Fig. 3.

β-arrestin1/2 allosterically enhance the rate of ERK2 autophosphorylation. (A and B) show ERK2 autophosphorylation kinetics assessed using an in vitro real-time fluorescence-based kinase assay, in the absence or presence of basal state βarr1 (A) or βarr2 (B) or each in their active state forms bound to either V₂Rpp or pβ₂V₂R together with Nb32. Kinase reactions were carried out with ERK2 (15 nM) and βarr1/2 (300 nM). Graphs (Top) represent the time course transitions of ERK2 autophosphorylation in relative fluorescence units (RFUs). Each experiment included control wells lacking ERK2 (with βarr 1 or 2, and ATP), and fluorescence signal data points from control reactions were subtracted to obtain corrected RFUs. (A and B, Lower) show quantification of ERK autophosphorylation presented as fold-enhancement of initial rates from each profile relative to vehicle control (ERK2 alone treated as onefold). Mean values are plotted, with error bars representing SEM (N = 3). Significances by one-way ANOVA, followed by Dunnett's multiple comparison test. *P ≤ 0.05; **P ≤ 0.01; and ***P ≤ 0.001.