GPCR Activation and β-Arrestin Recruitment

β-arrestin 2 recruitment was determined as previously described [17 (link),36 (link)]. Briefly, BRET experiments were performed in HEK-293T cells 48 h after transfection with the cDNA corresponding to A_2AR–YFP or A₃R–YFP (0.5 μg cDNA each) and 1 μg cDNA corresponding to β-arrestin 2-Rluc. Cells (20 μg protein) were distributed in 96-well microplates (Corning 3600, white plates with white bottom) and were incubated with antagonists (SCH 442416 for A_2AR and/or PSB-10 for A₃R) for 15 min and stimulated with agonists (CGS 21680 for A_2AR and/or IB-MECA for A₃R) for 10 min prior to the addition of 5 μM coelenterazine H (Molecular Probes, Eugene, OR, USA). One minute after coelenterazine H addition, BRET between β-arrestin 2-Rluc and receptor-YFP was determined and quantified. The readings were collected using a Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany) that allows the integration of the signals detected in the short-wavelength filter at 485 nm and the long-wavelength filter at 530 nm. To quantify protein-Rluc expression, luminescence readings were also performed 10 min after adding 5 μM coelenterazine H.