β-Arrestin recruitment assays

Upon GPCR activation, β-arrestins (βarr) are recruited to stop G protein signaling and to initiate clathrin-mediated receptor internalization. During this process, the release of the C-terminal domain of βarrs is associated with the binding of βarrs to the adaptor protein 2 (AP2). This interaction can be measured using the HTRF technology (PerkinElmer CisBio) based on the use of two specific antibodies, one directed against βarr2, and the second one specific for AP2. In this assay (βarr2 recruitment kit, PerkinElmer CisBio), the AP2 antibody is labeled with a Europium (Eu) cryptate fluorescent donor, and the one against βarr2 is labeled with a d2 fluorescent acceptor, with their proximity being detected by FRET signals. The specific signal is positively modulated in proportion with the recruitment of βarr2 to AP2 upon V2R activation by AVP. Briefly, HEK cells were plated at a seeding density of 2.5 × 10⁴ cells per well in white-walled 96-well plates (CELLSTAR plate, Sigma-Aldrich) precoated with poly-l-ornithine (14 μg/ml; Sigma-Aldrich) for 24 hours, in DMEM (GIBCO) complemented with 10% fetal bovine serum (Eurobio), 1% nonessential amino acids (GIBCO), and 1% penicillin-streptomycin antibiotic solution (GIBCO). To produce the V2R, the cells were transfected with 30 ng of the pRK5-Flag-Snap-V2R plasmid (coding for the cleaved V2R construct used in cryo-EM studies) using X-tremeGENE 360 (Merck), according to the manufacturer’s recommendations. After a 24-hour culture, cells were used to evaluate the recruitment of βarr2 to AP2 upon V2R activation with the βarr2 recruitment kit (PerkinElmer CisBio), following the manufacturer’s recommendation. Briefly, the cells were first washed once with DMEM and incubated for 2 hours at RT with 100 μl per well of stimulation buffer containing various concentrations of the ligand AVP (ranging from 10⁻⁶ to 10⁻¹² M). The medium was then replaced by 30 μl per well of stabilization buffer for 15 min at RT. The cells were then washed three times with 100 μl per well of wash buffer before adding 100 μl per well of a premix of Eu cryptate and d2 antibodies in detection buffer. Following overnight incubation at RT, 80 μl of medium was removed from each well before reading the 96-well plates on a Spark 20M multimode microplate reader (Tecan) or a PHERAstar FS (BMG Labtech) by measuring the signals of the donor (Europium cryptate–labeled AP2 antibody) at a wavelength of 620 nm and of the acceptor at 665 nm (d2-labeled βarr2). Last, the results were expressed as the FRET ratio [(665/620) × 10,000] and plotted using GraphPad 9.1.1 (GraphPad Prism Software Inc.). Experiments were repeated at least three times on different cultures, with each condition in triplicate. Data are presented as means ± SEM (fig. S2).