| Abstract No.: | A-B1068 |
| Country: | Canada |
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| Title: | DYSTROGLYCAN RECRUITS THE PI3K/AKT PATHWAY BY CIS INTERATIONS AT THE CELL SURFACE IN LAMININ-INDUCED ACETYLCHOLINE RECEPTOR AGGREGATION |
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| Authors/Affiliations: | 1 Mathieu R. Tremblay*; 1 Rami Massie; 1 Huashan Peng; 1 Salvatore Carbonetto;
1 McGill University, Montreal, QC, Canada
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| Content: | Alpha- and beta-dystroglycan (α/β-DG) are peripheral and transmembrane protein subunits of a complex of proteins linking laminin in the extracellular matrix (ECM) to dystrophin in skeletal muscle. Dysfunction of this complex leads to muscular dystrophies and mental retardation. α- and β-DG coordinate assembly of extracellular and cytoskeletal matrices of proteins necessary to stabilize acetylcholine receptors (AChRs) at the neuromuscular synapse induced by agrin/MuSK signaling. OBJECTIVES: We wish to test the notion that α- and β-DG function as a single-pass transmembrane receptor where the extracellular ligands binding to α-DG transduce conformational changes intracellularly via β-DG. In addition, we wish to assess how the α/β-DG complex mediates signaling to function in cell survival and synapse formation.
MATERIALS AND METHODS: Cell Culture: Cultured myotubes were derived from C2C12 myoblasts or differentiated from DG +/+ and -/- embryonic stem cells. Expression of DG constructs: DG cDNA constructs were generated by PCR and were fused at their C-terminus to the Enhanced Green Fluorescent Protein (eGFP). All constructs were expressed via adenoviruses by infection of cultured myotubes. AChR Aggregation: Cultured myotubes were treated with 1 nM agrin or with 100 nM laminin and labeled for surface AChRs using rhodamine-conjugated α-bungarotoxin. Aggregates of AChRs were visualized by epifluorescence. Pharmacological Studies: Cultured myotubes were incubated with kinase inhibitors: Wortmannin (100然); Akt Inh (10 然); GSK3β Inh (10 然); PP1 (20 然); PP2 (10 然); U0126 (60 然) to screen for their effect on agrin- and laminin-mediated AChR aggregation. Phosphatidyl-inositol-3 kinase (PI3K) Activity: Mutant constructs of PI3K (dominant-negative or constitutively active) were expressed in C2C12 myotubes by infection with adenoviruses, and their effect on AChR aggregation in infected myotubes was assessed. To assay for PI3K activation, myotubes were stimulated with agrin or laminin; cellular proteins were extracted in 1x RIPA lysis buffer, subjected to SDS-PAGE, and electrotransferred onto nitrocellulose membranes. Membranes were immunoblotted with antisera to phosphorylated Akt (serine473) and to total Akt. Immunoprecipitations: Native proteins were immunoprecipitated from C2C12 myotubes that were left untreated, or stimulated with 1 nM agrin, 100 nM laminin, or 400 ng/mL neuregulin. Pulled-down protein complexes were eluted from agarose beads in 1x SDS buffer, and samples were subjected to SDS-PAGE. Nitrocellulose membranes were immunoblotted for phosphotyrosine (PY20), PI3K (p85), α-DG (VIA4), β-DG, ErbB2, ErbB3, and Grb2.
RESULTS: We show that extracellular interactions of α-DG are sufficient to mediate laminin-induced AChR aggregation, and this requires signaling via Src-family kinases, protein kinase A, and PI3K/Akt. We present evidence that laminin-DG activates the PI3K/Akt signaling pathway to aggregate AChRs. In addition, laminin-DG interactions facilitate the assembly of an ErbB2/ErbB3 complex that function in AChR aggregation.
CONCLUSION: Dystroglycan in muscle acts as a receptor via the independent functions of its α and β subunits, and not as a single-pass transmembrane protein. α-DG initiates cis interactions within a complex of ECM to recruit multiple transmembrane pathways relevant to the functions of DG in muscle cell survival, synapse formation and ECM assembly. |
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