4.3. β-. Arrestin Assay

The β-Arrestin recruitment assay system PathHunter® developed by DiscoverX (Fremont, CA, USA, https://www.discoverx.com/arrestin, accessed on 30 July 2021) detects GPCR activation following ligand stimulation. The assay is based on enzyme fragment complementation of β-galactosidase. The assay is performed using a cell line expressing an Enzyme Acceptor (EA) which is fused to the β-Arrestin. The second part of the enzyme (ProLink/ PL) is fused to the C terminus of the GPCR of interest. EA and PL are inactive as single fragments. When a ligand binds and activates the GPCR of interest, the β-arrestin-2 protein is recruited to the GPCR since it is involved in receptor desensitizing and recycling. The recruitment of the β-arrestin leads to the complementation of the β-galactosidase. The active enzyme can catalyze hydrolysis and generate chemiluminescence when an appropriate substrate is proved. Thus, measured chemiluminescence (or β-galactosidase activity) correlates with receptor activation. In this study, the recruitment assay was performed using engineered CHO cell lines stably expressing the β-Arrestin protein linked to the EA fragment. The cell lines were transfected with the GPCR cDNA of interest fused to the ProLink-tag.

At 48 h post-transfection, co-transfected CHO β-Arrestin DRD₂ and DRD₃ cells were seeded in 96 well-plates (NunclonTM F96 MicroWellTM, Thermo Fischer Scientific, Scwerte, Germany). On the day of the assay, the medium was changed for 90 µl of F12 with 100 U/mL penicillin G, 100 µg/mL streptomycin, and the cells were incubated for at least 2 h at 37 °C with 5% CO₂. Then, 10 µl of DMSO-diluted dopamine was added. Unsupplemented F12 medium was used as negative control. The final dimethyl sulfoxide (DMSO) concentration per well was 1%. After 90 min, the PathHunter® detection reagent (DiscoverX, Fremont, CA, USA) was added to the cell plate (50 µL/well) and incubated 1 h at room temperature in the dark, and finally chemiluminescence was detected by using the MikroWin2000 software and a multimode microplate reader (Mithras LB 940, Berthold Technologies, Bad Wildbad, Germany).