Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

The overall goal of this experiment is to provide an efficient and easily-interpretable method to detect the subcellular localization and internalization of expressed GPCR fusion proteins in mammalian cells. This method can help answer the key questions in the subcellular protein localization field, such as receptor internalization and recycling. The main advantage of this technique is that it can be used easily and efficiently.

So this method can provide insight in monitoring and locating GPCRs. And it can also apply to other systems, such as cytoplasmic and nuclear protein. After recovering and passaging HEK293 cells according to the text protocol, when the cells have grown to near confluence, discard the equilibrated growth medium, add two milliliters of PBS, and swirl the dish to wash the cells.

Discard the PBS, and add one milliliter of trypsin EDTA. To completely digest the cells, carefully swirl the dish and aspirate off the solution. Then, put the dish in this 37 degrees Celsius water-jacketed C02 incubator for three minutes.

While the cells are incubating, prepare a six well plate by adding two milliliters of equilibrated growth medium to each well. Then, when the cells have lifted from the bottom of the dish, add one milliliter of equilibrated growth medium to the dish. Mix by pipetting and immediately transfer 0.1 milliliters of cell suspension into each well of the prepared six well plate.

Gently pipette the suspension in the wells, and incubate the cells at 37 degrees Celsius and 5%CO2. The following day, ensure that the cells are 85-95%confluent. Then, to two sterile microcentrifuge tubes, add three micrograms of plasmid DNA and 100 microliters of reduced serum medium to one tube.

Meanwhile, add nine microliters of reagent one and 100 microliters of reduced serum medium to another tube. Incubate the tubes at room temperature for five minutes. Combine the diluted plasmid mixture with diluted reagent one.

Then, gently mix the solution, and incubate it at room temperature for 20 minutes. Aspirate the medium of the cells and add 1.5 milliliters of fresh DMEM without FBS. Then, add dropwise 215 microliters of DNA transfection reagent mixture to each well, and gently mix by swirling the plate.

Incubate the plate at 37 degrees Celsius and 5%CO2 for four to eight hours. Then, replace the medium with fresh DMEM with 10%FBS. Incubate the cells for another 24 to 48 hours before running any experiments.

Transfection efficiency may vary greatly in different cell lines. Therefore, the optimization of the sit and stab in deposition may be necessary depending on the particular cell type used. Remove the growth medium from the six well culture plate.

Then, use one milliliter of PBS to wash the cell monolayer. Add 0.5 milliliters of trypsin EDTA, swirl the dish, and aspirate off the solution. Then, incubate the culture plate at 37 degrees Celsius for three minutes.

During the incubation, insert 15 millimeter diameter sterile coverslips into each well of a 12 well plate. Resuspend the cells in a small volume of fresh complete growth medium, and transfer the required number of cells to the 12 well plate with coverslips. Then, incubate the cells for 16 to 24 hours.

After 16 to 24 hours of incubation, when the cells have grown to near confluence, add five micromolar of the membrane probe dill to each well, and place the cells back in the incubator for 30 minutes. Remove the equilibrated growth medium and use cold PBS to wash the cells twice. Then, add one milliliter of 4%PFA to each well, and agitate the cells for 15 minutes to fix the cells.

With PBS, wash the fixed cells twice, then add 200 to 300 microliters of DAPI to each well, and incubate the plate for 10 minutes at room temperature. After washing the cells with PBS twice, carefully remove the coverslips from the plate, and wick off the excess buffer. Invert the coverslips onto microscope slides loaded with one drop of mounting medium.

Then, remove the excess mounting medium from the slides. Using cells that have grown to near confluence, remove the equilibrated growth medium and add fresh warmed DMEM. Incubate the cells for one hour.

Treat the cells with different concentrations of ligand for 30 minutes. And treat the cells with 10 to the negative six molar ligand for the different times shown here. Use cold PBS to wash the cells twice, then add one milliliter of 4%PFA in PBS to each well, before gently agitating the cells for 15 minutes.

After washing the cells twice in PBS, carefully remove the coverslips from the wells of the plate, and mount them as just demonstrated earlier in this video. To carry out confocal microscopy, turn on the hardware of the confocal microscope, including the mercury lamp power, PC microscope, scanner, and laser. Launch the software, then check the configuration and select the laser.

Turn on the different units of confocal microscopy system according to the instructions. Place a prepared slide on the microscope stage, and use the stage controller to position the sample over the objective lens. For an oil immersion lens, add one drop of cedar oil on to the coverslip, and orient the slide with a coverslip facing the lens.

Under fluorescence, find the cells of interest, then switch to scan mode, and choose the laser power and spectral range of the emission fluorophore. Capture high-quality images by tuning the laser intensity and other parameters. This figure illustrates the expression of pEGFP-N1 in HEK293 cells.

The GFP signal was detected in the cytoplasm and the nucleus. The subcellular localization in HEK293 cells of gonadotropin-releasing hormone receptors from S-Japonica, or SjGnRHR, is shown here. EGFP receptors are in green, and Dil staining, which highlights the cell membrane, is in red.

The receptors are seen localizing to the plasma membrane. To visualize the internalization of SjGnRHRs, cells were treated with gonadotropin-releasing hormone at different concentrations. As seen here, the internalization of the receptors proceeded in a dose-dependent manner, and the receptor was completely internalized from the cell's surface with the 10 to the minus six molar ligand.

When treated with 10 to the minus six molar ligand for different time periods, internalized SjGnRHRs were detectable 10 minutes after agonist simulation, and extreme internalization occurred by 60 minutes. As demonstrated in HEK293 cells, in both experiments, the fluorescent SjGnRHR EGFP fusion protein primarily localized to the plasma membrane, and was dramatically and rapidly internalized into the cells. While attempting this procedure, it's important to remember to keep the cells under the right conditions without bacteria, fungi, and mycoplasma contamination.

Following this procedure, and the methods like cell checking and immobilization can be performed in order to answer additional questions like the second in pass V of the protein receptors. After each development, the technology paved the way for researchers in the field of the cell biology and biochemistry to explore the localization and the internalization of transmembrane receptors in various cell lines.