FIGURE 6.

C terminus-independent GRK interaction and rescue of K315P internalization. A, HEK 293 cells stably expressing HA-tagged B₂Rwt or G327* were transiently transfected with GRK3 and stimulated (or not) with 1 μm BK for 2 or 30 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 GRK3 (top panel). Blots were stripped and re-probed to confirm receptor expression (middle panel), and lysates were probed for GRK expression (bottom panel). IP, immunoprecipitation; IB, immunoblot. B, cells stably expressing GRK3wt, GRK3-5A, or GRK3-K220R were transiently transfected with G327*, and internalization was monitored with 1 nm [³H]BK for 15 min at 37 °C. Significance was determined by one-way ANOVA using Bonferroni's multiple comparison test. ***, p < 0.001. C, a representative immunoblot of cell lysates using ∼2 μg protein of cells (over)expressing GRK3wt, GRK3-K220R, and GRK3-5A is shown. D and E, HEK 293 cells stably (over)expressing the indicated β-arrestin subtypes or GRK3 constructs were transiently transfected with either K315P or K315P-G327*. Internalization was monitored for 15 min at 37 °C with 1 nm ³H. Significance was determined by one-way ANOVA using Bonferroni's multiple comparison test. ***, p < 0.001