Fig 2: Arrb2 deficiency causes prolongation of NMDA-induced allodynia.(a) Prolongation of NMDA (1 nmol, i.t.)-induced mechanical allodynia in Arrb2 KO mice. *P<0.05, versus baseline (BL), #P<0.05 (KO versus WT), Two-Way ANOVA, n=5, 7 mice per group. (b) GluN2A antagonist TCN-201 (10 nmol, i.t.) fails to reduce the NMDA-induced allodynia. (c) GluN2B antagonist Ro25-6091 (10 nmol, i.t.) reduces the NMDA-induced allodynia in WT and KO mice. *P<0.05, versus pre-NMDA injection baseline (6 d), One-Way ANOVA, n=6 mice per group. #P<0.05 (WT versus KO), Two-Way ANOVA, n=6 mice per group. BL, baseline. The antagonists were given 6 d after i.t. NMDA injection. Note that the difference between WT and KO mice is abolished by the GluN2B inhibition. All the data are mean±s.e.m. d, day.

Fig 3: 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 4: Beta-arrestin-1 and 2 protein expression in lung ADC and SCC samples in the in-house and the amsbio TMAs. Scatter plots represent quantitative analysis (VIS score) of IHC results in sections of a–c the in-house TMA and of d the amsbio TMA. Sections of the in-house TMA were incubated with an antibody that (a) recognises both beta-arrestin-1 and 2 (ARRB1-2), or specifically detects either (b) beta-arrestin-1 (ARRB1) or c beta-arrestin-2 (ARRB2). d Sections of the amsbio TMA were incubated with the anti-beta-arrestin-1-2 antibody or with the specific anti-beta-arrestin-1 antibody and “n” indicates the number of subjects in each group. Data points and their median are shown. ****P < 0.0001 **P < 0.01 and *P < 0.05 using Mann–Whitney rank sum test

Fig 5: ß-arrestins and Nedd4 regulate endocytosis of mGlu7 in neurons.(A) The endocytosis of mGlu7 was analyzed by an antibody uptake internalization assay. myc-mGlu7 was co-transfected with Nedd4 WT, C867S mutant, or vector control (Vec) in cultured hippocampal neurons. Two days after transfection, neurons were labeled with anti-myc antibody for 10 min, and returned to conditioned media for 15 min at 37°C. Neurons were fixed and incubated with Alexa Fluor 568-conjugated secondary antibody (red) to label surface-expressed receptors before permeabilization. After permeabilization with 0.25% Trion X-100 for 5 min, neurons were then incubated with Alexa Fluor 488-conjugated secondary antibody (green) to label the internalized receptors. Merged images are presented in which the red signal represents the surface mGlu7 and the green signal represents the internalized mGlu7. Scale bar, 20 µm. (B) Summary histograms quantifying the internalized mGlu7 from panel A are present as the ratio of the internalized population compared with total (surface + internalized) population measured using Metamorph software. Scatter plots show mean ± SEM (Vec, 1.00 ± 0.04; Nedd4 WT, 1.23 ± 0.04; Nedd4 C867S, 1.02 ± 0.04; n > 35, **p<0.01, one-way ANOVA). (C) myc-mGlu7 was co-transfected with pSuper-Ctl shRNA (Ctl KD) or pSuper-Nedd4 shRNA (Nedd4 KD) in cultured hippocampal neurons. Internalization of mGlu7 was analyzed in the absence or presence of 400 µM L-AP4 for 15 min at 37°C. (D) Summary histograms quantifying the internalized mGlu7 from panel C. Scatter plots show mean ± SEM (Vec, 1.00 ± 0.06; Vec + L-AP4, 1.39 ± 0.14; Nedd4 KD, 0.86 ± 0.06; Nedd4 KD + L-AP4, 0.94 ± 0.09; n > 10, *p<0.05, n.s. indicates p>0.05, one-way ANOVA). (E) myc-mGlu7 and ß-arrestin 1 or 2 were co-expressed and the internalized mGlu7 was analyzed as above. (F) Summary histograms quantifying the internalized mGlu7 from panel E. Scatter plots show mean ± SEM (Vec, 1.00 ± 0.05; ß-arr1, 1.26 ± 0.07; ß-arr2, 1.30 ± 0.07; n > 30, **p<0.01, one-way ANOVA).10.7554/eLife.44502.020Figure 5—source data 1.Endocytosis of mGlu7 by beta-arrestins and Nedd4.