Fig 2: Critical roles of Y86 phosphorylation in activating Rapsn E3 ligase activity and AChR clustering.(A) Reduced E3 ligase activity of Rapsn by Y86F mutation in transfected HEK293T cells. (B) Quantitative data of neddylated δ-AChR in (A) (mean ± SEM), ***, p<0.001, One-way ANOVA, n = 3. (C) Reduced Rapsn tyrosine phosphorylation in Y86F mt myotubes, compared with WT controls. (D) Quantitative data of tyrosine phosphorylation of Rapsn in (C) (mean ± SEM), *, p<0.05, unpaired t-test, n = 3. (E) Fewer Agrin-induced AChR clusters in Y86F mt C2C12 myotubes, compared with WT controls. Arrows, AChR clusters. (F) Quantitative data in (E), (mean ± SEM), **, p<0.01, ***, p<0.001, Two-way ANOVA, n = 20 cells. (G) Reduced E3 ligase activity of Rapsn in Y86F mt myotubes. (H) Quantitative data in (G) (mean ± SEM), ***, p<0.001, unpaired t-test, n = 3. (I) Agrin treatment increased Rapsn self-association. Cultured C2C12 cells were transfected with HA- and EGFP-tagged Rapsn, respectively. Resulting myotubes were treated with Agrin. Cell lysates were subjected to co-immunoprecipitation with anti-HA beads, and probed with anti-GFP antibody to reveal the association between Rapsn-HA and Rapsn-EGFP. (J) Quantitative analysis of MuSK and Rapsn tyrosine phosphorylation in Figure 6A, neddylated δ-AChR in Figure 6B, and Rapsn self-association in Figure 7I. Data were shown as mean ± SEM, **, p<0.01, ***, p<0.001 (compared with time 0), One-way ANOVA, n = 3. (K–N) Reduced Rapsn self-association by Y86F (K) or N88K (M) mutation revealed by co-immunoprecipitation in HEK293T cells. Quantitative data of WT or Y86F Rapsn self-association(L); WT or N88K Rapsn self-association (M) (mean ± SEM), *, p<0.05; ***, p<0.001, unpaired t-test, n = 3. Also see Figure 7—figure supplement 1.10.7554/eLife.49180.025Figure 7—source data 1.Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 7B, D, F, H, J, L and N.

Fig 3: Impaired ability of N88K mt Rapsn in AChR clustering in HEK293T cells and in cultured muscle cells.(A) Impaired ability of N88K mt Rapsn to induce AChR clustering in HEK293T cells. HEK293T cells were transfected with AChR subunits (α, β, γ, δ), along with EGFP empty vector, Rapsn-EGFP, or N88K-EGFP. After 36 hr, live, unfixed cells were incubated with Flour 594-α-BTX (red) to label surface AChRs. Arrows, AChR clusters colocalized with Rapsn clusters. (B) Quantitative data of (A) (mean ± SEM). ***, p<0.001, unpaired t-test, n = 20 cells. (C) Comparable amount of AChRs and Rapsn in (A). Total levels of α-AChR, β-AChR, Rapsn, and surface α-AChR, β-AChR in the parallel experiment of (A) were examined by western blotting, using GAPDH and Transferrin (Trf) as lysate and surface protein loading controls, respectively. (D) Comparable total Rapsn and AChR, and surface AChR expression in N88K mt cultured myotubes. Total α-AChR, β-AChR, Rapsn and surface α-AChR, β-AChR from WT or N88K mt C2C12 myotubes were examined by western blotting, using GAPDH and Trf as lysate and surface protein loading controls, respectively. (E) Fewer Agrin-induced AChR clusters in N88K mt C2C12 myotubes, compared with WT controls. Myotubes were treated with or without 50 ng/ml Agrin for 12 hr. Arrows, AChR clusters. (F) Quantitative data of (E) (mean ± SEM), ***, p<0.001, Two-way ANOVA, n = 20 cells. Also see Figure 4—figure supplement 1 and Figure 4—figure supplement 2.10.7554/eLife.49180.015Figure 4—source data 1.Sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 4B and F.

Fig 4: Reduced E3 ligase activity in N88K mt Rapsn.(A) Schematic diagram of extraction of surface AChRs from C2C12 myotubes. Live C2C12 myotubes were incubated with biotin-α-BTX at 4°C for 1 hr to capture AChR complex, and then were lysed. Resulting biotin-α-BTX-AChR complex in lysates were precipitated by streptavidin-coupled agarose beads. Cell lysates, precipitated AChRs and AChR-associated proteins were examined by western blotting. (B) Comparable amounts of Actin and Rapsn were co-precipitated by surface AChR between WT and N88K mt C2C12 myotubes. Surface AChRs from WT or N88K mt myotubes were isolated, and associated Rapsn and actin were examined by western blotting. (C) Reduced Rapsn E3 ligase activity by N88K mutation in HEK293T cells. HEK293T cells were transfected with HA tagged WT, C366A, or N88K mt Rapsn, along with Flag-δ-AChR and Nedd8-Myc. After 48 hr, cells were lysed and precipitated with anti-Flag beads to pull down δ-AChR. The precipitated δ-AChR was blotted with anti-Nedd8 antibody to examine its neddylation. (D) Quantitative data of neddylated δ-AChR in (C) (mean ± SEM), ***, p<0.001, One-way ANOVA, n = 3. (E–H) Reduced Rapsn E3 ligase activity in N88K mt cultured myotubes and in mt mice. (E, F) WT and N88K mt cultured myotubes were treated with Agrin for 2 hr. Myotubes were lysed and precipitated with anti-Nedd8 antibody. The resulting lysates and precipitated proteins were blotted with indicated antibodies to reveal neddylated δ-AChR, readout for E3 ligase activity of Rapsn (E). (F) Quantitative data in (E) (mean ± SEM), ***, p<0.001, unpaired t-test, n = 3. (G) Neddylated δ-AChR was examined in WT or N88K mt mice. (H) Quantitative data in (G) (mean ± SEM), ***, p<0.001, unpaired t-test, n = 3. Also see Figure 5—figure supplement 1.10.7554/eLife.49180.019Figure 5—source data 1.Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 5D, F and H.

Fig 5: SNX17 overexpression increased cell surface LRP4 expression, MuSK phosphorylation, and AChR aggregation. (A) Western blotting and qPCR detection of SNX17 protein (n = 3/group) and mRNA (n = 9/group) expression, respectively, in lentivirus-infected cells. (B) Myotube cells were treated with 10 μg/mL anti-LRP4 antibody for 30 min to induce internalization of LRP4 protein on the membrane, following which the anti-LRP4 antibody was removed and the cells were cultured for 30 min, and then the content of LRP4 protein in the membrane protein was analyzed by Western Blotting. n = 3/group. (C) Myotube cells were treated with 10 μg/mL anti-LRP4 antibody for 30 min, after which the anti-LRP4 antibody was removed, the cells were incubated with 10 ng/mL Agrin for 16 h, and the expression of MuSK and p-MuSK in total protein was analyzed by Western Blotting. n = 3/group. (D) AChR clusters (red) on lentivirus-infected C2C12 myotubes were labeled with R-BTX after application of separate or mixed Agrin and anti-LRP4 antibody, the nuclei were stained with DAPI (blue), and the number of AChR clusters on myotubes was counted and the length of AChR clusters was analyzed by ImageJ software. n = 3/group; Scale bar = 50 μm. * represents SNX17 OE-con vs. SNX17 OE. All data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ## P<0.01. AChR, acetylcholine receptor; EAMG, experimental autoimmune myasthenia gravis; LRP4, low-density lipoprotein receptor-related protein 4; MuSK, muscle-specific receptor tyrosine kinase; R-BTX, rhodamine-labeled bungarotoxin; SEM, Standard Error of Mean; SNX17, sorting nexin 17. ns, no significance.