GHSR Activity Assay Using Tango β-Arrestin Recruitment

GHSR activity was examined using the Tango β-arrestin recruitment assay that was developed by B.L. Roth (89 (link)). HTLA cells stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene were a gift from B.L. Roth (University of North Carolina, Chapel Hill, North Carolina, USA). HTLA cells were maintained in DMEM with 10% FBS (Thermo Fisher Scientific), 100 U/mL penicillin/100 μg/mL streptomycin (Thermo Fisher Scientific), 2 μg/mL puromycin (Tocris Bioscience), and 100 μg/mL hygromycin B (Corning) at 37°C with 5% CO₂. Cells were transfected with a GHSR-Tango plasmid expressing human GHSR and a vasopressin receptor (V₂) tail (66293, Addgene) using the calcium-phosphate method and maintained for 24 hours prior to the next step. Transfected cells were transferred into a poly-D-lysine–coated 96-well plate (Corning) and maintained for 24 hours before treatment. Cells were then treated with an appropriate amount of human ghrelin peptide (Phoenix Pharmaceuticals, 031-30), LEAP2 peptide (Phoenix Pharmaceuticals, 075-40), or combinations of the 2 (LEAP2 was added 2 hours prior to ghrelin) at different ratios and doses for 24 hours to allow the expression of luciferase. Treated cells were incubated with Bright-Glo Luciferase Assay solution (Promega, E2610), and the signal was detected using a Biotek Neo2 microplate reader.