We investigated NE’s and isoproterenol’s effects on β-arrestin trafficking at wild-type and mutant α1B-ARs and β2-ARs, respectively, using a BRET assay. The yellow fluorescent protein (YFP)–β-arrestin-2 construct was provided by M. Caron (Duke University, NC, USA), and the Rluc8/pcDNA3.1 construct for cloning of the α1B- and β2-AR cDNA was provided by A. Christopoulos (Monash Institute of Pharmaceutical Sciences, Melbourne, Australia). HEK293 cells were transfected with Rluc8-tagged wild-type or mutant receptors and YFP–β-arrestin-2. Agonist-induced BRET responses were performed 48 hours after transfection. On the day of the assay, cells were allowed to equilibrate in Hank’s balanced salt solution containing 0.05% (w/v) bovine serum albumin at 37°C before incubating with coelenterazine-h (5 μM) for 10 min, before reading BRET signals at 475 ± 30 nM and 535 ± 30 nM in response to increasing concentrations of NE (α1B-AR) or isoproterenol (β2-AR) (100 pM to 100 μM). The BRET signal was determined by calculating the ratio of the light emitted at 515 to 555 nm over the light emitted at 465 to 505 nm. Net BRET signals were determined by subtracting the BRET signal obtained with cells only expressing Rluc8-tagged α1B-AR or β2-AR, respectively, from BRET signals obtained with cells co-expressing Rluc8-tagged α1B-AR or β2-AR, respectively, and YFP-tagged β-arrestin-2.

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