Résumé :
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Dystroglycan (DG) is a transmembrane glycoprotein, which in skeletal muscle forms a central role in the dystrophin-associated glycoprotein complex (DGC). Thiscomplex links dystrophin to the extracellular matrix, helping to provide stability to muscle fibres. Loss of functional components of the DGC or dystrophin leads tomuscular dystrophy. However, presumably because of the importance of DG in early embryonic membrane formation, no pathogenic mutations in DG have to date beenobserved. Post-translational processing of DG is important to its cellular function. Modifications include, cleavage into _ and _ subunits, phosphorylation and extensiveglycosylation. Here we focus on the role of phosphorylation of _-DG in muscular dystrophy. Recent work has shown that phosphorylation of a Cterminal tyrosine in _-DGcan cause redistribution of the protein from the cell membrane to endosomal compartments, possibly as a prerequisite to degradation. In normal muscle, binding ofdystrophin to this tyrosine would block phosphorylation, meaning that phosphorylation of _-DG could be key in the disease process in DMD. By ES cell targeting we havegenerated a knock-in mouse in which _DG cannot be phosphorylated at this tyrosine. We have confirmed through DNA sequencing and protein immunoblotting that theknockin has been successful. The homozygote mice are healthy with no overt phenotype or detectable muscle pathology, indicating that the mutation is not deleterious.Having crossed these mice with the mdx model of DMD, we have analysed muscle pathology by assessing levels of central nucleation and serum creatine kinase. Ourpreliminary analysis of the double mutants compared to mdx show a reduction in these markers of disease pathogenesis. We are currently furthering our analysis byassessing the localisation of DGC markers and several other measures of muscle pathology such as Evans blue dye uptake and changes in muscle fibre diameter. Inparallel to this we are using a zebrafish model system to dissect more finely the mechanisms involved. Finally, we have also used FRAP and surface biotinylation assaysin myoblast cells to characterise the dynamics of _-DG in response to phosphorylation to allow us to better model this effect.
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