Competition binding assays that measure the affinity of ISO for β2AR WT and β2AR Y219A were performed in a 250-μl reaction volume consisting of 60 pM [125I]-CYP, a serial dilution of ISO, and 60 fmol of functionally active receptors [from HEK 293 cell membranes or purified receptors reconstituted in high-density lipoprotein (HDL) particles] in assay buffer [20 mM Hepes (pH 7.4), 100 mM NaCl, and 0.2% bovine serum albumin (BSA)]. For experiments with transducers, 1 μM β-arrestin1 was added within the total volume of 250 μl. Nonspecific binding was measured with 10 μM propranolol, and total binding was determined in the absence of ISO. To ensure that the binding reaction reached equilibrium, the mixed components were incubated at room temperature for 90 min. Unbound [125I]-CYP was separated by filtration onto GF/B glass microfiber filters treated with 0.3% polyethylenimine using a 96-well-format Brandel harvester. The GF/B filters were then rapidly washed three times with ice-cold buffer (20 mM Hepes and 100 mM NaCl). Bound [125I]-CYP was quantified with a 2470 automatic gamma counter (PerkinElmer). The affinity of [125I]-CYP for β2AR WT and β2AR Y219A was determined by saturation binding (fig. S4B). All data represent at least three independent experiments; SE and analysis were performed in GraphPad Prism. For Fig. 4 (C and D), the difference in log (IC50) of ISO between a control curve (receptor only, closed circles) and a β-arrestin1 curve (open squares) was verified by Welch’s unpaired t test.

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