Mice deficient in src and fyn or src and yes move and breathe poorly and die perinatally, consistent with defects in neuromuscular function. Src and Fyn are associated with acetylcholine receptors (AChRs) in muscle cells, and Src and Yes can act downstream of ErbB2, suggesting roles for Src family kinases in signaling pathways regulating neuromuscular synapse formation. We studied neuromuscular synapses in src Ϫ/Ϫ ; fynand src Ϫ/Ϫ ; yes Ϫ/Ϫ mutant mice and found that muscle development, motor axon pathfinding, clustering of postsynaptic proteins, and synapse-specific transcription are normal in these double mutants, showing that these pairs of kinases are not required for early steps in synapse formation. We generated muscle cell lines lacking src and fyn and found that neural agrin and laminin-1 induced normal clustering of AChRs and that agrin induced normal tyrosine phosphorylation of the AChR ␤ subunit in the absence of Src and Fyn. Another Src family member, most likely Yes, was associated with AChRs and phosphorylated by agrin in myotubes lacking Src and Fyn, indicating that Yes may compensate for the loss of Src and Fyn. Nevertheless, PP1 and PP2, inhibitors of Src-class kinases, did not inhibit agrin signaling, suggesting that Src class kinase activity is dispensable for agrin-induced clustering and tyrosine phosphorylation of AChRs. AChR clusters, however, were less stable in myotubes lacking Src and Fyn but not in PP1-or PP2-treated wild-type cells. These data show that the stabilization of agrin-induced AChR clusters requires Src and Fyn and suggest that the adaptor activities, rather than the kinase activities, of these kinases are essential for this stabilization.

Duchenne muscular dystrophy is caused by deficiency of dystrophin and leads to progressive weakness. It has been proposed that the muscle degeneration occurring in this disease is caused by increased Ca2+ influx due to enhanced activity of cationic channels that are activated either by stretch of the plasma membrane (stretch-activated channels) or by Ca2+-store depletion (store-operated channels). Using both cytosolic Ca2+ measurements with Fura-2 and the manganese quench method, we show here that store-operated Ca2+ entry is greatly enhanced in dystrophic skeletal flexor digitorum brevis fibers isolated from mdx5cv mice, a mouse model of Duchenne muscular dystrophy. Moreover, we show for the first time that store-operated Ca2+ entry in these fibers is under the control of the Ca2+-independent phospholipase A2 and that the exaggerated Ca2+ influx can be completely attenuated by inhibitors of this enzyme. Enhanced store-operated Ca2+ entry in dystrophic fibers is likely to be due to a near twofold overexpression of Ca2+-independent phospholipase A2. The Ca2+-independent phospholipase A2 pathway therefore appears as an attractive target to reduce excessive Ca2+ influx and subsequent degeneration occurring in dystrophic fibers.

During neuromuscular synaptogenesis, neurally released agrin induces aggregation and tyrosine phosphorylation of acetylcholine receptors (AChRs) by acting through both the receptor tyrosine kinase MuSK (muscle-specific kinase) and the AChR-associated protein, rapsyn. To elucidate this signaling mechanism, we examined tyrosine phosphorylation of AChR-associated proteins, particularly addressing whether agrin activates Src family kinases bound to the AChR. In C2 myotubes, agrin induced tyrosine phosphorylation of these kinases, of AChR-bound MuSK, and of the AChR ␤ and ␦ subunits, as observed in phosphotyrosine immunoblotting experiments. Kinase assays revealed that the activity of AChR-associated Src kinases was increased by agrin, whereas phosphorylation of the total cellular kinase pool was unaffected. In both rapsyn-deficient myotubes and staurosporine-treated C2 myotubes, where AChRs are not clustered, agrin activated MuSK but did not cause either Src family or AChR phosphorylation. In S27 mutant myotubes, which fail to aggregate AChRs, no agrin-induced phosphorylation of AChR-bound Src kinases, MuSK, or AChRs was observed. These results demonstrate first that agrin leads to phosphorylation and activation of AChR-associated Src-related kinases, which requires rapsyn, occurs downstream of MuSK, and causes AChR phosphorylation. Second, this activation intimately correlates with AChR clustering, suggesting that these kinases may play a role in agrininduced AChR aggregation by forming an AChR-bound signaling cascade.A common feature of most synapses is the local accumulation of proteins regulating synaptic responses in the postsynaptic membrane. At the neuromuscular junction, a model system for synaptogenesis, aggregation of acetylcholine receptors (AChRs) 1 is an early sign of postsynaptic differentiation during development and occurs at sites of nerve-muscle contact (1, 2).Clusters of AChRs and further synaptic components are induced by neurally released agrin, an essential factor for synaptogenesis (3). Accordingly, mice deficient for agrin lack differentiated synapses and nerve-associated clusters of postsynaptic proteins (4). Neurons or recombinant agrin induce aggregation of AChRs and other muscle components when added to cultured myotubes (5, 6), a process that mimics events at developing endplates, as it is blocked by antibodies specific for nerve-released agrin (7).Little is known about the signaling pathway of agrin, although a muscle-specific kinase, MuSK, is part of the agrin receptor (8, 9), and proteoglycans are thought to play an accessory role in presenting agrin to its receptor (10). Agrin causes tyrosine phosphorylation of MuSK, and this kinase is essential for postsynaptic specialization, as shown in MuSK-deficient mice, in which the phenotype is similar to agrinϪ/Ϫ animals (11). Furthermore, myotubes lacking MuSK fail to respond to agrin (9, 11). Agrin also causes tyrosine phosphorylation of the ␤ subunit of the AChR, which may play a role in AChR clustering because, among other observations...

We have investigated the role of acetylcholine receptors (AChRs) in an early step of postsynaptic assembly at the neuromuscular synapse, the clustering of postsynaptic proteins induced by nerve-released agrin. To achieve this, we used two variants of C2 myotubes virtually lacking AChRs and C2 cells in which surface AChRs were down-regulated by AChR antibodies. In all cases, agrin caused normal clustering of the agrin receptor component MuSK, a-dystrobrevin and utrophin, but failed to aggregate AChRs, a-and b-dystroglycan, syntrophin isoforms and rapsyn, an AChR±anchoring protein necessary for postsynaptic assembly and AChR clustering. In C2 variants, the stability of rapsyn was decreased, whereas in antibody-treated cells, rapsyn ef®ciently co-localized with remaining AChRs in microaggregates. Upon ectopic injection into myo®bers in vivo, rapsyn did not form clusters in the absence of AChRs. These results show that AChRs and rapsyn are interdependent components of a pre-assembled protein complex that is required for agrin-induced clustering of a full set of postsynaptic proteins, thus providing evidence for an active role of AChRs in postsynaptic assembly.