FIGURE 5.

Subtype-specific role of β-arrestin2 for the internalization of B₂Rwt and G327*. A, HEK 293 cells stably expressing B₂Rwt or G327* were transfected with the indicated siRNAs. 72 h later internalization was determined with 5 nm [³H]BK (B₂Rwt) or 1 nm [³H]BK (G327*) after 5 min (B₂Rwt) or 15 min (B₂Rwt) as described under “Experimental Procedures.” B, shown is a representative immunoblot of endogenous β-arrestin levels in HEK 293 cells silenced with the indicated siRNAs and probed with a monoclonal β-arrestin1 antibody recognizing both subtypes. C, HEK 293 cells stably expressing β-arrestin1 or β-arrestin2 were transfected with B₂Rwt or G327*, and internalization was determined after 48 h with 5 nm [³H]BK (B₂Rwt) or 1 nm [³H]BK (G327*) after 5 min (B₂Rwt) or 15 min (G327*) at 37 °C. Where indicated, cells were pretreated with β-arrestin2 siRNA. D, shown is a representative immunoblot of cell lysates using ∼2 μg of protein of cells overexpressing β-arrestin1 and -2. Statistical analysis was done with one-way ANOVA using Bonferroni's multiple comparison test. ***, p < 0.001, **, p < 0.01; *, p < 0.05. ctl, control. E, HEK 293 cells stably expressing HA-tagged B₂Rwt_H or G327*_H were transiently transfected with β-arrestin1 (left) or β-arrestin2 (right) and stimulated (or not) with 1 μm BK for 5 min at 37 °C. After cross-linking with DSP and cell lysis, the protein complexes were precipitated with anti-HA-matrix. Precipitates were separated by reducing SDS-PAGE, transferred onto nitrocellulose membranes, and probed for β-arrestin1 (left) or β-arrestin2 (right) (top panels). Blots were stripped and re-probed to confirm receptor expression (middle panels), and lysates were tested for β-arrestin expression (bottom panels). IP, immunoprecipitation; IB, immunoblot.