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J. Biol. Chem. 276 (17): 14505-14513

© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Peggy Mittaud, P. Angelo Marangi, Susanne Erb-Vögtli, and Christian Fuhrer§

From the Brain Research Institute, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland

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 beta  and delta  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 agrin-induced AChR aggregation by forming an AChR-bound signaling cascade.

* This work was supported by the Kanton Zürich and the Dr. Eric Slack-Gyr Foundation and by grants from the Swiss National Science Foundation and the Swiss Foundation for Research on Muscle Diseases (to C. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: +41-1-635-33-10; Fax: +41-1-635-33-03; E-mail: chfuhrer@hifo.unizh.ch.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

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