The Study Of PTPRR Inhibits The Cluster Of ACHRs By Dephosphorylating MuSK

Objective: Seeking for the tyrosine phosphatase which dephosphorylate MuSK,then explore the tyrosinephosphorylation sites of MuSK and tyrosine sites dephosphorylated by the tyrosine phosphatase,preliminary discussion on the regulation of tyrosine phosphatase for acetylcholine receptor(AChR)cluster.This study will help us deepen our understanding of the pathogenesis of MG,provide new targets for the diagnosis and treatment.Methods: MuSK and 10 tyrosine phosphatase plasmids were co-transfected into HEK293 cells,western blot and immunoprecipitation were performed to screen tyrosine phosphatase that dephosphorylate MuSK.Then,co-immunoprecipitation were performed to detected whether the tyrosine phosphatase could form protein complex with MuSK.Flag-MuSK plasmids were transfected into HEK293 cells,Flag Beads were used to enrich and purify MuSK,the protein of MuSK was recovered through SDS-PAGE and silver-staining.Mass spectrometry was performed using the recoverd protein sample to detect tyrosine phosphorylation sites of MuSK.Single or multiple point mutant plasmids of MuSK were constructed by point mutation technique.To detect the tyrosine sites of MuSK dephosphorylated by the screened tyrosine phosphatase,plasmids of mutant MuSK and screened tyrosine phosphatase were co-transfected into HEK293 cells followed by western blot.Lentivirus packaging technology was used to build the wild type/mutant tyrosine phosphatase overexpressed C2C12 cell lines.To differentiate C2C12 cells and induce them to form myotubes,growth medium was changed to differential medium as cells reached approximately 90%.C2C12 myotubes were treated with Agrin for 24 hr to induce AChR clusters on the surface.To examine AChR clusters on the surface of C2C12 myotubes,the culture was fixed with 4% paraformaldehyde and incubated with 1 mM of AlexaFluor 555-conjugated alpha-bungarotoxin(?-BTX)at 4? overnight.Then,aBTX-stained myotubes were visualized with a laser confocal fluorescence microscopy.For quantification of AChR clusters,the length of clusters was measured and the number was counted.Results: MuSK was significantly dephosphorylated by tyrosine phosphatase PTPRR,and immunoprecipitation showed that MuSK could form protein complex with PTPRR.Mass spectrometry analysis showed that 4 tyrosine residues(Y553,Y750,Y754 and Y755)of MuSK could be phosphorylated,and PTPRR could dephosphorylate Y754.Immunofluorescence showed that,compared with wild-type C2C12 cells,C2C12 cells overexpressed wild-type PTPRR had less AChR clusters and shorter clusters length,while C2C12 cells overexpressed D554 A mutant-PTPRR had more AChR clusters and longer clusters length(P < 0.05).Conclusion: Tyrosine phosphatase PTPRR can significantly dephosphorylate MuSK,and this effect is achieved by dephosphorylating the tyrosine site of MuSK Y754.PTPRR is involved in regulating MuSK signaling pathway which lead to the ihibition of AChR clusters,preliminarily shows that PTPRR is a potential target for the diagnosis and treatment of MG.