TITLE:  Whole-cell radioligand binding for receptor internalization
AUTHORS: Angus Li, Samuel Liu, Rennica Huang, Seungkirl Ahn, Robert J Lefkowitz

[DESCRIPTION]
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.

[STEPS]
SECTION: Day 1
1. Grow cells on 150 mm dish to ~70% confluency
SECTION: Day 1
2. Wash twice with 10 mL PBS
SECTION: Day 1
3. 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
SECTION: Day 1
4. Count cells with hematocytometer and dilute collected cells to 164,000 cells/mL
SECTION: Day 1
5. Pipet 1 mL into each well of poly-D-lysine coated 12-well plate
SECTION: Day 2
6. Add 250 mL of 5x doxycycline for all desired concentrations to cells 14 hours before assay
SECTION: Day 3
7. Prepare > 300 uL ligand-containing media per well (e.g. 10 uM AngII) for stimulation
SECTION: Day 3
8. Remove media from plate wells by vacuum in cell culture hood
SECTION: Day 3
9. Add 300 uL ligand-containing media per well and return to incubator for 1 hour
SECTION: Day 3
10. 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
SECTION: Day 3
11. 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
SECTION: Day 3
12. 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.
SECTION: Day 3
13. 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
SECTION: Day 3
14. Remove acid wash buffer by vacuum and wash 2-3 times with 1 mL ice-cold PBS with MgCl2 and CaCl2 per well.
SECTION: Day 3
15. 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
SECTION: Day 3
16. 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
SECTION: Day 3
17. Remove radioligand media into dedicated radioactive liquid waste vial using P1000 on ice. Remove nonspecific binding media last
SECTION: Day 3
18. 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
SECTION: Day 3
19. 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
SECTION: Day 3
20. 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
SECTION: Day 3
21. Wipe vials with moist KimWipe and place in racks for scintillation counter. Place racks in counter and count using 3h_cpm_3_min protocol
SECTION: Day 3
22. Perform second count using different protocol flag on next day
SECTION: Analysis
23. Copy binding counts into Excel spreadsheet
SECTION: Analysis
24. Calculate nonspecific counts for each receptor concentration
SECTION: Analysis
25. Calculate specific counts for each condition by averaging duplicates and subtracting nonspecific count
SECTION: Analysis
26. Calculate percent internalization as percent reduction in specific counts for ligand-stimulated condition compared to control