(short)-CASE protein sensor, a concentration-dependent decrease in the BRET signal was observed, indicating activation of the Gs protein (Figure 3B). ΔBRET values decreased upon ligand concentration until saturation was reached, with a plateau observed approximately 5 min after ligand stimulation. In contrast, HEK wt cells transfected with the empty pcDNA3.1 plasmid and the Gs(short)-CASE protein sensor exhibited an inverse but low and non-significant response to isoproterenol stimulation (Figure 3B). In this protocol, values obtained after 15 min of ligand stimulation are plotted. To ensure results consistency and independence from time selection, other time points should be tested, ensuring that equilibrium has been reached. Additionally, to compare results across different experiments, the time selection should be adjusted based on the activation kinetics of each experiment.

To generate the sigmoidal dose-response curves shown in Figure 3C, the ΔBRET values measured at 15 min post-stimulation (when the plateau is stabilized) were plotted against the different ligand concentrations. The observed EC50 (9.4 nM ± 2.1 nM) is in the range values of the receptor's known Kd for isoproterenol (13 nM ± 6 nM)13,14 (Figure 3C,D). These results demonstrate the efficiency of the G-CASE sensors in evaluating G protein activation with an observed response specifically associated with the presence of the β2-AR.

Figure 3: Assessing Gs protein sensor in HEK wt cells. Kinetic ΔBRET readings upon addition of the agonist isoproterenol to HEK wt cells transfected with Gs protein sensor and β2-AR (A) or pcDNA3.1 (B). (C) Dose-response curves obtained by fitting the ΔBRET (%) values after 15 min ligand stimulation at varying ligand concentrations. (D) ΔBRET maximal values comparison between the control pcDNA3.1 and β2-AR transfected cells stimulated with isoproterenol. Statistical difference to pcDNA3.1 condition was tested using a non-parametric Mann-Whitney test (**p < 0.01). Data show mean ± SEM of three independent experiments performed in duplicate. Please click here to view a larger version of this figure.

Secondly, the biosensors were tested in HEK CB1 cells, a HEK 293T cell line stably expressing the CB1R. These cells were transiently transfected with the Gi3-CASE protein sensor. Stimulation with the CB1R agonist WIN-55,212-2 induced a concentration-dependent ΔBRET signal, indicating activation of the Gi3 pathway (Figure 4A). In contrast, HEK wt cells transfected with the Gi3-CASE protein sensor and stimulated under the same conditions did not show any response, confirming the specificity of CB1R activation (Figure 4B). A plateau was reached at approximately 5 min after ligand activation. The corresponding dose-response curve highlighted the concentration-dependent ΔBRET response induced by WIN-55,212-2 stimulation in HEK-CB1 cells, whereas no such response was observed in HEK wt cells (Figure 4C). The observed EC50 (112 nM ± 25 nM) is in the range values of the receptor's known EC50 for WIN-55,212-2 (354 nM ± 62 nM)15 (Figure 4C, D). Finally, a comparison of ΔBRET values obtained 15 min upon agonist stimulation with 10 µM of WIN-55,212-2 in HEK CB1, and HEK wt cells illustrate the CB1R-dependent activation of the Gi3 signaling pathway (Figure 4D).

Figure 4: Assessing Gi3 protein sensor in HEK CB1 cells. Kinetic ΔBRET readings upon addition of the agonist WIN-55,212-2 to HEK CB1 (A) or HEK wt cells (B). Dose-response curves obtained by fitting the ΔBRET (%) values after 15 min of ligand stimulation against ligand concentration (C). ΔBRET maximal values comparison between the control HEK wt and HEK CB1 cells stimulated with WIN-55.212-2 (D). Statistical difference to HEK wt condition was tested using a non-parametric Mann-Whitney test (****p < 0.0001). Data show mean ± SEM of five independent experiments performed in duplicate. Please click here to view a larger version of this figure.

The present protocol describes the use of BRET biosensors for real-time measurement of G protein activation in living cells in response to GPCR stimulation with specific ligands. By using the bioluminescent donor-acceptor energy transfer principle, this approach allows for the detection of conformational changes and dissociation of heterotrimeric G proteins upon receptor activation, providing a robust and sensitive platform for studying GPCR signaling. This protocol can be adapted and applied to various GPCR families, including class B and class C GPCRs as well as orphan GPCRs^10,12. As examples, results with the class-A GPCRs, β2-AR, and CB1R, are reported herein.

GPCR stimulation with its corresponding agonist induces G protein recruitment, ultimately leading to its dissociation into two subunits: G_α on one side and the obligate G_βγ dimer on the other. In this protocol, the GPCR activation can be monitored through G protein dissociation, facilitated by the BRET partners attached to the different G protein components.

The obtained results demonstrate that the BRET-based G-CASE biosensors are effective in detecting activation of either G_s or G_i proteins upon ligand stimulation of β2-AR or CB1R, respectively, and enable precise analysis of GPCR-mediated signaling pathway activation. The use of appropriate agonists, such as isoproterenol for β2-AR and WIN-55,212-2 for CB1R, confirms the specificity of ligand-receptor interactions.

Results obtained with G_s and G_i3 protein sensors highlight the sensor's ability to provide dynamic insights into G protein signaling. Stimulation with increasing concentrations of the agonist, isoproterenol, in HEK wt cells transiently expressing β2-AR led to receptor activation and subsequent G_s protein dissociation, translated into a decrease in the BRET signal. Importantly, the observation of an opposite but negligible response in HEK wt cells transfected with the empty plasmid and stimulated with isoproterenol underscores the specificity of the BRET response.

Similarly, in HEK CB1 cells transiently transfected with the G_i3-CASE biosensor, agonist stimulation induced a pronounced BRET decrease, indicative of G_i3 activation. The absence of a BRET response in HEK wt cells not transfected with CB1R further confirms the receptor-specific that G_i3 pathway activation is specifically mediated by CB1R.

Similar results have also been reported for other GPCRs combined with G-CASE variants for G_i1, G_i2, G_o1, G_z, G_q, G₁₅, G₁₂ and G₁₃^10,16. Screening all of the above with diverse ligands can be performed to achieve the discovery of novel therapeutic compounds. These assays can reveal significant differences in ligand affinity EC₅₀ and/or receptor maximal activity E_max, which may vary depending on the specific G-protein subtype involved. Such observations are key to understanding how ligands selectively activate specific signaling pathways through biased signaling mechanisms. This contributes to the development and implementation of targeted therapeutics strategies by enabling the identification of ligands with improved efficacy, selectivity, and safety profiles. To perform ligand screening, this protocol can be carried out in high throughput. To achieve this, eliminating the washing step by using pre-coated plates is recommended. Additionally, the use of an automated injector or syringe system present in several plate readers is advised to avoid manual addition. Full white well plates can also be used to avoid the need for stickers, but they may obstruct the ability to visualize the cells to check their growth.

Antagonists can be tested in a competition assay format, following an initial stimulation with an agonist, to assess their ability to block receptor activation. This assay is also compatible with the use of an inverse agonist, in a dose-dependent manner, to identify constitutively active GPCRs, as shown by Schihada et al.¹⁰.

The BRET-based G-CASE biosensors provide significant advantages over conventional FRET-based assays. By utilizing an intrinsic bioluminescent donor, BRET eliminates the need for an external light source, thereby minimizing autofluorescence, photobleaching, phototoxicity, and light scattering. This reduction in background noise enhances BRET assay sensitivity, enabling the detection of subtle biological interactions, an essential feature for pharmacological studies requiring precise receptor activity measurements. It also bypasses the use of radioactivity and biochemical methods such as [³⁵S]GTPγS binding or cAMP accumulation assays. Data acquisition is carried out using a standard fluorescence plate reader, commonly available in many laboratories, reinforcing the accessibility and simplicity of this approach.

Additionally, the single-plasmid nature of the G-CASE biosensors simplifies experimental design, reducing variability associated with co-transfection protocols. The validation of G-CASE biosensors in both transient and stable expression systems highlights their versatility for different experimental conditions. A variation of this protocol can also be implemented by transfecting cells in suspension on day one before plating, potentially optimizing transfection efficiency and experimental reproducibility. This protocol can also be applied to membrane fragments, as reported by Scott-Dennis et al., in studies on both cannabinoid receptors type 1 and type 2¹². The plate reading setup during data acquirement offers a sufficient temporal resolution of a few milliseconds between duplicates, which ensures accurate detection of dynamic changes. Combined with the short total reading time, this method ensures sufficient throughput and sensitivity for BRET assays.

The data obtained also highlight the importance of selecting appropriate ligand concentrations, as dose-response curves are well-fitted around the known K_d values of the ligands, enabling accurate determination of pharmacological parameters such as EC₅₀ and E_max values.

Overall, these findings reinforce the potential of BRET sensors as powerful tools for investigating GPCR signaling. The approach presented here is highly adaptable, enabling quantitative and kinetic analyses of GPCR signaling in living cells with minimal background interference.