Laminin-1 redistributes postsynaptic proteins and requires rapsyn, tyrosine phosphorylation, and Src and Fyn to stably cluster acetylcholine receptors.
Clustering of acetylcholine receptors (AChRs) is a critical step in neuromuscular synaptogenesis, and is induced by agrin and laminin which are thought to act through different signaling mechanisms. We addressed whether laminin redistributes postsynaptic proteins and requires key elements of the agrin signaling pathway to cause AChR aggregation. In myotubes, laminin-1 rearranged dystroglycans and syntrophins into a laminin-like network, whereas inducing AChR-containing clusters of dystrobrevin, utrophin, and, to a marginal degree, MuSK. Laminin-1 also caused extensive coclustering of rapsyn and phosphotyrosine with AChRs, but none of these clusters were observed in rapsyn -/- myotubes. In parallel with clustering, laminin-1 induced tyrosine phosphorylation of AChR beta and delta subunits. Staurosporine and herbimycin, inhibitors of tyrosine kinases, prevented laminin-induced AChR phosphorylation and AChR and phosphotyrosine clustering, and caused rapid dispersal of clusters previously induced by laminin-1. Finally, laminin-1 caused normal aggregation of AChRs and phosphotyrosine in myotubes lacking both Src and Fyn kinases, but these clusters dispersed rapidly after laminin withdrawal. Thus, laminin-1 redistributes postsynaptic proteins and, like agrin, requires tyrosine kinases for AChR phosphorylation and clustering, and rapsyn for AChR cluster formation, whereas cluster stabilization depends on Src and Fyn. Therefore, the laminin and agrin signaling pathways overlap intracellularly, which may be important for neuromuscular synapse formation.
Marangi
P Angelo
PA
Department of Neurochemistry, Brain Research Institute, University of Zürich, CH-8057, Switzerland.
Wieland
Simon T
ST
Fuhrer
Christian
C
D000818:Animals; D016227:Benzoquinones; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D049030:Dystroglycans; D049029:Dystrophin-Associated Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047029:Lactams, Macrocyclic; D007797:Laminin / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism*; D009469:Neuromuscular Junction / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D011809:Quinones / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism*; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology; D013569:Synapses / Q000201:enzymology*; D014443:Tyrosine / Q000378:metabolism; D049411:Utrophin; D019061:src-Family Kinases / Q000378:metabolism*
D016227:Benzoquinones; D003598:Cytoskeletal Proteins; C483081:DAG1 protein, human; C490915:DTNA protein, human; D049029:Dystrophin-Associated Proteins; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D007797:Laminin; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009124:Muscle Proteins; D009479:Neuropeptides; D011518:Proto-Oncogene Proteins; D011809:Quinones; D011950:Receptors, Cholinergic; C483600:Utrn protein, mouse; D049411:Utrophin; C121346:laminin 1; C051609:peripheral membrane protein 43K; C091824:syntrophin; D049030:Dystroglycans; D017828:Rifabutin; D014443:Tyrosine; C020754:herbimycin; C095714:MUSK protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; D019311:Staurosporine