The neuromuscular junction is a complex structure resulting from the interaction of an innervating neuron and skeletal muscle fiber. The neuron releases a molecule called agrin which acts via a muscle specific tyrosine kinase, MuSK, to initiate the localized differentiation and specialization of the muscle membrane at the synapse. A defining characteristic of the postsynaptic aspect neuromuscular junction is the concentration of acetylcholine receptors (AChRs) at the crests of junctional folds. Also clustered to these sites is the dystrophin associated protein (DAP) complex, a collection of proteins previously associated with muscular dystrophy and synapse formation. A central component of this complex, dystroglycan, had previously been shown to bind to agrin with high affinity. Inhibition studies that blocked agrin binding to dystroglycan indicated that dystroglycan might be required for synapse formation. In addition, several properties of dystroglycan, including its ability to co-cluster with the AChR and to bind both the neural and muscle forms of agrin, made it an obvious candidate as the putative co-receptor for MuSK. Based on these results we have attempted to define the role of dystroglycan in synaptogenesis. / We studied nerve muscle co-cultures derived from Xenopus embryos and found that dystroglycan is present at almost all neural agrin and AChR clusters. This holds true even in developing synapses as well as in extrasynaptic clusters of AChRs. AChR and dystroglycan aggregates can be induced in vitro, by treating myotubes with agrin and/or exogenous laminin. The AChR clusters formed by laminin application were larger and more dense then those formed by agrin treatments and could be inhibited by the addition of anti-laminin antiserum or laminin fragments that do not self-polymerize. In addition, laminin, unlike agrin, was found to induce AChR and dystroglycan clustering independent of MuSK activation. The introduction of an antisense dystroglycan construct into the C2C12 muscle cell line and the resulting reduction in expressed dystroglycan on myotubes also had little effect on phosphorylation but instead reduced the number of agrin induced AChR clusters on myotubes. These finding suggested that dystroglycan is not the co-receptor for MuSK but rather functions at a point in AChR clustering downstream of agrin-MuSK signaling. When similar experiments were conducted on dystroglycan null myotubes we found that these myotubes respond to agrin in a manner similar to wild-type myotubes but that the AChR clusters formed on null myotubes were two to three times larger, half as dense and significantly less stable. Synapses in chimeric mice with dystroglycan deficient muscle were similarly affected. In culture and in vivo the absence of dystroglycan also resulted in the disruption of laminin, perlecan and acetylcholinesterase localization to AChR clusters but did not affect rapsyn or agrin localization. Finally, I present unpublished observations that suggest that the close association of dystroglycan with rapsyn and AChRs may not result from a direct interaction between dystroglycan and rapsyn. From these results we propose that dystroglycan is a key element of a "trap" requir

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, August 2000. / Includes bibliographical references. Also available on the Internet.

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Dystroglycan is a ubiquitous protein that links the extracellular matrix to the cytoskeleton and is the central unit of the dystrophin glycoprotein complex (DGC), a membrane complex that connects the cytoskeleton to the extracellular matrix (ECM). Dystroglycan is composed of two subunits that are tightly but non-covalently linked. alpha Dystroglycan (alpha DG) is located extracellularly and it is the only component of the DGC linked to the ECM, while beta Dystroglycan (beta DG) spans the plasma membrane and has both an extracellular and a cytoplasmic domain. The DGC is involved in skeletal muscle maintenance and viability, and in the organization and stabilization of the neuromuscular junction, but its function in brain is poorly understood. DGC components are target of several protein kinases, indicating that they are involved in cell signalling pathways. The finding of new dystroglycan interacting proteins could help to obtain some insights in its function in brain tissues. Previous immunoprecipitation and pull down experiments have been used to identify proteins interacting with the cytoplasmic tail of beta DG in brain tissues. Here, we attempt to extend the use of these techniques by using pull down experiments performed with the Glutathione-S-transferase (GST) fusion expression system as a tool for the proteomic analysis of Dystroglycan interacting proteins in the brain.

The cytoskeletal component of the muscle membrane, dystrophin and its associated proteins (DAPs), are essential for the maintenance of muscle integrity, since the absence of these molecules results in a variety of muscular dystrophies. The purpose of this work was to create and characterize monoclonal antibodies (mAbs) designed to recognize components of the DAP complex (DAPC), in order to provide tools for the study of its structure and function. / The first mAb generated, 1137, was raised against a 33 amino acid sequence of the core protein at the c-terminus of alpha-dystroglycan (alpha DG), a cell surface member of the DAPC linked to dystrophin via its co-transcript, the transmembrane protein, beta-dystroglycan. 1B7 was used to perform a comparative study in denervated rat muscle tissue in parallel with IIH6, a mAb which recognizes a different, more glycosylated form of alpha DG. The second and third mAbs were raised against a complex of proteins purified by succinylated Wheat Germ Agglutinin (sWGA) following extraction from rabbit skeletal muscle. (Abstract shortened by UMI.)

Thesis (M.Sc.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2009/13/07). Includes bibliographical references.

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