Fig. 3
From: Structural basis of arrestin-3 activation and signaling
IP6 mediated trimerization and receptor-independent activation in cells. a-b Sedimentation velocity analytical ultracentrifugation (SVAUC) of arrestin-3. Measurements were performed in triplicate. a Representative SVAUC run in the absence of IP6 predicts a molecular weight matching a monomer. b In the presence of 100 µM IP6, the predicted molecular weight is consistent with a trimer. c Representative size exclusion chromatograms of arrestin-3 (Arr3) in the presence and absence of IP6. In the absence of IP6, 60 µM arrestin-3 (black) elutes at volume corresponding to the Stokes radius of a globular protein with a molecular mass of 64 ± 5.9 kDa. In the presence of 100 μM IP6, the elution volume is consistent with a globular protein of molecular mass 169.6 ± 9.7 kDa. The ratio of these molecular weights is consistent with IP6-dependent trimer formation. Measurements were performed in triplicate, errors are ± SEM. d Plot of the probability of the distances between spin labels at S13 and A392 for 100 µM arrestin-3 in the presence of the indicated molar ratios of IP6; inset shows the location of the spin labeled sites in basal arrestin-3 (PDB entry 3P2D6). e Comparison of JNK3 activation by GFP-arrestin-3 and Cys-less mutant. GFP-tagged arrestin allowed comparison of the expression levels of wild-type and variant arrestin-3, as described14, 33. JNK3 activation (mean ± SEM) was assessed by measuring the pp-JNK3 levels in COS7 cells co-transfected with HA-ASK1, HA-JNK3 and GFP or Venus-tagged wild-type and Cys-less arrestin-3. Assay was repeated five times and JNK3 phosphorylation was compared by one-way ANOVA followed by Bonferroni post hoc test with correction for multiple comparisons. ***p < 0.001
