Figure 3: Barbadin specifically blocks the interaction between β2-adaptin and β-arrestin. | Nature Communications

Figure 3: Barbadin specifically blocks the interaction between β2-adaptin and β-arrestin.

From: A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

Figure 3

(a–c) BRET-based assay monitoring the interaction between β2-adaptin-YFP and either β-arrestin1-RlucII or β-arrestin2-RlucII. HEK293T cells were pre-incubated with DMSO or Barbadin (100 μM) for 30 min before 45 min receptor stimulation with AVP (100 nM, a), ISO (10 μM, b) or AngII (100 nM, c). Data are the mean±s.e.m. of at least three independent experiments and unpaired t-test were used to assess statistical significance (***P<0.001; **P<0.005). (d,e) BRET-based kinetics monitoring the interaction between β-arrestin1-RlucII and β2-adaptin-YFP (d) or V2R-YFP (e) in HEK293T cells pretreated with DMSO or Barbadin (100 μM) for 30 min before receptor stimulation with AVP (100 nM) for the indicated times. Data are the mean±s.e.m. of three independent experiments. (f) Effect of Barbadin on the co-immunoprecipitation between β-arrestins and AP2. HEK293SL cells expressing V2R and Flag-β-arrestin2 were pretreated for 20 min with DMSO or Barbadin (50 μM) before stimulation by AVP (1 μM) for 2.5 or 5 min. Endogenous AP2 complexes (using the AP1/2 antibody) were immunoprecipitated (IP) from total cell lysates (TCL), and then analysed by western blot using anti-Flag, anti-epsin or anti-adaptin antibodies as described in the Material and Methods. TCLs represent 5% of input used for IP. IP were quantified over three independent experiments and statistical significance was assessed by a two-way ANOVA followed by Bonferroni’s post-hoc tests (*P<0.05). (g) Effects of Barbadin on the pull-down of β-arrestin1 and clathrin with GST-β2-adaptin. DMSO or Barbadin (100 μM) were incubated with GST-β2-adaptin (592–937) beads. HEK293T cells transfected with Flag-β-arrestin1 were lysed and added to the beads. The amounts of GST-β2-adaptin were detected by Coomassie, whereas β-arrestin1 and clathrin associated with GST-β2-adaptin were detected by western blot using anti-Flag and anti-clathrin (heavy chain) antibodies, respectively. Relative intensities were normalized to the GST input for each condition and densitometry data are the mean±s.e.m. of three independent experiments and analysed using a two-way ANOVA followed by Bonferroni’s post-hoc tests (NS, non-significant; ***P<0.001).

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