The overall goal of this procedure is to isolate cellular membranes that contain G-protein-coupled receptors or GPCRs and to study GPCR pharmacology using a functional GTP binding essay. This method can help answer key questions in the field of biochemistry regarding the pharmacologic properties of established or orphaned G-protein-coupled receptors. The main advantages are that it is simple, rapid and measures the most proximal event in G-protein-coupled receptors signaling.
First, plate human embryonic kidney cells onto a 10 centimeter tissue culture plate in 10 milliliters of complete DMEM. Incubate the cells at 37 degree celsius and 5%carbon dioxide, until 70%to 80%confluency is reached. Following incubation, transfect the cells with HAMOR1 using a suitable transfection reagent per the manufacturer's guidelines.
Then, incubate the cells as previously described for 36 to 48 hours. After removing the transfected cells from the incubator, aspirate the medium and rinse the cells with five milliliters of ice cold PBS. Then, aspirate the PBS and excess liquid from the plate.
Next, add 600 microliters of buffer 1 to the cells. Using a cell scraper, dislodge the cells from the plate surface and pipette the cell suspension into a 1.6 milliliter micro centrifuge tube. Immediately snap freeze the micro centrifuge tube in liquid nitrogen.
Once the sample is frozen, thaw the lysates on ice. Once the lysates have thawed, use a single pulse microtube homogenizer to break open the cells. Pulsing that homogenizer three to five times for 10 seconds.
Place the tube on ice for 30 seconds between pulses. Following this, centrifuge the sample for 10 minutes at 1, 000 times g and four degrees celsius. Then, transfer the supernatant to a new 1.6 milliliter micro centrifuge tube on ice.
Re-suspend the palate in 100 microliters of buffer 1. Re-homogenize the sample and pulse the homogenizer three to five times for five seconds, placing the tube on ice for 30 seconds between pulses. After centrifuging the sample a second time, combine the supernatants and centrifuge for 20 minutes at 11, 000 times g and four degree celsius.
Transfer the supernatant to a new 1.6 milliliter micro centrifuge tube. Re-suspend in 200 microliters of buffer 2. Then, homogenize the palate by triturating it three to five times.
After centrifuging the sample and aspirating the supernatant, re-suspend the palate containing the crude membrane faction in 50 microliters of buffer 2. Dilute stock S35GTP gamma S to 50 nanomolar and 10 millimolar trace HCl and 10 millimolar DTT. Make 250 microliter hour quarts for the binding experiments, and flash freeze for storage if desired.
Next, prepare complete binding buffer or CBB, by supplementing incomplete binding buffer to include a final concentration of one millimolar DTT, 0.1%BSA, 10 micromolar GTP and 0.1 nanomolar S35GTP gamma S.After thawing the membrane fraction on ice, dilute it to a concentration of 100 micrograms per milliliter and incomplete binding buffer. To essay the effect of the peptide DAMGO on GTP binding via MOR1, prepare a series of DAMGO dilutions and CBB to a final volume of 100 microliters. To evaluate basil GTP binding, prepare a 1.6 milliliter micro centrifuge tube with 100 microliters of CBB.
To evaluate non-specific binding, prepare a 1.6 milliliter micro centrifuge tube with 99 microliters of CBB supplemented with one microliter of two millimolar non-radial labeled GTP gamma S.Now, add 100 microliters of the diluted membrane solution to each sample. Incubate the samples for 30 minutes at 25 degrees celsius in a thermo-mixer at 300 rpm. Pre-soak glass fiber filters in distilled water for 10 minutes.
After removing the samples from the thermo-mixer, briefly pulse spin them for five seconds to collect each sample at the bottom of the tube. Next, remove the lid of a vacuum filtration apparatus and lay the pre-soaked filters onto the vacuum ports of the apparatus. Re-secure the apparatus lid to form a vacuum seal and turn on the vacuum.
Pipette 195 microliters of each sample onto the filters. Wash the filters three times with one milliliter of ice cold wash buffer. Place five milliliters scintillation counting vials in a counting rack.
Add five milliliters of scintillation fluid to each vial. Next, turn off the vacuum and remove the lid of the vacuum filtration apparatus. Using tweezers, pick up the filters from the vacuum ports of the filtration apparatus and drop each filter into a scintillation vial.
After capping each vial securely, incubate the samples on an orbital shaker at 25 degree celsius for 10 minutes. Following this, turn on the scintillation counter, program the counter to measure sulfur 35 isotope emission for five minutes per sample using the standard associated scintillation program and press start to take account. Self fractionation, cleanly separates membrane proteins from nuclear protein histone h2b and the cytoplasmic protein glycerol to hide three phosphate dehydrogenase.
Proteins are enriched in their respective sub-cellular fractions. A representative of Ponceau stain demonstrates equal protein loading in each fraction. Multiple pharmacological parameters can be drive to characterize a GPCR ligand interaction via GTP binding experiments such as the EC50 and hill coefficient.
Those responsive GTP binding to MOR1 after DAMGO treatment is demonstrated here. This plot illustrates the agonist activity of DAMGO in the antagonism of Naloxone on MOR1 G-protein signaling. Once mastered, this technique can be done in two to four hours, depending on the number of experimental conditions.
While attempting this procedure, it's important to remember to use GPCR ligand concentrations at least five orders of magnitude above and below the expected EC50. Following this procedure, assessing molecules that are structurally similar to your ligand of interest, can help define important structural moieties in the ligand that define a receptor's ligand profile. After its development, this technique has aided in drug discovery and paved the way for researchers to explore detailed aspects of GPCR signaling, such as biased agonism.
After watching watching this video, you should have a good understanding of how to isolate cellular membranes and measure pharmacologic properties of GPCRs using radio labeled GTP. Don't forget, that working with radiation can be extremely hazardous. Precaution should be taken, such as wearing appropriate personal protective equipment and working with radiation appropriate lab wear.
Institutional guidelines for working with radioactivity should be reviewed prior to performing this procedure.
