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Fig. 6. Y737 in β2-adaptin is a Src regulatory site for β-arrestin–AP-2 dissociation in cells. (A) Expression of β2-adaptin-YFP in HEK293 cells. Cells were transfected with pcDNA3 (Mock), non-silencing siRNA (siRNA-NS) or siRNA targeting β2-adaptin (siRNA-β-ad) and either β2-adaptin-YFP (WT) or β2-adaptin-Y737F-YFP (Y737F) as described in Materials and Methods. Shown is the expression of endogenous and transfected β2-adaptin, and ERK1/2. (B) Colocalization of β-arrestin2 with wild type and mutant β2-adaptin in HEK293 cells. Cells expressing HA-AT1R, β-arrestin2-CFP, and either β2-adaptin-YFP wild type or Y737F mutant were treated with Ang II (1 µM) for 2 minutes and imaged live by confocal microscopy as described in Materials and Methods. Shown are representative images of β-arrestin2-CFP (cyan) and β2-adaptin-YFP proteins (yellow). (C) Ang II-promoted BRET between β-arrestin2 and β2-adaptin. HEK293 cells expressing Flag-AT1R, β-arrestin2-Rluc and either β2-adaptin-YFP wild-type or Y737F mutant were stimulated with increasing concentrations of Ang II before BRET measurements. (D) Effects of Src on agonist-promoted maximal values of BRET (BRET_max). HEK293 cells expressing Flag-AT1R, β-arrestin2-Rluc and either β2-adaptin-YFP wild type or mutant, in absence (black bars) or presence (white bars) of Src were stimulated with Ang II (1 µM). BRET measurements were performed as described in Materials and Methods. Data represent the average of three to five independent experiments.

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