Feb 28, 2023
Whole-cell radioligand binding for receptor internalization
- Angus Li1,
- Samuel Liu1,
- Rennica Huang2,
- Seungkirl Ahn1,
- Robert J Lefkowitz1,2,3
- 1Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America;
- 2Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, United States of America;
- 3Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina, United States of America
External link: https://doi.org/10.1371/journal.pone.0283477
Protocol Citation: Angus Li, Samuel Liu, Rennica Huang, Seungkirl Ahn, Robert J Lefkowitz 2023. Whole-cell radioligand binding for receptor internalization. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9dz91g3e/v1
Manuscript citation:
Li A, Liu S, Huang R, Ahn S, Lefkowitz RJ (2023) Loss of biased signaling at a G protein-coupled receptor in overexpressed systems. PLoS ONE 18(3): e0283477. doi: 10.1371/journal.pone.0283477
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 28, 2023
Last Modified: February 28, 2023
Protocol Integer ID: 77746
Funders Acknowledgement:
Howard Hughes Medical Institute
National Institutes of Health
Grant ID: R01 HL16037
Abstract
This protocol details an experimental procedure used to generate results described in the manuscript Li, A., Liu, S., Huang, R., Ahn, S., & Lefkowitz, R. J. (2023). Loss of biased signaling at a G protein-coupled receptor in overexpressed systems.
Day 1
Day 1
Grow cells on 150 mm dish to ~70% confluency
Wash twice with 10 mL PBS
Detach cells with 1.5 mL trypsin-EDTA + 10.5 mL media. Trypsinize for 5 minutes and check under microscope for complete detachment; tap dish if necessary
Count cells with hematocytometer and dilute collected cells to 164,000 cells/mL
Pipet 1 mL into each well of poly-D-lysine coated 12-well plate
Day 2
Day 2
Add 250 mL of 5x doxycycline for all desired concentrations to cells 14 hours before assay
Day 3
Day 3
Prepare > 300 uL ligand-containing media per well (e.g. 10 uM AngII) for stimulation
Remove media from plate wells by vacuum in cell culture hood
Add 300 uL ligand-containing media per well and return to incubator for 1 hour
Prepare saturating concentration of membrane-impermeable radioligand in media on ice. A ligand with high specific activity is preferred. For example, 30 uL 3H-AngII in 12 mL media. Prepare at least 4 mL per plate. Vortex briefly to mix
Take 1/3 of media with radioligand and add high concentration (orders of magnitude above radioligand concentration) of nonlabelled antagonist (e.g. 200 uM candesartan final concentration) for nonspecific binding measurement. Vortex briefly to mix
After 1 hour ligand incubation, place plates on ice and remove media by vacuum. Wash twice with 1 mL ice-cold PBS with MgCl2 and CaCl2 per well.
Strip ligand for 5 minutes with 2 mL acid wash buffer (0.2 M acetic acid, 0.5 M NaCl, pH 2.5) per well
Remove acid wash buffer by vacuum and wash 2-3 times with 1 mL ice-cold PBS with MgCl2 and CaCl2 per well.
Remove wash by vacuum. Add 300 uL media with radioligand per well on ice. Use top two rows of plate for total binding duplicates and bottom row for nonspecific binding. Wait ≥ 1.5 hours on ice
Pre-chill 50 mL per plate PBS with MgCl2 and CaCl2 on ice and pre-warm 0.1% SDS + 0.5 N NaOH in 42 °C water bath
Remove radioligand media into dedicated radioactive liquid waste vial using P1000 on ice. Remove nonspecific binding media last
Wash wells with 1 mL cold PBS with MgCl2 and CaCl2 on ice and collect using P1000 into radioactive liquid waste vial. Wash 4 times total. Check plates under microscope after last wash
Remove plates from ice and add 500 uL warm 0.1% SDS + 0.5 N NaOH per well. If there is precipitate in the solution, allow it to warm for longer. After adding, shake for ≥ 15 minutes
Collect samples from wells using P1000 and add to labeled scintillation vials. Add 300 uL of each radioligand media mix to two scintillation vials each for total counts. Add 5 mL scintillation cocktail (Lefko-Fluor) to each vial. Cap vials
Wipe vials with moist KimWipe and place in racks for scintillation counter. Place racks in counter and count using 3h_cpm_3_min protocol
Perform second count using different protocol flag on next day
Analysis
Analysis
Copy binding counts into Excel spreadsheet
Calculate nonspecific counts for each receptor concentration
Calculate specific counts for each condition by averaging duplicates and subtracting nonspecific count
Calculate percent internalization as percent reduction in specific counts for ligand-stimulated condition compared to control