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

We are interested in understanding key questions in GPCR signaling that would allow us to identify new GPCR targets and ligand-biased signaling. Biomolecular and biochemical methods, cryo-EM, NMR and molecular dynamic simulations are used to understand the regulation of GPCR by ligands with different efficacy or bias. To begin, pour approximately six milliliters of sterile filtered 0.01%poly-L-lysine solution into a multi-channel reservoir.

Use a multi-channel pipette to dispense 60 microliters of this solution into each well of a 96-well white microplate. Incubate the microplate in the dark for 20 minutes. After incubation, aspirate the poly-L-lysine solution from each well using a multi-channel pipette and transfer it to a 15-milliliter tube for storage at four degrees Celsius.

Wash the plate three times with DPBS solution using a multi-channel pipette. For cell preparation and plating, discard the medium in the flask containing the HEK293 cells. Then rinse the cells with two milliliters of DPBS solution.

Now, pipette 0.5 milliliters of trypsin into the flask. Incubate the suspension for three to five minutes at 37 degrees Celsius to detach the cells. Neutralize the trypsin with 4.5 milliliters of DMEM.

Pipette the suspension up and down repeatedly to fully detach and resuspend the cells. Transfer the cell suspension to a 15-milliliter tube, then centrifuge at 1, 000 G for five minutes. After discarding the supernatant, resuspend the cell pellet in 2 milliliters of DMEM.

Pipette 40 microliters of the resuspended cells on a Malassez counting chamber. Now, count the cells using a Malassez counting chamber. Prepare 10 milliliters of the cell suspension at a concentration of 300, 000 cells per milliliter.

Pour this suspension into a multi-channel reservoir. Then use a multi-channel pipette to seed each well of the 96-well plate with 100 microliters of the solution. Incubate the microplate at 37 degrees Celsius with 5%carbon dioxide for approximately 24 hours.

For transfection, transfer 10 micrograms of plasma DNA and 20 microliters of Lipofectamine into separate tubes, each containing 500 microliters of Opti-MEM medium. After a five-minute incubation at room temperature, combine the solutions into a single tube. Mix gently to obtain a 1 milliliter transfection mixture, and incubate.

Add 10 microliters of the transfection mixture drop wise into each well of the 96-well plate. Gently rock the plate back and forth to ensure uniform distribution. Then incubate the plate in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for 24 hours.

The next day, prepare a ligand serial dilution of 10 microliters in the appropriate solubilization solvent, centered around the dissociation constant of the ligand. Store the serial dilution at 20 degrees Celsius. For each concentration and the vehicle control, perform a 1 in 100 dilution by adding 1.3 microliters of the stock to 128.7 microliters of HBSS.

Then prepare 980 microliters of furimazine at 1 in 100 dilution by adding 9.8 microliters of stock to 970.2 microliters of HBSS. To perform plate reading, aspirate the medium from the first four columns of the 96-well plate. Rinse each well with 100 microliters of HBSS.

Then add 80 microliters of HBSS to each well. Then stick a white sticker on the underside of the plate. Insert the 96-well plate into the plate reader to begin recording the cp Venus 173 emission spectrum.

Set the monochromator to 535 by 30 nanometers and measure luminescence emission between 500 and 600 nanometers with two nanometer resolution. Record the end Luca mission intensity by setting the monochrometer to four 50 by 40 nanometers and scanning between 400 and 600 nanometers with five nanometer resolution. Remove the plate from the reader.

Then add 10 microliters of the prepared furine solution to each well using a multi-channel pipette. To record G-protein basal BRET signal, measure both in Luke and CP venous 173 emissions using the 450 by 40 nanometer and 535 by 30 nanometer monochrometers before adding GPCR ligands. Then remove the plate and add 10 microliters of the prepared GPCR ligand dilution to the wells of one column using a multi-channel pipette.

Reinsert the plate into the reader to record ligand induced Brett by measuring nluc and CP venous 173 emissions with the same monochromator settings. A concentration dependent decrease in delta BRET values was observed in HEK 293 T cells expressing beta two adrenergic receptors upon isoproterenol stimulation reaching a plateau around five minutes. No significant BRET change was observed in HEK 293 T cells.

Transfected with the empty pcDNA3.1 vector upon isoproterenol stimulation. Sigmoidal dose response curves showed an EC 50 of approximately 9.4 nano molar for isoproterenol in beta two adrenergic receptor expressing cells. Delta BRET values measured at 15 minutes post stimulation with 10 micromolar isoproterenol were significantly lower in beta two adrenergic receptor expressing cells compared to PC DNA control cells.

A concentration dependent decrease in delta BRET was observed in HEK CB1 cells stimulated with WIN-55.212-2 stabilizing around five minutes. HEK wildtype cells did not show significant delta BRET changes upon WIN-55.212-2 stimulation confirming specificity of CB1 receptor activation. WIN-55-212-2 induced a sigmoidal dose response in HEK CB1 cells with an EC 50 of approximately 112 nano molar.

Delta BRET values at 15 minutes post stimulation with 10 micromolar WIN-55.212-2 were significantly lower in HEK CB1 cells compared to HEK wild type cells. GK's BRET sensors are trisistronic construct that allow us to measure precisely specific geo-protein activation in real time and in live cells. Discovery of bias ligands with IPU methods could open pharmacological studies for specific drug screening with improved efficacy.