FIGURE 4.

The role of Ubc9 on arrestin SUMOylation and receptor trafficking. A, HeLa cells transfected with control (CON, GAPDH) and Ubc9 siRNA plus His-tagged SUMO or empty vector (pcDNA) were solubilized in 2× sample buffer and subjected to IB to detect HA-tagged arrestin-3. SUMOylated arrestin is indicated. Shown are IBs from one of three independent experiments. B, the role of Ubc9 on GPCR internalization was examined in HeLa cells co-transfected with DNA encoding FLAG-β₂AR and control (Ctrl, GAPDH) or Ubc9 siRNA. The cells were treated with 10 μm isoproterenol for 30 min, and during the final 5 min of the incubation, the cells were treated with 25 μg/μl Alexa Fluor 594-conjugated transferrin (Tfn). The cells were processed as described under “Experimental Procedures.” Shown are representative confocal fluorescence microscopy images of cells analyzed from three independent experiments. Bar, 20 μm. C, shown is a graph representing FLAG-β₂AR internalization calculated in control (GAPDH) and Ubc9 siRNA-transfected cells shown in B. The percentage of receptor internalization was determined by counting cells showing punctate staining and no membrane staining in 10 randomly selected fields containing 5–10 cells/field. The data represent the averages from three independent experiments, and the error bars represent the standard error of the mean. The data were analyzed by a Student's t test (*, p < 0.0001). D, internalization of the transferrin receptor was determined in the same cells used for the analysis performed in C. Internalization of the transferrin receptor was calculated by measuring the fluorescence intensity of transferrin Alexa Fluor 594 using LSM 510 image analysis software, as described under “Experimental Procedures.” The percentage of fluorescence intensity in Ubc9 transfected cells was normalized to control (GAPDH) transfected cells. The error bars represent the standard error of the mean. E, levels of Ubc9 and actin were detected by immunoblotting whole cell lysates from control and Ubc9 siRNA-transfected cells. F, FLAG-β₂AR internalization was examined in HEK293 cells co-transfected with DNA encoding FLAG-β₂AR and control (GAPDH) or Ubc9 siRNA, as described under “Experimental Procedures.” The cells were treated with vehicle (0.01 mm ascorbic acid) and 10 μm isoproterenol for 15 min, and receptor internalization was measured by cell surface ELISA. The data represent the averages from three or four independent experiments, and the error bars represent the standard error of the mean. The data were analyzed by a Student's t test (*, p < 0.05). Shown in the inset are representative IBs to detect Ubc9 and actin levels.