Agrin-induced Activation of Acetylcholine Receptor-bound Src Family Kinases Requires Rapsyn and Correlates with Acetylcholine Receptor Clustering

Abstract

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...

Nicotinic α7 receptors: Synaptic options and downstream signaling in neurons

“…One candidate is the Src family of tyrosine kinases. Several studies have provided evidence that members of the Src family may be complexed with MuSK, rapsyn, and nAChRs in muscle, and that activation of the kinase produces receptor phosphorylation (Mohamed and Swope, 1999;Mittaud et al, 2001;Mohamed et al, 2001). The phosphorylation has been proposed as being important for receptor localization and stability, but this remains controversial (see article by S. Burden, this volume).…”

MuSK Glycosylation Restrains MuSK Activation and Acetylcholine Receptor Clustering

“…and Clustering-Agrin activation of MuSK leads to tyrosine phosphorylation of the AChR ␤-and ␦-subunits (13,26). The mechanisms that lead to tyrosine phosphorylation of the AChR are poorly understood and could, in principle, require glycosylation of MuSK.…”

Agrin and laminin induce acetylcholine receptor clustering by convergent, Rho GTPase-dependent signaling pathways

“…Although the signaling systems that induce AChR clustering in response to agrin and laminin have remained largely undefined, recent findings have suggested that formation of stable AChR clusters by both laminin and agrin require rapsyn, tyrosine phosphorylation of AChR ␤ and ␦ subunits and activation of Src-related kinases (Borges and Ferns, 2001;Ferns et al, 1996;Marangi et al, 2001;Marangi et al, 2002;Mittaud et al, 2001;Mohamed et al, 2001;Smith et al, 2001). A mechanism through which agrin and laminin couple these components to direct the lateral movement of AChR on the surface of postsynaptic muscle cells into nascent synapses, however, remains elusive.…”