This protocol has a high impact in the field of drug discovery and development, especially in the development of novel, first-in-class drugs with minimal side effects. The main advantage of this technique is that it can be applied to any GPCR system, and used to obtain accurate information about receptor pharmacology in real-time living systems. The implications of this technique are closely related to new therapies particularly where beta-arrestins are relevant.
These include opiod receptors in pain and dopamine receptors in schizophrenia. This method can be useful in many medical scientific research areas from neurology to cardiopulmonary disease. PCR demonstration of our method is important, because it allows for a detailed presentation of the critical steps in the protocol, which would not be possible with conventional publication.
Begin by designing primers to introduce genes of interest to the pBiT vectors. Due to the inframe stop codon that divides the multi-cloning site, select at least one of the three sites as one of the two unique restriction enzymes needed for directional cloning. Refer to the manuscript for nucleotide sequences to encode the linker residues and incorporate them into the primers.
For the pBiT1.1-C and pBiT2.1-C make sure that the forward primer contains an ATG codon and a potent Kozak consensus sequence. For the pBiT1.1-N and pBiT2.1-N make sure that the reverse primer contains a stop codon. Use the designed primers to amplify the insert DNA of the gene of interest.
Combine the PCR reagents according to manuscript directions, making sure to use a high-fidelity DNA polymerase to minimize mutations. Program the thermocycler and start the reaction. After completing PCR, digest the product in a 50-microliter digestion reaction.
In a 1.5-milliliter tube, combine 12 microliters of distilled water, five microliters of the appropriate 10X restriction digestion buffer, and 30 microliters of the PCR product. Then add 1.5 microliters of each restriction enzyme. Vortex the reaction mixture.
And incubate overnight at 37.5 degrees Celsius. Prepare a second reaction to digest the recipient plasmid. In a 1.5-milliliter tube, combine 23 microliters of water, five microliters of the appropriate 10X restriction digestion buffer, 15 microliters of the recipient plasmid, and 1.5 microliters of each restriction enzyme.
Briefly mix the tube. And incubate overnight at 37.5 degrees Celsius. On the day following the cloning and transformation, pick three to 10 individual bacterial colonies and transfer them to one milliliter of LB medium with ampicillin.
Incubate the cells for six hours. And then transfer 200 microliters of bacterial suspension to five milliliters of fresh LB medium with ampicillin. Incubate the culture at 37.5 degrees Celsius with shaking at 200 rpm overnight.
On the next day, isolate the plasmid with a mini-prep DNA purification kit. Screen the purified plasmid for successful ligation with PCR. One day before transfection, seed the cells on a poly-L-lysine coated 96-well plate using Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum, 100 units per milliliter of penicillin G, and 100 microliters per milliliter of streptomycin.
Only add cells to the 60 inner wells to minimize potential for thermal gradients across the plate and edge effects from evaporation. Add 200 microliters of sterile distilled water to the 36 outside wells, and 150 microliters into the spaces between the wells. Then incubate the plate overnight at 37.5 degrees Celsius and 5%carbon dioxide.
On the next day, perform transfection using a total of 100 nanograms DNA. Set up four different plasmid combinations according to manuscript directions. Using 20 microliters of modified Eagle's minimum essential media buffered with HEPES, and 0.3 microliters of lipidic transfection reagent per well.
Add 20 microliters of lipidic transfection reagent and DNA mixture to each well, and mix the plate in circles for 10 seconds. Incubate the plate at 37.5 degrees Celsius and 5%carbon dioxide for six hours. Refresh the medium.
And incubate for another 24 hours. After the incubation, aspirate the medium and add 100 microliters of modified Eagle's minimum essential media buffered with HEPES to each well. Allow the plate to stabilize at room temperature for 10 minutes.
Meanwhile, prepare the furimazine substrate by combining one volume of the 100X substrate with 19 volumes of LCS dilution buffer, creating a 5X stock to mix with the cell culture medium. When the cells are ready, add 25 microliters of the stock to each well, and gently mix the plate for 10 seconds. Then measure the luminescence for 10 minutes at room temperature for signal stabilization.
For experiments with ligand addition, prepare the 13.5X ligand solution in modified Eagle's minimum essential media buffered with HEPES, and add 10 microliters per well using a multichannel pipette. Then mix the plate by hand or with an orbital shaker. This protocol has been successfully used to study interactions between a prototypical GPCR and two beta-arrestin isoforms.
Four different plasmid combinations were screened, and the one with the highest luminescent signal was selected for further experiments. The EC50 values for each beta-arrestin isoform were determined using dose-response curves, which were obtained from the maximum response of each concentration from kinetic studies. It's critical to pay special attention when designing the PCR primers.
The development of the right constructs is essential to the success of these assays. Following this methodology, you will be able to monitor receptor-beta-arrestin interactions, as well as receptor interactions with other cytosolic proteins. This methodology will help to answer fundamental questions in the field of GPCR pharmacology by studying how GPCR-beta-arrestin interactions can be modulated by modifications in the ligand structure.
