Identification Of The Downstream Molecules Of Agrin/Dok-7 Signaling Pathway At The Neuromuscular Junction

BackgroundThe neuromuscular junction is the synaptic structure between motor neurons and skeletal muscles.Abnormal development or functional defects of this structure cause neuromuscular related diseases,such as myasthenia gravis and congenital myasthenia syndrome,seriously damaging people's health,even threatening lives.Current research shows that the most critical signal pathway in the development of neuromuscular junctions is the agrin/Lrp4/Mu SK/Dok-7 signaling pathway.The development of neuromuscular junctions depends on the signaling process of phosphorylation of various proteins in this pathway.Agrin,released by motor neurons,binds to the LRP4 receptor on muscle cells and stimulates the phosphorylation of Mu SK.When Mu SK is phosphorylated,it recruits Dok-7 and promotes tyrosine phosphorylation of Dok-7.The activated Dok-7 further transmits signals to the acetylcholine receptor,causing it to accumulate at the postsynaptic.Dok-7 plays a crucial role in this process.Tyrosine phosphorylation of Dok-7 is essential for the formation of mature and functional neuromuscular junctions,and as a tyrosine kinase,Dok-7 mediates signal transduction by phosphorylating its downstream molecules.However,there are few reports about the downstream of Dok-7 so far,and how Dok-7transmits signals remains an urgent problem to be solved.In this study,we combined the CRISPR/Cas9 technique and quantitative phosphoproteomics analysis to study the tyrosine phosphorylation events triggered by agrin/Dok-7.This study would provide new clues to understanding the complex signaling networks downstream of agrin/Dok-7.ObjectiveTo explore the downstream signaling pathway of Dok-7,we will generate a C2C12 cell line with mutated Dok-7 gene and study the tyrosine phosphorylation downstream of Dok-7 through quantitative phosphorylation proteomics analysis.Finally,we will select candidate genes identified by proteomics analysis to further verify gene function in vitro and in vivo.MethodsDok-7 mutant muscle cell line was generated by CRISPR/Cas9 technique;Omics analysis was performed by label-free;The localization analysis of Anxa3 was performed by immunofluorescence experiment,the interaction between Anxa3 and Dok-7 was performed by immunoprecipitation,the phosphorylation of Anxa3 was tested by western blot,and the loss-of-function of Anxa3 was analyzed with shRNAs.ResultsWe successfully constructed a Dok-7 gene mutant cell line;omics analysis identified13 kinds of Dok-7-dependent tyrosine phosphoproteins,including Crk-L and acetylcholine receptor subunit,as previously reported,as well as other phosphorylated proteins such as cytoskeletal proteins,receptors,and enzyme-regulating molecules.The tyrosine phosphorylation level of Anxa3 increased specifically by agrin stimulation in cultured myotubes,while the tyrosine phosphorylation of Anxa3 remains almost unchanged in the Dok-7 mutated cells even with agrin stimulation.Moreover,co-immunoprecipitation showed that Anxa3 could interact with Dok-7,and the immunofluorescence of frozen muscle sections showed that Anxa3 is enriched at the NMJ and close to ACh Rs.In addition,knocking down of Anxa3 in muscle cells inhibited the accumulation of agrin-induced acetylcholine receptors,while the reduction of Anxa3 in mouse muscles reduced the area of the postsynaptic motor endplates,leading to postsynaptic dysplasia.ConclusionsA series of downstream molecules of Dok-7 signaling pathway were identified by tyrosine phosphorylation proteomics experiments,and Anxa3,as a downstream molecule of Dok-7,plays an important role in the accumulation of acetylcholine receptors in muscle cells and the development of motor endplates in skeletal muscle of mice.