Conjugation of LG Domains of Agrins and Perlecan to Polymerizing Laminin-2 Promotes Acetylcholine Receptor Clustering

Abstract

Neuromuscular junction (NMJ) assembly is characterized by the clustering and neuronal alignment of acetylcholine receptors (AChRs). In this study we have addressed post-synaptic contributions to assembly that may arise from the NMJ basement membrane with cultured myotubes. We show that the cell surface-binding LG domains of non-neural (muscle) agrin and perlecan promote AChR clustering in the presence of laminin-2. This type of AChR clustering occurs with a several hour lag, requires muscle-specific kinase (MuSK), and is accompanied by tyrosine phosphorylation of MuSK and ␤AChR. It also requires conjugation of the agrin or perlecan to laminin together with laminin polymerization. Furthermore, AChR clustering can be mimicked with antibody binding to non-neural agrin, supporting a mechanism of ligand aggregation. Neural agrin, in addition to its unique ability to cluster AChRs through its B/z sequence insert, also exhibits laminin-dependent AChR clustering, the latter enhancing and stabilizing its activity. Finally, we show that type IV collagen, which lacks clustering activity on its own, stabilizes laminin-dependent AChR clusters. These findings provide evidence for cooperative and partially redundant MuSK-dependent functions of basement membrane in AChR assembly that can enhance neural agrin activity yet operate in its absence. Such interactions may contribute to the assembly of aneural AChR clusters that precede neural agrin release as well as affect later NMJ development. Laminin G-like (LG)2 domains are ␤-sandwich globular structures present in the C-terminal moieties of basement membrane laminins, agrins, and perlecan (1, 2). They serve as ligands for ␣-dystroglycan and integrins (3, 4) and in the case of laminins can be anchored to the cell surface through sulfated glycosphingolipids (5). Proteins bearing these domains are found in the basement membrane of the motoneuron endplate of skeletal muscle. With the exception of neural agrin that is released by pre-synaptic neuronal processes, they are substantially secreted by the skeletal muscle and deposited on the sarcolemmal surface.The basement membrane of the neuromuscular junction (NMJ) is enriched in laminins containing the ␣2 subunit, with predominance of ␣2␤2␥1 in the mature endplate (3). Laminins are critical for formation of basement membrane, self-assembling into cell-adherent polymers that serve as scaffolds for the tethering of agrins, nidogens, and perlecan, and are connected to a type IV collagen network (reviewed in Ref. 4). Laminins have also been found to play a role in NMJ assembly, with different subunits contributing to their size, pre-and post-synaptic alignment, infoldings, and degree of ensheathment (4). The ␣1-and ␣2-laminins have been found to induce AChR clustering in vitro (6 -9), an effect resulting in tyrosine phosphorylation of AChR subunits (6, 9, 10). Loss of the ␣2-laminin subunit is associated with reduced depths and complexity of the post-synaptic infoldings of the NMJ accompanied by defects of myelination and muscular dystro...

Chimeric protein repair of laminin polymerization ameliorates muscular dystrophy phenotype

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An Extracellular Pathway for Dystroglycan Function in Acetylcholine Receptor Aggregation and Laminin Deposition in Skeletal Myotubes

“…By extension, these data indicate that DG, and more broadly the DGC, may function extracellularly in formation of NMJs. Recent studies have reported that inactive muscle agrin can be potentiated to stimulate AChR aggregation when complexed with laminin into an ECM (57,74). The association of ␣-DG with laminin, agrin, and perlecan may similarly facilitate the assembly of endogenous ligands to stimulate AChR aggregation.…”

The developmental roles of the extracellular matrix: beyond structure to regulation

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