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

Scotton, Patrick; Bleckmann, Dorothée; Stebler, Michael; Sciandra, Francesca; Brancaccio, Andrea; Meier, Thomas; Stetefeld, Jörg; Rüegg, Markus A.

doi:10.1074/jbc.m607887200

Cited by 44 publications

(47 citation statements)

References 60 publications

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“…The binding of Ca 2ϩ , at a distinct site separate from this loop region, further stabilizes the active conformation(s) thereby increasing the activity of AgG3z8 protein. A similar role for these z8 residues in musclespecific receptor tyrosine kinase phosphorylation activity was observed by Scotton et al (49). However, in that study using a longer chick agrin C45 A4B8 fragment that contains two LG domains (LG2 and LG3), functional deficiency is apparent only when more than one B8 residue is mutated.…”

Section: Discussionsupporting

confidence: 75%

Asparagine of z8 Insert Is Critical for the Affinity, Conformation, and Acetylcholine Receptor-clustering Activity of Neural Agrin

Tseng

Zhang

et al. 2010

Journal of Biological Chemistry

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Agrin isoforms with different bioactivities are synthesized by the nerve and the muscle. Neural agrin containing an 8-amino acid insert (z8) introduced by alternative splicing is the active form that induces synaptic differentiation at the neuromuscular junction. In addition to alternative splicing, extracellular calcium is also required for the activity of neural agrin. To understand better how the activity of agrin is regulated by alternative splicing, we have applied alanine substitution mutagenesis to the z8 insert and the calcium binding site in the minimally functional AgG3z8 fragment. Single alanine substitutions in the 4th through the 7th amino acid of the z8 splice insert significantly reduced the function of agrin, in terms of acetylcholine receptor clustering activity and the affinity for binding to the muscle surface. Mutation of the asparagine at the 4th position drastically reduces bioactivity such that it is equivalent to that of muscle form AgG3z0. These reduced activity mutants also show reduced magnitudes of the calcium-induced CD spectrum change from that observed in AgG3z8 fragments, indicating that cross-talk between calcium and the z8 insert is critical for the normal activity of agrin. However, removal of Ca 2؉ binding via mutation of both aspartic acids in the calcium binding site did not totally eliminate the activity of AgG3z8. These results suggest a model wherein the z8 insert is a Ca 2؉ -responsive allosteric element that is essential in forming an active conformation in neuronal agrin.Agrin is a heparan sulfate proteoglycan first isolated from the electric organ of the marine ray Torpedo californica (1). Studies in the recent decades have established that agrin plays a critical role in directing the formation and stabilization of the mammalian neuromuscular junction. Agrin is secreted by the axonal terminals of motor neurons of the spinal cord and is highly concentrated in basal lamina of the synaptic cleft (2). Incubation of agrin with cultured myotubes or ectopic expression of agrin in adult skeletal muscles induces virtually all major aspects of postsynaptic specialization in the endplates, which include clustering of the nicotinic acetylcholine receptors (AChRs) 2 and recruitment of other major postsynaptically expressed proteins such as acetylcholine esterase, utrophin, and rapsyn (3-5). Mutant mice deficient in agrin are born with severe defects of the neuromuscular synapse and die shortly after birth due to respiratory failure (6). In response to agrin released from the motor neurons, muscle produces retrograde signals to immobilize growth cones and to induce accumulation of synaptic vesicles and voltage-gated calcium channels in the presynaptic nerve terminals (6 -9).Recent study indicates that the downstream signaling in the neuromuscular junction is mediated by a receptor complex in myotubes containing the muscle-specific receptor tyrosine kinase, MuSK, and LRP4, a member of the LDL receptor family. LRP4 knock-out mice exhibit severe neuromuscular defects similar to those ...

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Section: Discussionsupporting

confidence: 75%

Asparagine of z8 Insert Is Critical for the Affinity, Conformation, and Acetylcholine Receptor-clustering Activity of Neural Agrin

Tseng

Zhang

et al. 2010

Journal of Biological Chemistry

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“…12). That the miniaturized form of neural agrin consisting solely of the laminin-binding (30) and the MuSK-activating domain (31,32) can rescue the perinatal death caused by agrin deficiency strongly argues that none of the other interactions of full-length agrin are required for its function in the initial development of NMJs. Of particular interest is that our work provides in vivo evidence that induction of postsynaptic structures during development does not require binding of neural agrin to ␣-dystroglycan, unlike what was postulated previously (33).…”

Section: Discussionmentioning

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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“…Mouse monoclonal anti-GAPDH antibody (clone 6C5) and rabbit anti-MuSK antiserum for Western blot were purchased from Abcam. Rabbit antiserum to MuSK (194T) for immunofluorescence was a gift from M. Ruegg, University of Basel, Basel, Switzerland (Scotton et al, 2006). The mouse monoclonal anti-rapsyn (clone 1234) and anti-Flag M2 were obtained from Sigma-Aldrich.…”

Section: Methodsmentioning

confidence: 99%

ColQ Controls Postsynaptic Differentiation at the Neuromuscular Junction

Sigoillot¹,

Bourgeois²,

Lambergeon³

et al. 2010

J. Neurosci.

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

Cited by 44 publications

References 60 publications

Asparagine of z8 Insert Is Critical for the Affinity, Conformation, and Acetylcholine Receptor-clustering Activity of Neural Agrin

Asparagine of z8 Insert Is Critical for the Affinity, Conformation, and Acetylcholine Receptor-clustering Activity of Neural Agrin

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

ColQ Controls Postsynaptic Differentiation at the Neuromuscular Junction

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