Figure 1.

β-Arrestin2 binds to unliganded AT₁R upon PKC activation. a, PKC activation triggers β-arrestin2 binding to AT₁R. HEK 293T cells were co-transfected with plasmids encoding NES–BirA (biotin ligase), β-arrestin2–Cerulean, and/or AT₁R–YFP–BAP, as indicated. After a 20-min stimulation with 100 nm AngII or 100 nm PMA, the cells were lysed and biotin-labeled AT₁R was pulled down using NeutrAvidin beads. Left panel, YFP- and Cerulean fluorescence of the co-precipitated proteins. The values were normalized to the autofluorescence of the beads. n = 3, *, p < 0.05, analyzed with one-way ANOVA (repeated measures, versus control samples containing AT₁R–YFP–BAP) with Bonferroni post hoc test. Scatter dot plots with columns (mean ± S.E.) are shown. Right panel, representative images show the YFP (top) and Cerulean (bottom) fluorescence of the pulled-down proteins bound to the surface of the beads, recorded with confocal microscope. Scale bar represents 100 μm. b, schematic representation of the used BRET set-ups with the applied stimuli. c–f, kinetics of β-arrestin2 binding to AT₁R upon PKC activation. Intermolecular BRET was measured between AT₁R–Rluc8 and β-arrestin2–Venus after AngII (c–f), PMA (c and d), or α_1AAR agonist A61603 (1 μm, d), M₃AChR agonist carbachol (10 μm, e), or EGF (100 ng/ml, f) treatments in every 70 s in cells co-expressing the indicated constructs. Stimulus-induced BRET ratio change was calculated by subtracting the corresponding vehicle-stimulated control. Kinetic curves are shown in c (n = 6) and d–f (n = 3), data are mean ± S.E. of independent biological replicates.