β-Arrestin1 and 2 differentially regulate PACAP-induced PAC1 receptor signaling and trafficking

Figures

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  Fig 1. PAC1R interacts with and β-arrestin2 with similar PACAP-dose and time dependency. HEK293T cells were transfected with the indicated combinations of plasmid vectors. (A) Representative images of NanoBiT luminescence 15 min after addition of 100 μM PACAP. Scale bar, 1 mm. (B) The ratios of luminescence intensity compared with cells mock transfected with pFC36K empty vector. AVPR2-SmBiT serves as positive control. **p < 0.01 vs. mock, one-way ANOVA followed by Fisher-PLSD test. (C and D) PACAP-dose dependent increase in luminescence accumulation intensity which was determined 60 min after PACAP addition. *p < 0.05 vs. 0 nM PACAP, one-way ANOVA followed by Fisher-PLSD test. (E and F) Time course of changes in luminescence intensity for 60 min after addition of the indicated concentrations of PACAP. β-arr1, β-arrestin1; β-arr2, β-arrestin2. Values are mean ± SEM (B–D) or means (E and F) of three independent experiments each conducted in duplicate.

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  Fig 2. Time-lapse cell imaging showing PAC1R and β-arrestin coupling and translocation in HeLa cells. HeLa cells were transfected with the indicated combinations of plasmid vectors. (A and B) Representative images of NanoBiT luminescence at 3, 15, 30 and 60 min after stimulation with 1 μM PACAP. (C and D) Representative time-dependent changes of line-scan images for 60 min after stimulation with 1 μM PACAP. (E and F) Time course of changes in luminescence intensity at the vicinity of the plasma membrane (membrane) and the cytoplasm for 60 min after stimulation with 1 μM PACAP in HeLa cells. Scale bars, 10 μm. β-arr1, β-arrestin1; β-arr2, β-arrestin2. Values are mean ± SEM (n = 3–5). *p < 0.05 vs. cytoplasm, repeated measure two-way ANOVA followed by Fisher-PLSD test. See also S1 and S2 Movies.

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  Fig 3. PAC1R and β-arrestin coupling and translocation in primary cultured cortical neurons. Primary cultured cortical neurons were infected with the indicated combinations of lentiviruses. (A and B) PACAP-dose dependent increase in NanoBiT luminescence accumulation intensity which was determined 60 min after PACAP addition. (C and D) Time course of changes in luminescence intensity for 60 min after addition of the indicated concentrations of PACAP. (E and F) Representative images of NanoBiT luminescence at 3, 15, 30 and 60 min after stimulation with 1 μM PACAP. (G and H) Representative time-dependent changes of line-scan images for 60 min after stimulation with 1 μM PACAP. Scale bars, 10 μm. β-arr1, β-arrestin1; β-arr2, β-arrestin2. Values are mean ± SEM (A and B) or mean (C and D) of three independent experiments each conducted in duplicate. *P < 0.05, **p < 0.001 and ***p < 0.001 vs. 0 nM PACAP, one-way ANOVA followed by Fisher-PLSD test. See also S3 and S4 Movies.

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  Fig 4. Differential effects of β-arrestin1 and β-arrestin2 siRNA on PAC1R internalization. (A) Knockdown of endogenous β-arrestin1 and β-arrestin2 in HEK293T cells. The upper images, representative images of western blots. Values are mean ± SEM of three independent experiments. **p < 0.01 and ***p < 0.001, two-way ANOVA followed by Fisher-PLSD test. (B–E) Representative images of HEK293T cells that were transfected with PAC1R-Halo and the indicated siRNA, labeled with Alexa Fluor 488 HaloTag ligand and treated with 1 μM PACAP or saline for 30 min. (F) Quantification of PAC1R-Halo internalization. Scale bar, 10 μm. β-arr1, β-arrestin1; β-arr2, β-arrestin2. Values are mean ± SEM of 60 cells obtained from three independent experiments. *p < 0.05 and **p < 0.01, two-way ANOVA followed by Fisher-PLSD test.

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  Fig 5. Differential effects of silencing of β-arrestin1 and β-arrestin2 on PACAP-induced ERK1/2 activation. (A) Representative images of western blots for total and phosphorylated ERK1/2 in HEK293T cells transfected with the indicated siRNA and treated with 1 μM PACAP or saline for 3 or 25 min. (B) Quantification of ERK1/2 activation by normalizing phosphorylated ERK1/2 to total ERK1/2 levels analyzed by western blotting. β-arr1, β-arrestin1; β-arr2, β-arrestin2. Values are mean ± SEM of three independent experiments. *p < 0.05 and **p < 0.01, two-way ANOVA followed by Fisher-PLSD test.