FIGURE 4.

ARRDC3 regulates agonist-induced β₂AR recycling. A, rate of β₂AR internalization was evaluated by cell surface ELISA. Cells were transiently transfected with vector, ARRDC3, or ARRDC3-AASA/AALA in FLAG-β₂AR-stable HEK293 cells. n = 3. Data are represented as mean ± S.D. B, immunofluorescence staining and co-localization of the β₂AR and ARRDC3 or ARRDC3-AASA/AALA upon 20 min of treatment with 10 μm ISO in FLAG-β₂AR-stable HEK293 cells. C, total amount of β₂AR present in β₂AR-stable HEK293 cells at each ISO-treated time point was assessed by [¹²⁵I]iodocyanopindolol binding assay. The graph represents the mean ± S.D. from three independent experiments. D, immunofluorescence staining and co-localization analysis, demonstrated by Pearson's coefficient, of the β₂AR, ARRDC3, and the lysosomal marker LAMP1, upon a 4-h treatment with 10 μm ISO in β₂AR-stable HEK293 cells. Representative images are shown. Pearson's coefficient is the mean ± S.D. from n = 30. E, flow cytometric analysis of the β₂AR recycling by uptake and efflux of cell surface-labeled M1 anti-FLAG antibody in FLAG-β₂AR-stable HEK293 cells transiently transfected with vector, ARRDC3, or ARRDC3-AASA/AALA. The graph represents the mean ± S.D. from three independent experiments. F, representative immunofluorescence staining and co-localization of the β₂AR and ARRDC3 or ARRDC3-AASA/AALA upon 20 min of treatment with 10 μm ISO followed by a 30-min incubation with 10 μm propranolol. G, flow cytometric analysis of β₂AR recycling by uptake and efflux of cell surface-labeled M1 anti-FLAG antibody in FLAG-β₂AR-stable HEK293 cells transfected with siControl or siARRDC3. The graph represents the mean ± S.D. from three independent experiments. Unpaired t test. H, quantitation of fluorescence changes of SpH-β₂AR demonstrating a rapid loss of surface β₂AR level in response to 10 μm ISO treatment and recovery in response to washout with 10 μm alprenolol in SpH-β₂AR-stable HEK293 cells transfected with siControl or siARRDC3. Fluorescence values were normalized to initial values from multiple cells (n >1,500 cells analyzed). I, flow cytometric analysis of TfR recycling by uptake and efflux of cell surface-labeled transferrin. The graph represents the mean ± S.D. from three independent experiments. Scale bars, 5 μm.