Time course of isoproterenol-stimulated β₂AR ubiquitination in HEK-293 cells. A, HEK-293 cells stably expressing Flag-β₂AR (293^β2AR) or Flag-β₂AR-mYFP were stimulated with 1 μm isoproterenol for the indicated times, and receptors were isolated with anti-Flag-agarose affinity beads. Receptor ubiquitination was detected with a ubiquitin antibody conjugated to peroxidase (FK2-HRP). The lower panel shows the amounts of receptor as detected by a Flag antibody (M2). B, ubiquitin smears in each lane from the blot shown in A were quantified and plotted as bars. The graph shown represents a summary of four independent experiments. ^**, p < 0.01; ^***, p < 0.001 compared with non-stimulated samples; repeated measures ANOVA, Bonferroni post-test.

A dominant negative Nedd4 inhibits isoproterenol-stimulated β₂AR ubiquitination and degradation. A, 293^β2AR were transfected with vector or catalytically inactive Nedd4, Mdm2, or AIP4, and receptors were immunoprecipitated under unstimulated or agonist-stimulated conditions and probed for ubiquitination as in Fig. 1A. Receptor amounts in each sample were determined by reprobing the blot with a M2 Flag antibody (second panel from top). The expression levels of Nedd4-DN, Mdm2-DN, and AIP4-DN are shown in the respective lysate blots. In each case, the band corresponding to endogenous Nedd4, Mdm2, and AIP4 is also indicated. B, quantification of ubiquitination signals from three independent experiments is plotted as a bar graph, where maximum signal is set as 100%. #, p < 0.001 compared with the respective nonstimulated samples; ^**, <0.01, Nedd4 stimulated versus all other stimulated samples, ANOVA, Bonferroni post-test. C, 293^β2AR were transfected with vector, Nedd4-DN, Mdm2-DN, or AIP4-DN and stimulated with isoproterenol for 24 h or not. The receptor levels were determined by ¹²⁵I-CYP binding. The percent decrease in receptor amounts compared with levels under no stimulation was calculated and plotted as a bar graph, which summarizes data from 3–5 experiments. Data were analyzed by one-way ANOVA. ^**, p < 0.01.

Nedd4 RNAi inhibits agonist-stimulated β₂AR ubiquitination and degradation. A, 293^β2AR cells were transfected with siRNA that target nothing (CTL), Mdm2, Nedd4, or AIP4. Flag-β₂AR IPs were probed with ubiquitin antibody (FK2), shown in the upper panel and reprobed with a β₂AR antibody (H-20), displayed in the lower panel to detect ubiquitinated receptor and total receptor in each sample. B, ubiquitin signal in each sample is normalized to the respective receptor amount in the immunoprecipitate. All values are normalized to the maximum ubiquitin signal, which is set as 100%. The graph summarizes data from three independent experiments. ^**, p < 0.001, Nedd4+ versus CTL+ and Nedd4+ versus AIP4+; #, p < 0.01 Nedd4+ versus Mdm2+; repeated measures ANOVA, Bonferroni comparison. C, 20 μg of whole cell lysates from control, Mdm2, Nedd4, and AIP4 siRNA-treated cells were probed for Mdm2 (2A10 antibody) in the top panel, Nedd4 (anti-WW2 domain, Millipore) in the middle panel, and AIP4 (Santa Cruz Biotechnology) in the lowest panel. The extra cross-reactive bands in the Nedd4 blot are: ^*, Nedd4-2; ^**, unknown protein with WW domain. D, 293^β2AR cells transfected with siRNA targeting no known protein (CTL), Mdm2, Nedd4, or AIP4 siRNA were stimulated with 1 μm isoproterenol for 30 min, and receptor internalization was determined by radioligand binding as described under “Experimental Procedures.” The graph summarizes results from four independent experiments. ^**, p < 0.01, Mdm2 versus control and AIP4. E, 293^β2AR under control or depleted conditions for either Mdm2, Nedd4, or AIP4 were stimulated with vehicle or 1 μm isoproterenol for 24 h. The receptor levels under stimulated and unstimulated conditions were determined by radioligand binding. The bar graph depicts degraded receptors as a percentage of total receptors in each sample and is the summary of four independent experiments. ^**, p < 0.01 Nedd4 versus Mdm2; #, p < 0.05 Nedd4 versus Control and AIP4; one-way ANOVA, Bonferroni comparison.

Nedd4 is required for lysosomal targeting of the β₂AR. A, 293^β2AR cells were transfected with siRNA that target nothing, Nedd4, and Nedd4-2, stimulated for 6 h with 1 μm isoproterenol for the indicated times, fixed, permeabilized, and immunostained for the β₂AR (green) and LAMP2 (red). Colocalization is seen in the overlay panels (yellow). The data shown are from one of four independent experiments with identical results. B, whole cell lysates (20 μg) were probed with anti-WW2 domain antibody that recognizes both Nedd4 and Nedd4-2. The predicted molecular weight of human Nedd4 is 104,086 (NP_006145) and Nedd4-2 is 109,891 (NP_056092). C, 293^β2AR cells were transfected with siRNA targeting Mdm2 (top row) or AIP4 (bottom row), stimulated with 1 μm isoproterenol for indicated times and analyzed for receptor and LAMP2 distribution by immunostaining as described in A. The confocal panels displayed are representative of four independent experiments with identical results.

Time course of isoproterenol-stimulated β-arrestin interaction with Nedd4, Mdm2, and AIP4. HEK-293 cells were transiently transfected with HA-β₂AR and either β-arrestin1-Flag or β-arrestin2-Flag. After 1 μm isoproterenol stimulation for the indicated times, followed by DTME cross-linking, β-arrestins were immunoprecipitated and further analyzed by Western blots for Nedd4 binding (upper panel), and the amounts of β-arrestin in the immunoprecipitate (middle panel). The lower panel displays lysate levels of Nedd4 in each lane. Under these experimental conditions, we do not observe a clear separation of the three WW-positive proteins (see “Experimental Procedures”). B, Nedd4 bands detected in A were quantified, and the graph represents the mean from three independent experiments. ^*, p < 0.01 two-way ANOVA. C and D, HA-β₂AR and β-arrestin2-Flag were transiently expressed in HEK-293 cells and Flag β-arrestin precipitates were isolated after DTME cross-linking and analyzed for bound Mdm2 in C and AIP4 in D. Data are representative blots from one of three separate experiments.

β-Arrestin2 expression regulates Nedd4 binding to activated β₂AR. A, HEK-293 cells stably expressing Flag-β₂AR (293^β2AR) plated on 100-mm Biocoat dishes were serum-starved for 1 h, stimulated with 1 μm isoproterenol for indicated times, subjected to chemical cross-linking (DTME), receptors solubilized in radioimmune precipitation assay buffer, and immunoprecipitated with anti-Flag affinity beads. As shown in the top panel, the immunoprecipitates (IP) were probed with an antibody specific for Nedd4 (WW2 domain, Millipore). The levels of Nedd4 in the lysates are shown in the second panel. The third panel displays the amount of receptors in the IPs as detected by a β₂AR antibody (H-20, Santa Cruz Biotechnology). B, Nedd4 bands in receptor IP were quantified and displayed in the bar graph. ^**, p < 0.01 one-way ANOVA, Bonferroni comparisons. C, 293^β2AR cells were transfected with either control or β-arrestin2-specific siRNA with GeneSilencer reagent (Genlantis). 48 h after transfection, cells were replated on Biocoat dishes, and 24 h after this were serum-starved and stimulated with isoproterenol as indicated and subjected to cross-linking as described under “Experimental Procedures.” The top three panels show the Western blotting of IP with Nedd4 antibody (top panel), anti-β-arrestin1/2 antibody (2nd panel), and anti-β₂AR antibody (3rd panel). The levels of Nedd4 and β-arrestin in whole cell extracts are also shown in the lysate blots displayed in the lower panels. D, the bar graph represents quantification of Nedd4 in receptor IPs from eight independent experiments. The amount of Nedd4 in the control-unstimulated sample was assigned as 1. ^***, p < 0.01 control Iso versus control NS; ##, p < 0.01; control Iso versus β-arr2 Iso.

Roles of ubiquitination in the life cycle of agonist-stimulated β₂AR. Step 1, within seconds of agonist exposure, β₂ARs stimulate G_s and adenylyl cyclase, increasing cellular cAMP. Step 2, agonist-occupied receptors are also phosphorylated by GRKs on cytoplasmic domain seryl and/or threonyl residues, within seconds to minutes of agonist exposure. Step 3, cytosolic β-arrestin2 (βarr2) translocates to phosphorylated receptors within 1–5 min after agonist treatment. Agonist-dependent β-arrestin ubiquitination (U) occurs immediately upon β-arrestin recruitment and is mediated by Mdm2 that is bound to β-arrestin. β-Arrestin recruitment prevents further G protein coupling and β-arrestin ubiquitination allows it to form signaling and endocytic complexes, facilitating both receptor endocytosis and MAPK signaling. Step 4, β-arrestin conformational changes that occur upon receptor binding allow its interaction with Nedd4, which probably displaces Mdm2 from β-arrestin (5–15 min after agonist treatment). Step 5, by interacting simultaneously with β₂AR, clathrin, and AP-2, β-arrestin2 facilitates β₂AR endocytosis. Step 6, β-arrestin2 is deubiquitinated by an as-yet unidentified process, leading to its disengagement from the receptor complex (10–15 min after agonist treatment). Nedd4 mediates ubiquitination of the endosomally located β₂AR. Step 7, ubiquitinated β₂ARs move on into early endosomes (at >15 min after activation). Step 8, β₂AR ubiquitination persists until about 6 h after agonist stimulation, when β₂ARs move into late endosomal/lysosomal compartments. Step 9, level of ubiquitinated β₂ARs decreases, as ubiquitinated receptors are degraded in lysosomes (6–24 h or more after agonist stimulation). Step 10, from the early endosomes, receptors may take up an alternate path and enter recycling endosomes (<15–30 min after activation), in which β₂ARs become dephosphorylated and perhaps deubiquitinated, and return to the plasma membrane as “naïve receptors” (Step 11).