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Fig. 7. Y737 in β2-adaptin regulates the dissociation of β-arrestin–AP-2 complexes in clathrin-coated pits and the internalization of AT1R. (A) Representative image of a cell expressing β-arrestin2 and β2-adaptin that was analyzed by ICCS. HEK293 cells transfected with HA-AT1R, β-arrestin2-CFP, and β2-adaptin-YFP wild-type were treated with Ang II (1 µM) and imaged live by confocal microscopy as described in Materials and Methods. Shown is a representative overlay confocal image taken after 1 minute of Ang II treatment and illustrated as pseudo color of β-arrestin2-CFP (green), β2-adaptin-YFP (red), and the colocalization of both proteins (yellow) (left panel). The region that was analyzed by ICCS is outlined in the white rectangle. The right panel represents the spatial autocorrelation (solid) and best-fit function (mesh) calculated for the image region outlined in the left panel. (B) Fraction of colocalization between β-arrestin2 and β2-adaptin wild type or its mutant was calculated from several image regions for different time points after Ang II treatment (1 µM) using ICCS as described in Materials and Methods. The dissociation rates were calculated from linear fits to the decay of the interaction fraction after reaching its maximum for five different cells from at least three different sets of experiments. (C) AT1R internalization was performed in HEK293 cells transfected with HA-AT1R and either β2-adaptin wild-type or Y737F mutant using [125I]-Ang II. Percent of receptor internalization was calculated as described in Materials and Methods. Data represent the mean ± s.e.m. of at least eight independent experiments.
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