A minigene of neural agrin encoding the laminin‐binding and acetylcholine receptor‐aggregating domains is sufficient to induce postsynaptic differentiation in muscle fibres

Meier, Thomas; Marangi, P. Angelo; Moll, Joachim; Hauser, D. M.; Brenner, Hans Rudolf; Rüegg, Markus A.

doi:10.1046/j.1460-9568.1998.00320.x

Cited by 26 publications

(21 citation statements)

References 74 publications

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“…We have previously shown that a miniaturized form of neural agrin (miniagrin) containing the 8-aa insert at the B/z site is sufficient to induce postsynapse-like structures when expressed in nonsynaptic regions of the adult soleus muscle (23). To further study this phenomenon, we generated several transgenic mouse lines expressing miniagrin derived from full-length chick or mouse agrin (see Fig.…”

Section: Transgenic Expression Of a Miniaturized Form Of Neural Agrin Inmentioning

confidence: 99%

Muscle-wide secretion of a miniaturized form of neural agrin rescues focal neuromuscular innervation in agrin mutant mice

Lin¹,

Maj²,

Bezakova³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Agrin and its receptor MuSK are required for the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). In the current model the local deposition of agrin by the nerve and the resulting local activation of MuSK are responsible for creating and maintaining the postsynaptic apparatus including clusters of acetylcholine receptors (AChRs). Concomitantly, the release of acetylcholine (ACh) and the resulting depolarization disperses those postsynaptic structures that are not apposed by the nerve and thus not stabilized by agrin-MuSK signaling. Here we show that a miniaturized form of agrin, consisting of the laminin-binding and MuSK-activating domains, is sufficient to fully restore NMJs in agrin mutant mice when expressed by developing muscle. Although miniagrin is expressed uniformly throughout muscle fibers and induces ectopic AChR clusters, the size and the number of those AChR clusters contacted by the motor nerve increase during development. We provide experimental evidence that this is due to ACh, because the AChR agonist carbachol stabilizes AChR clusters in organotypic cultures of embryonic diaphragms. In summary, our results show that agrin function in NMJ development requires only two small domains, and that this function does not depend on the local deposition of agrin at synapses. Finally, they suggest a novel local function of ACh in stabilizing postsynaptic structures.acetylcholine ͉ MuSK ͉ synapse formation O ne of the fundamental questions in neuroscience is how synapse formation between neurons and their targets is controlled during development. Current evidence indicates that initial stages of target recognition and synapse formation are driven by cell-adhesive interactions, and that later stages require electrical activity (1). The easy accessibility of the neuromuscular junction (NMJ) for experimental manipulation has allowed investigating synapse formation at both the molecular and physiological levels. NMJ formation critically depends on agrin, an extracellular matrix molecule released by the nerve (2, 3); the muscle-specific receptor tyrosine-kinase MuSK, which is activated by agrin (4); the low-density lipoprotein receptor-related protein 4 (5); and two intracellular adaptors, Dok-7 (6) and rapsyn (7), which bind to activated MuSK and the acetylcholine receptor (AChR), respectively. Of these, agrin and MuSK are the most upstream components, because forced expression in nonsynaptic regions of the muscle is sufficient to induce postsynaptic structures (8-11). Only certain splice variants of agrin, which differ in expression and in inserts localized in the most Cterminal laminin G (LG)-like domain, are capable of inducing AChR aggregation and MuSK activation (12).In contrast to postsynaptic differentiation, the molecular mechanisms that initiate presynaptic differentiation are less well understood. In addition, the role of agrin at early stages of NMJ formation is still debated (13-15), because developing muscle forms AChR clusters without nerve contact (16, 17), possibly by...

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Section: Transgenic Expression Of a Miniaturized Form Of Neural Agrin Inmentioning

confidence: 99%

Muscle-wide secretion of a miniaturized form of neural agrin rescues focal neuromuscular innervation in agrin mutant mice

Lin¹,

Maj²,

Bezakova³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…Thus, agrin binding to the myotube surface seems largely driven by its binding to ␣-dystroglycan and this increases its efficacy of activating MuSK. Similarly, the LG3 B8 domain is much more efficient in inducing postsynaptic specializations in vivo if the NtA domain, which confers binding to laminins, is included (59). Thus, the capturing of agrin to the muscle surface by its binding to either basement membrane or to the plasma membrane appears to have an important auxiliary function for the induction of postsynaptic structures.…”

Section: Tablementioning

confidence: 99%

Activation of Muscle-specific Receptor Tyrosine Kinase and Binding to Dystroglycan Are Regulated by Alternative mRNA Splicing of Agrin

Scotton

Bleckmann

Stebler

et al. 2006

Journal of Biological Chemistry

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Agrin induces the aggregation of postsynaptic proteins at the neuromuscular junction (NMJ). This activity requires the receptor-tyrosine kinase MuSK. Agrin isoforms differ in short amino acid stretches at two sites, called A and B, that are localized in the two most C-terminal laminin G (LG) domains. Importantly, agrin isoforms greatly differ in their activities of inducing MuSK phosphorylation and of binding to ␣-dystroglycan. By using site-directed mutagenesis, we characterized the amino acids important for these activities of agrin. We find that the conserved tripeptide asparagineglutamate-isoleucine in the eight-amino acid long insert at the B-site is necessary and sufficient for full MuSK phosphorylation activity. However, even if all eight amino acids were replaced by alanines, this agrin mutant still has significantly higher MuSK phosphorylation activity than the splice version lacking any insert. We also show that binding to ␣-dystroglycan requires at least two LG domains and that amino acid inserts at the A and the B splice sites negatively affect binding.

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“…1b). NA has previously been shown to possess AChR clustering and NMJ assembly properties of full-length agrin, whereas nNA was found to substantially ameliorate the dystrophic muscle histology and phenotype, prolonging animal survival when overexpressed in the muscle of laminin ␣2-deficient mice (25,26).…”

Section: Resultsmentioning

confidence: 99%

“…24), and type IV collagen. For this study we have used "minigene" versions of non-neural (nNA) and neural agrin (NA) that consist of the N-terminal (NtA) laminin-binding domain and the C-terminal LG domains that have been shown to possess biological activity (25,26) and that were chosen to simplify the analysis of domain contributions. We have also evaluated full-length perlecan and a protein consisting of the NtA domain fused to the LG domains of perlecan (NP).…”

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

Smirnov

Barzaghi

McKee

et al. 2005

Journal of Biological Chemistry

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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...

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A minigene of neural agrin encoding the laminin‐binding and acetylcholine receptor‐aggregating domains is sufficient to induce postsynaptic differentiation in muscle fibres

Cited by 26 publications

References 74 publications

Muscle-wide secretion of a miniaturized form of neural agrin rescues focal neuromuscular innervation in agrin mutant mice

Muscle-wide secretion of a miniaturized form of neural agrin rescues focal neuromuscular innervation in agrin mutant mice

Activation of Muscle-specific Receptor Tyrosine Kinase and Binding to Dystroglycan Are Regulated by Alternative mRNA Splicing of Agrin

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

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