Fig 2: Arrb2 regulates sLTP and DAMGO-induced analgesia and allodynia.(a) Normalized amplitudes of dorsal root stimulation evoked EPSCs (eEPSCs) in lamina IIo neurons before, during and after DAMGO (500 nM) perfusion in spinal cord slices in wild-type (WT) and Arrb2-knockout (KO) mice. In WT mice, DAMGO produces initial suppression of EPSCs followed by long-term potentiation (LTP) of eEPSCs during withdrawal phase. Spinal LTP (sLTP) is abolished in KO mice, only showing prolonged suppression of eEPSCs. *P<0.05, Two-Way ANOVA (WT. versus KO), n=5, 6 neurons per group. (b) von Frey test shows acute analgesia and late-onset mechanical allodynia following i.t. DAMGO (1 μg) in WT and KO mice and the reversal of allodynia by MK-801 (i.t., 10 nmol). *P<0.05, versus baseline (BL), #P<0.05 (WT versus KO). Two-Way ANOVA, n=8 mice per group. All the data are mean±s.e.m. d, day.

Fig 3: Depletion of β-arrestin2 aggravates apoptosis of astrocytes stimulated by IL-6. A Cell viability was detected treated with different concentration of IL-6 (0, 100, 200, 300, 400, 500 ng/mL) for primary astrocytes. B, D Apoptosis rate of WT and Arrb2−/− astrocytes were detected by flow cytometry (n = 3). C, E PI/Hoechst staining to observe cell apoptosis stimulated by IL-6 (300 ng/ml). Scale bar: 20 μm n = 7. F Protein levels of Bcl-2 and BAX were detected in WT and Arrb2−/− astrocytes. G, H Protein levels of p-JAK/JAK and p-STAT3/STAT3 were detected in WT and Arrb2−/− astrocytes or transfected with Arrb2 siRNA. I After extraction of nucleus protein from astrocytes, the levels of p-STAT3/STAT3 were detected. J Immunofluorescence staining p-STAT3 in WT and Arrb2−/− astrocytes. p-STAT3: Red; Hoechst: Blue; Scale bar: 20 μm. A Bars and error flags represent the means ± SEM of at least three independent experiments; statistically significant by Student t test; *P < 0.05, **P < 0.01, ***P < 0.001 VS WT Control group or Arrb2−/− untreated group. #P < 0.05, ##P < 0.01 VS corresponding IL-6 stimulated group. D, E Data were denoted as mean ± SEM using two-way ANOVA, then combined with Tukey to assess the differences between groups. ***P < 0.001 VS WT Control group; ###P < 0.001

Fig 4: β-arrestin 1 (β-arr1), β-arrestin 2 (β-arr2), and Nedd4 are recruited to mGlu7 by agonist stimulation within 5 min.(A–F) Time-course interaction among mGlu7, Nedd4, and β-arrestins. HEK 293 T cells were co-transfected with FLAG-mGlu7, HA-Nedd4, and β-arrestin 1-GFP (A) or β-arrestin 2-GFP (B). In panel E, myc-mGlu7, HA-Nedd4, and β-arrestin 1-FLAG were utilized. L-Glu (1 mM) was administered for the indicated time before cell lysis. Cell lysates were immunoprecipitated with anti-FLAG antibody and western blotting was carried out with the indicated antibodies. Time-course binding of β-arrestins to mGlu7 (C), Nedd4 to mGlu7 (D), β-arrestin 1 to Nedd4 (F) were quantified and presented as mean ± SEM (C, β-arr1, 5 min, 1.79 ± 0.31; 15 min, 1.11 ± 0.23; 30 min, 0.73 ± 0.12; 60 min, 0.52 ± 0.17; β-arr2, 5 min, 1.36 ± 0.10; 15 min, 1.36 ± 0.15; 30 min, 1.01 ± 0.18; 60 min, 0.96 ± 0.38; D, β-arr1, 5 min, 2.36 ± 0.57; 15 min, 1.16 ± 0.31; 30 min, 0.61 ± 0.14; 60 min, 0.72 ± 0.49; β-arr2, 5 min, 2.20 ± 0.36; 15 min, 1.97 ± 0.68; 30 min, 1.96 ± 0.62; 60 min, 1.83 ± 0.49; F, β-arr1, 1 min, 1.67 ± 0.12; 10 min, 1.72 ± 0.19; 30 min, 1.11 ± 0.17; 120 min, 0.71 ± 0.13; n = 3‒4, *p<0.05 versus 0 min, Student’s t-test). (G) Endogenous β-arrestin 1, Nedd4, and mGlu7 are present in the same complex. After cortical neurons were treated with 400 μM L-AP4 for 5 min, the lysates were immunoprecipitated with anti-β-arrestin 1 antibody or anti-GFP antibody as a control. Arrow and arrowhead indicate β-arrestin 1 and immunoglobulin heavy chain (IgH), respectively. (H) Bar graph represents mean ± SEM showing quantification of mGlu7 or Nedd4 binding levels to β-arrestin 1 (mGlu7 L-AP4, 1.56 ± 0.19; Nedd4 L-AP4, 1.46 ± 0.13; n = 5, *p<0.05 versus vehicle, Student’s t-test). (I) myc-mGlu7 and HA-Nedd4 were co-transfected either with pSuper β-arrestin 1, 2 shRNA or pSuper control shRNA in HEK 293 T cells. Three days after transfection, 1 mM L-Glu was treated for 5 min and cell lysates were immunoprecipitated using anti-myc antibody. Immunoprecipitates were resolved by SDS-PAGE and analyzed by western blotting using the indicated antibodies. (J) Bar graph represents mean ± SEM showing Nedd4 binding to mGlu7 levels normalized to untreated Ctl KD (Ctl KD + L-Glu, 1.98 ± 0.22; β-arr1 KD, 1.46 ± 0.23; β-arr1 KD + L-Glu, 1.51 ± 0.39; β-arr2 KD, 1.19 ± 0.13; β-arr2 KD + L-Glu, 0.97 ± 0.25; n = 3‒5, *p<0.05, n.s. indicates p>0.05, one-way ANOVA). (K) β-arrestins are involved in agonist-induced Nedd4 recruitment to mGlu7 in neurons. Cultured cortical neurons were infected with lentiviruses harboring β-arrestin 1 or 2 shRNA (β-arr1 or 2 KD), or non-related target shRNA (Ctl KD) for 7 days. Both β-arrestin 1 and 2 shRNA were co-infected for β-arr1/2 KD lane. After treatment with 400 μM L-AP4 for 5 min, the lysates were immunoprecipitated with anti-mGlu7 antibody and endogenous Nedd4 binding was analyzed by western blotting. (L) Bar graph represents mean ± SEM showing quantification of Nedd4 binding levels to mGlu7 (Ctl KD + L-AP4, 1.54 ± 0.11; β-arr1 KD + L-AP4, 1.99 ± 0.29; β-arr2 KD + L-AP4, 1.73 ± 0.16; β-arr1/2 KD, 1.15 ± 0.07; n = 4, *p<0.05 versus vehicle in Ctl KD, Student’s t-test). (M) β-arrestins mediate agonist-induced ubiquitination of mGlu7 in neurons. Cultured cortical neurons were infected with β-arrestins shRNA lentiviruses and then incubated with L-AP4 (400 μM) as panel K. Cell lysates were immunoprecipitated with anti-ubiquitin antibody (FK2) and western blotting was performed with anti-mGlu7 antibody. (N) Bar graph represents mean ± SEM showing mGlu7 ubiquitination levels normalized to Ctl KD (β-arr1 KD, 0.77 ± 0.06; β-arr2 KD, 0.74 ± 0.08; β-arr1/2 KD, 0.50 ± 0.03; n = 4, **p<0.01, *p<0.05, Student’s t-test).10.7554/eLife.44502.015Figure 3—source data 1.Binding analysis of mGlu7, Nedd4, and beta-arrestins complex.

Fig 5: Arrb2 deficiency increases synaptic GluN2B in SDH neurons.(a,b) Expression of GluN2A and GluN2B in synaptosome-containing P2 fraction of spinal cord dorsal horn (SDH) of WT and Arrb2-KO mice. (a) Enrichment of synaptic proteins PSD-95 and synaptophysin (SYN) in synaptosome-contacting P2 fraction but not cytosol fraction. (b) Quantification of GluN2A and GluN2B expression. *P<0.05, Student t-test, n=4 mice per group. (c) Surface expression of GluN2B and Arrb2 in HeLa cells transfected with GluN1/GluN2B and GluN1/GluN2B/Arrb2. (d) Relative expression levels of GluN2B and Arrb2, normalized with N-cadherin, a positive control for surface expression. *P<0.05, Student’s t-test, n=3 cultures per group. (e) Pull down assay showing Co-IP of Arrb2 with GluN2B in HeLa cells. All the data are mean±s.e.m. Gel images have been cropped for presentation. Full size images are presented in Supplementary Fig. 8a.