Contributions of the LG modules and furin processing to laminin-2 functions.
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The alpha2-laminin subunit contributes to basement membrane functions in muscle, nerve, and other tissues, and mutations in its gene are causes of congenital muscular dystrophy. The alpha2 G-domain modules, mutated in several of these disorders, are thought to mediate different cellular interactions. To analyze these contributions, we expressed recombinant laminin-2 (alpha(2)beta(1)gamma(1)) with LG4-5, LG1-3, and LG1-5 modular deletions. Wild-type and LG4-5 deleted-laminins were isolated from medium intact and cleaved within LG3 by a furin-like convertase. Myoblasts adhered predominantly through LG1-3 while alpha-dystroglycan bound to both LG1-3 and LG4-5. Recombinant laminin stimulated acetylcholine receptor (AChR) clustering; however, clustering was induced only by the proteolytic processed form, even in the absence of LG4-5. Furthermore, clustering required alpha(6)beta(1) integrin and alpha-dystroglycan binding activities available on LG1-3, acting in concert with laminin polymerization. The ability of the modified laminins to mediate basement membrane assembly was also evaluated in embryoid bodies where it was found that both LG1-3 and LG4-5, but not processing, were required. In conclusion, there is a division of labor among LG-modules in which (i) LG4-5 is required for basement membrane assembly but not for AChR clustering, and (ii) laminin-induced AChR clustering requires furin cleavage of LG3 as well as alpha-dystroglycan and alpha(6)beta(1) integrin binding. (+info)
Vascular endothelial cells that express dystroglycan are involved in angiogenesis.
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We have earlier shown that dystroglycan (DG) is a lamininbinding protein and as such is a cell adhesion molecule. DG is a heterodimer of alpha and beta DG subunits. beta-dystroglycan (betaDG) is the membrane spanning subunit, whereas the alpha subunit is bound to the extracellular domain of betaDG. To study physiological function of the protein, we expressed full-length DG (FL-DG) and the cytoplasmic domain of betaDG (deltabetaDG) in bovine aortic endothelial cells (BAE) and examined their effects on cell adhesion, migration, proliferation and tube formation. FL-DG enhanced adhesion of BAE to laminin-1, whereas deltabetaDG inhibited it. Cell migration was inhibited by overexpressing deltabetaDG in these cells, although it was not affected by FL-DG overexpression. In a proliferation assay, FL-DG decreased the growth rate, and it also caused cells to extensively spread. deltabetaDG caused opposite effects; it increased the growth rate and reduced the cell surface area. In a tube formation assay on Matrigel, FL-DG caused an obvious inhibition, whereas deltabetaDG accelerated tube formation. These results suggest a potential role of endothelial dystroglycan in the control of angiogenesis. Anti-betaDG immunohistochemistry indicated increased expression of DG in vascular endothelial cells within malignant tumors. By contrast, the antibody failed to stain endothelial cells in normal tissues. In cultured BAE, the level of betaDG was low in serum-deprived quiescent cells and was upregulated in proliferating cells. Our results suggest that the expression of DG in endothelial cells is under a dynamic regulation and may play a role in angiogenesis. (+info)
Overexpression of the cytotoxic T cell GalNAc transferase in skeletal muscle inhibits muscular dystrophy in mdx mice.
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Duchenne muscular dystrophy (DMD) is a congenital X-linked myopathy caused by lack of dystrophin protein expression. In DMD, the expression of many dystrophin-associated proteins (DAPs) is reduced along the sarcolemmal membrane, but the same proteins remain concentrated at the neuromuscular junction where utrophin, a dystrophin homologue, is expressed [Matsumura, K., Ervasti, J. M., Ohlendieck, K., Kahl, K. D. & Campbell, K. (1992) Nature (London) 360, 588-591]. This outcome has led to the concept that ectopic expression of a "synaptic scaffold" of DAPs and utrophin along myofibers might compensate for the molecular defects in DMD. Here we show that transgenic overexpression of the synaptic CT GalNAc transferase in the skeletal muscles of mdx animals (mdx/CT) increases the expression of utrophin and many DAPs, including dystroglycans, sarcoglycans, and dystrobrevins, along myofibers. Protein expression of utrophin and DAPs was equal to or above that of wild-type mice. In addition, alpha-dystroglycan was glycosylated with the CT carbohydrate antigen in mdx/CT but not in mdx muscles. mdx/CT mice have little or no evidence of muscular dystrophy by several standard measures; Serum creatine kinase levels, percentage of centrally located myofiber nuclei, and variance in myofiber diameter in mdx/CT muscles were dramatically reduced compared with mdx mice. These data suggest that ectopic expression of the CT GalNAc transferase creates a functional dystrophin-related complex along myofibers in the absence of dystrophin and should be considered as a target for therapeutic intervention in DMD. (+info)
New World arenavirus clade C, but not clade A and B viruses, utilizes alpha-dystroglycan as its major receptor.
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Alpha-dystroglycan (alpha-DG) has been identified as a major receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa virus, two Old World arenaviruses. The situation with New World arenaviruses is less clear: previous studies demonstrated that Oliveros virus also exhibited high-affinity binding to alpha-DG but that Guanarito virus did not. To extend these initial studies, several additional Old and New World arenaviruses were screened for entry into mouse embryonic stem cells possessing or lacking alpha-DG. In addition, representative viruses were further analyzed for direct binding to alpha-DG by means of a virus overlay protein blot assay technique. These studies indicate that Old World arenaviruses use alpha-DG as a major receptor, whereas, of the New World arenaviruses, only clade C viruses (i.e., Oliveros and Latino viruses) use alpha-DG as a major receptor. New World clade A and B arenaviruses, which include the highly pathogenic Machupo, Guanarito, Junin, and Sabia viruses, appear to use a different receptor or coreceptor for binding. Previous studies with LCMV have suggested the need for a small aliphatic amino acid at LCMV GP1 glycoprotein amino acid position 260 to allow high-affinity binding to alpha-DG. As reported herein, this requirement appears to be broadly applicable to the arenaviruses as determined by more extensive analysis of alpha-DG receptor usage and GP1 sequences of Old and New World arenaviruses. In addition, GP1 amino acid position 259 also appears to be important, since all arenaviruses showing high-affinity alpha-DG binding possess a bulky aromatic amino acid (tyrosine or phenylalanine) at this position. (+info)
Laminin-1 redistributes postsynaptic proteins and requires rapsyn, tyrosine phosphorylation, and Src and Fyn to stably cluster acetylcholine receptors.
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Clustering of acetylcholine receptors (AChRs) is a critical step in neuromuscular synaptogenesis, and is induced by agrin and laminin which are thought to act through different signaling mechanisms. We addressed whether laminin redistributes postsynaptic proteins and requires key elements of the agrin signaling pathway to cause AChR aggregation. In myotubes, laminin-1 rearranged dystroglycans and syntrophins into a laminin-like network, whereas inducing AChR-containing clusters of dystrobrevin, utrophin, and, to a marginal degree, MuSK. Laminin-1 also caused extensive coclustering of rapsyn and phosphotyrosine with AChRs, but none of these clusters were observed in rapsyn -/- myotubes. In parallel with clustering, laminin-1 induced tyrosine phosphorylation of AChR beta and delta subunits. Staurosporine and herbimycin, inhibitors of tyrosine kinases, prevented laminin-induced AChR phosphorylation and AChR and phosphotyrosine clustering, and caused rapid dispersal of clusters previously induced by laminin-1. Finally, laminin-1 caused normal aggregation of AChRs and phosphotyrosine in myotubes lacking both Src and Fyn kinases, but these clusters dispersed rapidly after laminin withdrawal. Thus, laminin-1 redistributes postsynaptic proteins and, like agrin, requires tyrosine kinases for AChR phosphorylation and clustering, and rapsyn for AChR cluster formation, whereas cluster stabilization depends on Src and Fyn. Therefore, the laminin and agrin signaling pathways overlap intracellularly, which may be important for neuromuscular synapse formation. (+info)
Dystroglycan is selectively associated with inhibitory GABAergic synapses but is dispensable for their differentiation.
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The dystrophin glycoprotein complex (DGC) is a multimolecular complex that links the extracellular matrix to the cytoskeleton. The DGC is present at the skeletal neuromuscular junction and required for its maturation and maintenance. Members of the DGC are also expressed in brain. We used cultured hippocampal neurons to analyze the distribution, regulation, and role in synaptogenesis of the major transmembrane component of the DGC, dystroglycan; one of its extracellular ligands, agrin; and one of its cytoskeletal binding partners, dystrophin. alpha-Dystroglycan, beta-dystroglycan, and dystrophin clustered at a subset of inhibitory synapses containing GABA(A)R subunits alpha1, alpha2, and gamma2, and the inhibitory receptor anchoring protein gephyrin. DGC components were not detected at excitatory glutamatergic synapses. Dystroglycan is the first identified adhesive macromolecule at mature GABA synapses. Developmentally, dystroglycan clustered at synaptic loci after synaptic vesicles, GABA(A)R, and gephyrin, the latter being closely associated with GABA(A)R at all stages of synaptogenesis analyzed. Analysis of gephyrin -/-, agrin -/-, and mdx mouse hippocampal neurons in culture indicated that synaptic clustering of dystroglycan occurs independently of gephyrin, agrin, and dystrophin. In dystroglycan-deficient neurons, cultured from a conditional mutant strain, GABAergic synapses differentiated with clusters of gephyrin and GABA(A)R apposed to synaptic terminals, but these synapses did not contain detectable dystrophin. Thus the DGC is not essential for GABAergic synaptogenesis but is likely to function in modulating inhibitory synapses or conferring specialized properties on a subset of them. (+info)
Matrix assembly, regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation.
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Laminin-1 is essential for early embryonic basement membrane assembly and differentiation. Several steps can be distinguished, i.e., the expression of laminin and companion matrix components, their accumulation on the cell surface and assembly into basement membrane between endoderm and inner cell mass, and the ensuing differentiation of epiblast. In this study, we used differentiating embryoid bodies derived from mouse embryonic stem cells null for gamma1-laminin, beta1-integrin and alpha/beta-dystroglycan to dissect the contributions of laminin domains and interacting receptors to this process. We found that (a) laminin enables beta1-integrin-null embryoid bodies to assemble basement membrane and achieve epiblast with beta1-integrin enabling expression of the laminin alpha1 subunit; (b) basement membrane assembly and differentiation require laminin polymerization in conjunction with cell anchorage, the latter critically dependent upon a heparin-binding locus within LG module-4; (c) dystroglycan is not uniquely required for basement membrane assembly or initial differentiation; (d) dystroglycan and integrin cooperate to sustain survival of the epiblast and regulate laminin expression; and (e) laminin, acting via beta1-integrin through LG1-3 and requiring polymerization, can regulate dystroglycan expression. (+info)
Removal of dystroglycan causes severe muscular dystrophy in zebrafish embryos.
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Muscular dystrophy is frequently caused by disruption of the dystrophin-glycoprotein complex (DGC), which links muscle cells to the extracellular matrix. Dystroglycan, a central component of the DGC, serves as a laminin receptor via its extracellular alpha subunit, and interacts with dystrophin (and thus the actin cytoskeleton) through its integral membrane beta subunit. We have removed the function of dystroglycan in zebrafish embryos. In contrast to mouse, where dystroglycan mutations lead to peri-implantation lethality, dystroglycan is dispensable for basement membrane formation during early zebrafish development. At later stages, however, loss of dystroglycan leads to a disruption of the DGC, concurrent with loss of muscle integrity and necrosis. In addition, we find that loss of the DGC leads to loss of sarcomere and sarcoplasmic reticulum organisation. The DGC is required for long-term survival of muscle cells in zebrafish, but is dispensable for muscle formation. Dystroglycan or the DGC is also required for normal sarcomere and sarcoplasmic reticulum organisation. Because zebrafish embryos lacking dystroglycan share several characteristics with human muscular dystrophy, they should serve as a useful model for the disease. In addition, knowing the dystroglycan null phenotype in zebrafish will facilitate the isolation of other molecules involved in muscular dystrophy pathogenesis. (+info)