A. Active (phosphorylated at T183 and Y185 by MEK1) or inactive ERK2 (30 pmol) was pre-incubated with or without 30 pmol of indicated arrestin for 20 min at 30°C, then phosphorylated rhodopsin (50 pmol) was added and incubated in the light (to produce P-Rh*) in 0.1 ml for 5 min. Rhodopsin-containing membranes were pelleted through 0.2 M sucrose cushion and dissolved in SDS sample buffer. ERK2 in the pellet (1/300 of each sample) was quantified by Western blot using anti-ERK antibodies (Cell Signaling) and known amounts of purified ERK2 to generate calibration curve. Abbreviations: Arr1, visual arrestin-1, Arr2, arrestin-2, Arr3, arrestin-3. Representative blot is shown. B. Quantification of ERK2 binding to P-Rh*-associated arrestins. C. CNBr-activated Sepharose (30 µl) containing 9 µg of covalently attached active phosphorylated (without or with 1 mM ATP) or inactive ERK2 was incubated with 3 µg of indicated purified arrestin in 60 µl of binding buffer (50 mM Tris-HCl, pH 7.4, 100 mM KCl, 1 mM EGTA, 1 mM DTT) for 20 min at 30°C. The beads were washed twice with 1 ml of ice-cold binding buffer supplemented with 0.01 mg/ml BSA. Bound arrestins were eluted with SDS sample buffer and quantified by Western blot, where known amounts of respective arrestins were run alongside samples to generate calibration curves. Means ± SD of three independent experiments are shown in panels B and C.

A, B. ERK2 (12 pmol) was incubated with MEK1 (2 pmol) in 0.1 ml of 50 mM Hepes-Na, pH 7.2, 100 mM NaCl, and 0.1 mM [γ-³²P]ATP in the absence (control) or presence of 4.4 pmol of arrestin-2 (Arr2), arrestin-3 (Arr3), or arrestin-3-(1–393) (Arr3-(1–393)) for 30 min at 30°C. The reaction was stopped by MeOH-precipitation of the proteins. The pellet was dissolved in SDS sample buffer and subjected to SDS-PAGE. The gels were stained, dried, and exposed to X-ray film to visualize radiolabeled bands (panel A). ERK2 bands were cut out and ³²P incorporation was quantified by scintillation counting (panel B). Means ± SD of four independent experiments are shown. (**) p<0.01, as compared to control.

COS-7 cells were transfected with WT, 3A, or Δ7 mutant forms of Flag-tagged arrestin-2 (A) or arestin-3 (B), along with ERK2-HA, with or without HA-β2AR. Cells were serum starved overnight 24 hours post-transfection and treated for 10 min at 37°C with or without 10 µM β2AR agonist isoproterenol. Cells were lysed, and arrestins were immunoprecipitated with anti-Flag antibody, and co-immunoprecipitated ERK2 and β2AR were detected with anti-HA antibody. Bar graphs show the ratio of co-immunoprecipitated ERK2 to immunoprecipitated arrestin. The data from three independent experiments were statistically analyzed by ANOVA. The significance of the differences is indicated, as follows: * or ^&, p<0.05; ** or ^&&, p<0.01, as compared to corresponding within group basal level of ERK2 co-immunoprecipitation (black bars); ^a or ^$ or ^#, p<0.05 compared to WT control (black bar in WT group).

COS-7 cells were transfected with WT, 3A, or Δ7 mutant forms of Flag-tagged arrestin-2 (A) or arrestin-3 (B), along with MEK1-HA, with or without HA-β2AR. Cells were serum starved overnight 24 hours post-transfection and treated for 10 min at 37°C with or without 10 µM β2AR agonist isoproterenol. Cells were lysed, and arrestins were immunoprecipitated with anti-Flag antibody, and co-immunoprecipitated MEK1 and β2AR were detected with anti-HA antibody. Bar graphs show the ratio of co-immunoprecipitated MEK1 to immunoprecipitated arrestin. The data from three independent experiments were analyzed by ANOVA. ^&, p<0.05, as compared to corresponding within group basal level of MEK1 co-immunoprecipitation (black bars).

COS-7 cells were transfected with WT, 3A, or Δ7 mutant forms of Flag-tagged arrestin-2 (A) or arestin-3 (B), along with c-Raf1-HA, with or without HA-β2AR. Cells were serum starved overnight 24 hours post-transfection and treated for 10 min at 37°C with or without 10 µM β2AR agonist isoproterenol. Cells were lysed, and arrestins were immunoprecipitated with anti-Flag antibody, and co-immunoprecipitated c-Raf1 and β2AR were detected with anti-HA antibody. Bar graphs show the ratio of co-immunoprecipitated c-Raf1 to immunoprecipitated arrestin. The data from three independent experiments were analyzed by ANOVA. * or ^& or ^a, p<0.05, as compared to corresponding within group basal level of c-Raf1 co-immunoprecipitation (black bars); ^$ or ^#, p<0.05, compared to WT control (black bar in WT group).

HA-tagged ERK2 was co-expressed with Flag-tagged WT arrestin-2 (A,B,C), or arrestin-3 (D,E,F) in COS-7 cells. Cells were serum starved 24 hours after transfection and stimulated for 10 min at 37°C with 10 µM of indicated β2AR ligands. Arrestins were immunoprecipitated with anti-Flag antibody, and co-immunoprecipitated ERK2 was visualized with anti-HA antibody. The binding of ERK2 to arrestin-2 (B) or arrestin-3 (E) was significantly increased by treatment with ligands. C,D. ERK1/2 activation in cell lysates was determined by Western blot with anti phospho-ERK1/2 antibody. Means ± SD of 3–4 independent experiments are shown in bar graphs; representative blots are shown in panels A and D. ANOVA with Bonferroni post-hoc test revealed the following differences: *, p<0.05; **, p<0.01; ***, p<0.001, as compared to untreated cells.

DKO MEFs were infected with retrovirus encoding GFP (control, -), or untagged WT arrestin-2 (A2-WT), arrestin-2-3A (A2-3A), or arrestin-2-Δ7 (A2-Δ7). The cells were serum-starved 48 hours post-infection for 2 hours, stimulated with 1 µM ICI118551 for 10 min at 37°C, lysed, and analyzed by Western blot. Means ± SD of 3–4 independent experiments are shown in bar graphs; representative blots are shown below. *, p<0.05; **, p<0.01.

A. DKO MEFs were infected with retrovirus encoding GFP, untagged WT arrestin-2 (A2-WT), arrestin-2-3A (A2-3A), or arrestin-2-Δ7 (A2-Δ7). Serum-starved cells were stimulated with indicated β2AR ligands, lysed, and analyzed by Western blot. Representative blots are shown. The expression of different forms of arrestin-2 is compared in the blot below. B. Phospho-ERK1/2 bands were quantified. Means ± SD of 3 independent experiments are shown. C. Comparison of arrestin expression levels in COS-7 cells (5 µg protein/lane) and DKO MEFs (10 µg protein/lane) was performed by Western blot with anti-arrestin antibody. Standards containing indicated amounts of purified arrestin-2 were run along with cell lysates to generate calibration curve. Arrestin expression was measured by quantitative Western in COS-7 cells: A2-WT, 100.1 pmol/mg; A2-3A, 81.1 pmol/mg; A2-Δ7, 92.8 pmol/mg. Arrestin expression in DKO MEFs was much lower: A2-WT, 13.2 pmol/mg; A2-3A, 12.3 pmol/mg; A2-Δ7, 21.7 pmol/mg.