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 betaAChR. 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.
Highlights
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]
We show that the cell surface-binding LG domains of non-neural agrin and perlecan promote acetylcholine receptors (AChRs) clustering in the presence of laminin-2
Both stages of AChR assembly occur in a muscle basement membrane zone enriched in ␣2-laminins, nidogens, non-neural agrin, perlecan, and type IV collagen
Summary
Chick Minigene Neural—Chick minigene neural (containing the A4B8 insertions) agrin (pCepN25C954,8His) and non-neural (A0B0) (pCepN25C950,0His) agrin plasmids were used to express protein with an N-terminal sequence containing a His tag, NtA domain, and the first follistatin repeat fused to the C-terminal neural or non-neural three G-repeats [11, 26]. Agrin NtA-FS.1—The minigene sequence between the first follistatin repeat and stop codon was removed from plasmid pCepN25C950,0His by PCR with the reverse primer 5Ј-AAATATGCGGCCGCATTCCCCTCTGCACAG-3Ј and forward primer 5Ј-GTGAACCGTCAGATCTCTAG-3Ј. Clones stably expressing recombinant agrin proteins were expanded and secreted protein was affinity purified by His-Select HC Ni-affinity Gel (Sigma) chromatography. Nidogen-1—A pCIS vector encoding full-length mouse nidogen-1 (gift of Rupert Timpl, MPI for Biochemistry, Martinsried, Germany) was used to stably transfect 293 cells. Secreted protein was purified from medium by metal chelating chromatography [27]
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