Muscle specific kinase: organiser of synaptic membrane domains

Muscle Specific Kinase (MuSK) is a transmembrane tyrosine kinase vital for forming and maintaining the mammalian neuromuscular junction (NMJ: the synapse between motor nerve and skeletal muscle). MuSK expression switches on during skeletal muscle differentiation. MuSK then becomes restricted to the postsynaptic membrane of the NMJ, where it functions to cluster acetylcholine receptors (AChRs). The expression, activation and turnover of MuSK are each regulated by signals from the motor nerve terminal. MuSK forms the core of an emerging signalling complex that can be acutely activated by neural agrin (N-agrin), a heparin sulfate proteoglycan secreted from the nerve terminal. MuSK activation initiates complex intracellular signalling events that coordinate the local synthesis and assembly of synaptic proteins. The importance of MuSK as a synapse organiser is highlighted by cases of autoimmune myasthenia gravis in which MuSK autoantibodies can deplete MuSK from the postsynaptic membrane, leading to complete disassembly of the adult NMJ.     

Phosphoinositide 3-kinase acts through RAC and Cdc42 during agrin-induced acetylcholine receptor clustering

The formation of the neuromuscular junction (NMJ) is regulated by the nerve-derived heparan sulfate proteoglycan agrin and the muscle-specific kinase MuSK. Agrin induces a signal transduction pathway via MuSK, which promotes the reorganization of the postsynaptic muscle membrane. Activation of MuSK leads to the phosphorylation and redistribution of acetylcholine receptors (AChRs) and other postsynaptic proteins to synaptic sites. The accumulation of high densities of AChRs at postsynaptic regions represents a hallmark of NMJ formation and is required for proper NMJ function. Here we show that phosphoinositide 3-kinase (PI3-K) represents a component of the agrin/MuSK signaling pathway. Muscle cells treated with specific PI3-K inhibitors are unable to form full-size AChR clusters in response to agrin and AChR phosphorylation is reduced. Moreover, agrin-induced activation of Rac and Cdc42 is impaired in the presence of PI3-K inhibitors. PI3-K is localized to the postsynaptic muscle membrane consistent with a role during agrin/MuSK signaling. These results put PI3-K downstream of MuSK as regulator of AChR phosphorylation and clustering. Its role during agrin-stimulated Rac and Cdc42 activation suggests a critical function during cytoskeletal reorganizations, which lead to the redistribution of actin-anchored AChRs.     

Agrin triggers the clustering of raft-associated acetylcholine receptors through actin cytoskeleton reorganization

Background information. Cholesterol/sphingolipid‐rich membrane microdomains or membrane rafts have been implicated in various aspects of receptor function such as activation, trafficking and synapse localization. More specifically in muscle, membrane rafts are involved in AChR (acetylcholine receptor) clustering triggered by the neural factor agrin, a mechanism considered integral to NMJ (neuromuscular junction) formation. In addition, actin polymerization is required for the formation and stabilization of AChR clusters in muscle fibres. Since membrane rafts are platforms sustaining actin nucleation, we hypothesize that these microdomains provide the suitable microenvironment favouring agrin/MuSK (mu scle‐s pecific k inase) signalling, eliciting in turn actin cytoskeleton reorganization and AChR clustering. However, the identity of the signalling pathways operating through these microdomains still remains unclear. Results. In this work, we attempted to identify the interactions between membrane raft components and cortical skeleton that regulate, upon signalling by agrin, the assembly and stabilization of synaptic proteins of the postsynaptic membrane domain at the NMJ. We provide evidence that in C2C12 myotubes, agrin triggers the association of a subset of membrane rafts enriched in AChR, the ‐MuSK and Cdc42 (cell division cycle 42) to the actin cytoskeleton. Disruption of the liquid‐ordered phase by methyl‐β‐cyclodextrin abolished this association. We further show that actin and the actin‐nucleation factors, N‐WASP (neuronal Wiscott—Aldrich syndrome protein) and Arp2/3 (actin‐related protein 2/3) are transiently associated with rafts on agrin engagement. Consistent with these observations, pharmacological inhibition of N‐WASP activity perturbed agrin‐elicited AChR clustering. Finally, immunoelectron microscopic analyses of myotube membrane uncovered that AChRs were constitutively associated with raft nanodomains at steady state that progressively coalesced on agrin activation. These rearrangements of membrane domains correlated with the reorganization of cortical actin cytoskeleton through concomitant and transient recruitment of the Arp2/3 complex to AChR‐enriched rafts. Conclusions. The present observations support the notion that membrane rafts are involved in AChR clustering by promoting local actin cytoskeleton reorganization through the recruitment of effectors of the agrin/MuSK signalling cascade. These mechanisms are believed to play an important role in vivo in the formation of the NMJ.     

Distinct domains of MuSK mediate its abilities to induce and to associate with postsynaptic specializations.

Agrin released from motor nerve terminals activates a muscle-specific receptor tyrosine kinase (MuSK) in muscle cells to trigger formation of the skeletal neuromuscular junction. A key step in synaptogenesis is the aggregation of acetylcholine receptors (AChRs) in the postsynaptic membrane, a process that requires the AChR-associated protein, rapsyn. Here, we mapped domains on MuSK necessary for its interactions with agrin and rapsyn. Myotubes from MuSK(-/)- mutant mice form no AChR clusters in response to agrin, but agrin-responsiveness is restored by the introduction of rat MuSK or a Torpedo orthologue. Thus, MuSK(-/)- myotubes provide an assay system for the structure-function analysis of MuSK. Using this system, we found that sequences in or near the first of four extracellular immunoglobulin-like domains in MuSK are required for agrin responsiveness, whereas sequences in or near the fourth immunoglobulin-like domain are required for interaction with rapsyn. Analysis of the cytoplasmic domain revealed that a recognition site for the phosphotyrosine binding domain-containing proteins is essential for MuSK activity, whereas consensus binding sites for the PSD-95/Dlg/ZO-1-like domain-containing proteins and phosphatidylinositol-3-kinase are dispensable. Together, our results indicate that the ectodomain of MuSK mediates both agrin- dependent activation of a complex signal transduction pathway and agrin-independent association of the kinase with other postsynaptic components. These interactions allow MuSK not only to induce a multimolecular AChR-containing complex, but also to localize that complex to a primary scaffold in the postsynaptic membrane.     

The Ig1/2 domain of MuSK binds to muscle surface and is involved in acetylcholine receptor clustering

The neuromuscular junction, the synapse between motor neurons and muscle cells, serves as an excellent model for studying synapse formation. Agrin is believed to be released by motor neurons to induce postsynaptic differentiation at the neuromuscular junction. MuSK, a receptor tyrosine kinase, appears to be a key component of the agrin receptor complex. However, how agrin activates MuSK remains unclear. To address this question, we characterized the binding of the MuSK extracellular region to the muscle cell surface. The MuSK ectodomain was found to bind to muscle cells in a manner dependent on stimulation with neural agrin. Moreover, the binding was myotube specific and appeared to be mediated by two regions in the MuSK: one region containing the first and second immunoglobin domains and the other containing the cysteine-rich domain. Importantly, recombinant proteins containing the binding activity can block full-length MuSK binding to muscle cells and agrin-induced AChR clustering. These results suggest that the Ig1/2 domain of MuSK is involved in AChR clustering by binding to the muscle surface.     

Role of lipid rafts in agrin-elicited acetylcholine receptor clustering

Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.     

Sialic acid inhibits agrin signaling in C2 myotubes

Acetylcholine receptor (AChR) clustering is an early event in neuromuscular synapse formation that is commonly studied using muscle cell culture. Motor neuron-derived agrin induces the postsynaptic tyrosine phosphorylation of both a muscle-specific kinase (MuSK) and the AChR beta-subunit. These phosphorylation events are required for AChR clustering, suggesting an agrin-driven signaling pathway. Both the phosphorylation events and AChR clustering can also be induced by neuraminidase, an enzyme that cleaves sialic acid from glycoconjugates, suggesting that neuraminidase is able to activate the agrin signaling pathway. A postulated signal for postsynaptic differentiation at sites of nerve-muscle contact during vertebrate development is the enzymatic removal of basal lamina components. We show here that bath-applied sialic acid has an effect directly opposite that of agrin or neuraminidase. Sialic acid not only decreases AChR clustering but also diminishes the tyrosine phosphorylation of MuSK and the AChR beta-subunit signal-transduction events normally driven by agrin. However, sialic acid does not prevent agrin-binding molecules from colocalizing with the decreased number of AChR clusters that do form, suggesting that sialic acid is acting to inhibit the agrin signaling pathway downstream of agrin binding to the muscle cell membrane. We propose a regulatory role for sialic acid in the signal transduction events of neuromuscular synapse formation, in which agrin or neuraminidase can overcome this sialic acid repression, resulting in the clustering of AChRs and other postsynaptic molecules.     

The role of nitric oxide signaling in the formation of the neuromuscular junction

The formation of the vertebrate neuromuscular junction (NMJ) depends on the action of neural agrin on the muscle cell. The requirement for agrin and its receptor, muscle-specific kinase (MuSK), has been well established over the past 20 years. However, the signaling mechanisms through which agrin and MuSK cause synaptic differentiation are not well understood. New evidence from studies of muscle cells in culture and in embryos indicates that nitric oxide (NO) is an effector of agrin-induced postsynaptic differentiation at the NMJ. Cyclic GMP (cGMP) production by guanylate cyclase appears to be an important downstream step in this pathway. Nitric oxide and cGMP regulate the activity of several kinases, some of which may influence interaction of dystrophin and utrophin with the actin cytoskeleton to mediate or modulate postsynaptic differentiation in muscle cells. These signaling molecules could also play a role in retrograde signaling to influence differentiation of presynaptic nerve terminals.     

Synapse formation and agrin expression in stratospheroid cultures from embryonic chick retina

Stratospheroids are three-dimensional cellular spheres which develop in vitro through the proliferation and differentiation of retinal neuroepithelial precursor cells. We investigated synapse formation in stratospheroids by analyzing the development of aggregates of synapse-associated molecules and of electron microscopically identifiable synaptic specializations. Our results show that the first aggregates of the GABA(A) receptor, the glycine receptor, and gephyrin appear in the inner plexiform layer after 8 days in culture simultaneously with the development of the first active zones and postsynaptic densities. In contrast, presynaptic molecules including synaptophysin could be detected in the inner plexiform layer before synaptogenesis, suggesting functions for these molecules in addition to neurotransmitter exocytosis at mature synapses. Similar to the retina in vivo, synapses were not found in the nuclear layers of stratospheroids. We also analyzed the isoform pattern, expression, and distribution of the extracellular matrix molecule agrin, a key regulator during formation, maintenance, and regeneration of the neuromuscular junction. In stratospheroids, several agrin isoforms were expressed as highly glycosylated proteins with an apparent molecular weight of approximately 400 kDa, similar to the molecular weight of agrin in the retina in vivo. The expression specifically of the neuronal isoforms of agrin was concurrent with the onset of synaptogenesis. Moreover, the neuronal agrin isoforms were exclusively found in the synapse-containing inner plexiform layer, whereas other agrin isoforms were associated also with the inner limiting membrane and with Müller glial cells. These results show that synapse formation is very similar in stratospheroids and in the retina in vivo, and they suggest an important role for agrin during CNS development.     

A role for acetylcholine receptors in their own aggregation on muscle cells

Both neurotrophic factors and activity regulate synaptogenesis. At neuromuscular synapses, the neural factor agrin released from motor neuron terminals stimulates postsynaptic specialization by way of the muscle specific kinase MuSK. In addition, activity through acetylcholine receptors (AChRs) has been implicated in the stabilization of pre- and postsynaptic contacts on muscle at various stages of development. We show here that activation of AChRs with specific concentrations of nicotine is sufficient to induce AChR aggregation and that this induction requires the function of L-type calcium channels (L-CaChs). Furthermore, AChR function is required for agrin induced AChR aggregation in C2 muscle cells. The same concentrations of nicotine did not induce observable tyrosine phosphorylation on either MuSK or the AChR beta subunit, suggesting significant differences between the mechanisms of agrin and activity induced aggregation. The AChR/L-CaCh pathway provides a mechanism by which neuromuscular signal transmission can act in concert with the agrin-MuSK signaling cascade to regulate NMJ formation.     

LRP4 serves as a coreceptor of agrin

Neuromuscular junction (NMJ) formation requires agrin, a factor released from motoneurons, and MuSK, a transmembrane tyrosine kinase that is activated by agrin. However, how signal is transduced from agrin to MuSK remains unclear. We report that LRP4, a low-density lipoprotein receptor (LDLR)-related protein, is expressed specifically in myotubes and binds to neuronal agrin. Its expression enables agrin binding and MuSK signaling in cells that otherwise do not respond to agrin. Suppression of LRP4 expression in muscle cells attenuates agrin binding, agrin-induced MuSK tyrosine phosphorylation, and AChR clustering. LRP4 also forms a complex with MuSK in a manner that is stimulated by agrin. Finally, we showed that LRP4 becomes tyrosine-phosphorylated in agrin-stimulated muscle cells. These observations indicate that LRP4 is a coreceptor of agrin that is necessary for MuSK signaling and AChR clustering and identify a potential target protein whose mutation and/or autoimmunization may cause muscular dystrophies.     

Clustering of nicotinic acetylcholine receptors: From the neuromuscular junction to interneuronal synapses

Fast and accurate synaptic transmission requires high-density accumulation of neurotransmitter receptors in the postsynaptic membrane. During development of the neuromuscular junction, clustering of acetylcholine receptors (AChR) is one of the first signs of postsynaptic specialization and is induced by nerve-released agrin. Recent studies have revealed that different mechanisms regulate assembly vs stabilization of AChR clusters and of the postsynaptic apparatus. MuSK, a receptor tyrosine kinase and component of the agrin receptor, and rapsyn, an AChR-associated anchoring protein, play crucial roles in the postsynaptic assembly. Once formed, AChR clusters and the postsynaptic membrane are stabilized by components of the dystrophin/utrophin glycoprotein complex, some of which also direct aspects of synaptic maturation such as formation of postjunctional folds. Nicotinic receptors are also expressed across the peripheral and central nervous system (PNS/CNS). These receptors are localized not only at the pre- but also at the postsynaptic sites where they carry out major synaptic transmission. In neurons, they are found as clusters at synaptic or extrasynaptic sites, suggesting that different mechanisms might underlie this specific localization of nicotinic receptors. This review summarizes the current knowledge about formation and stabilization of the postsynaptic apparatus at the neuromuscular junction and extends this to explore the synaptic structures of interneuronal cholinergic synapses.     

MuSK Myasthenia Gravis IgG4 Disrupts the Interaction of LRP4 with MuSK but Both IgG4 and IgG1-3 Can Disperse Preformed Agrin-Independent AChR Clusters

A variable proportion of patients with generalized myasthenia gravis (MG) have autoantibodies to muscle specific tyrosine kinase (MuSK). During development agrin, released from the motor nerve, interacts with low density lipoprotein receptor-related protein-4 (LRP4), which then binds to MuSK; MuSK interaction with the intracellular protein Dok7 results in clustering of the acetylcholine receptors (AChRs) on the postsynaptic membrane. In mature muscle, MuSK helps maintain the high density of AChRs at the neuromuscular junction. MuSK antibodies are mainly IgG4 subclass, which does not activate complement and can be monovalent, thus it is not clear how the antibodies cause disruption of AChR numbers or function to cause MG. We hypothesised that MuSK antibodies either reduce surface MuSK expression and/or inhibit the interaction with LRP4. We prepared MuSK IgG, monovalent Fab fragments, IgG1-3 and IgG4 fractions from MuSK-MG plasmas. We asked whether the antibodies caused endocytosis of MuSK in MuSK-transfected cells or if they inhibited binding of LRP4 to MuSK in co-immunoprecipitation experiments. In parallel, we investigated their ability to reduce AChR clusters in C2C12 myotubes induced by a) agrin, reflecting neuromuscular development, and b) by Dok7- overexpression, producing AChR clusters that more closely resemble the adult neuromuscular synapse. Total IgG, IgG4 or IgG1-3 MuSK antibodies were not endocytosed unless cross-linked by divalent anti-human IgG. MuSK IgG, Fab fragments and IgG4 inhibited the binding of LRP4 to MuSK and reduced agrin-induced AChR clustering in C2C12 cells. By contrast, IgG1-3 antibodies did not inhibit LRP4-MuSK binding but, surprisingly, did inhibit agrin-induced clustering. Moreover, both IgG4 and IgG1-3 preparations dispersed agrin-independent AChR clusters in Dok7-overexpressing C2C12 cells. Thus interference by IgG4 antibodies of the LRP4-MuSK interaction will be one pathogenic mechanism of MuSK antibodies, but IgG1-3 MuSK antibodies will also contribute to the reduced AChR density and neuromuscular dysfunction in myasthenia patients with MuSK antibodies.     

Analysis of a Shc family adaptor protein, ShcD/Shc4, that associates with muscle-specific kinase

Shc family proteins serve as phosphotyrosine adaptor molecules in various receptor-mediated signaling pathways. In mammals, three distinct Shc genes have been described that encode proteins characterized by two phosphotyrosine-interaction modules, an amino-terminal phosphotyrosine binding (PTB) domain and a carboxy-terminal Src homology 2 domain. Here, we report the analysis of an uncharacterized fourth Shc family protein, ShcD/Shc4, that is expressed in adult brain and skeletal muscle. Consistent with this expression pattern, we find that ShcD can associate via its PTB domain with the phosphorylated muscle-specific kinase (MuSK) receptor tyrosine kinase and undergo tyrosine phosphorylation downstream of activated MuSK. Interestingly, additional sites of tyrosine phosphorylation, including a novel Grb2 binding site, are present on ShcD that are not found in other Shc family proteins. Activation of MuSK upon agrin binding at the neuromuscular junction (NMJ) induces clustering and tyrosine phosphorylation of acetylcholine receptors (AChRs) required for synaptic transmission. ShcD is coexpressed with MuSK in the postsynaptic region of the NMJ, and in cultured myotubes stimulated with agrin, expression of ShcD appears to be important for early tyrosine phosphorylation of the AChR. Thus, we have characterized a new member of the Shc family of docking proteins, which may mediate a specific aspect of signaling downstream of the MuSK receptor.     

Association of muscle-specific kinase MuSK with the acetylcholine receptor in mammalian muscle.

During synaptogenesis at the neuromuscular junction, a neurally released factor, agrin, causes the clustering of acetylcholine receptors (AChRs) in the muscle membrane beneath the nerve terminal. Agrin acts through a specific receptor which is thought to have a receptor tyrosine kinase, MuSK, as one of its components. In agrin-treated muscle cells, both MuSK and the AChR become tyrosine phosphorylated. To determine how the activation of MuSK leads to AChR clustering, we have investigated their interaction in cultured C2 myotubes. Immunoprecipitation experiments showed that MuSK is associated with the AChR and that this association is increased by agrin treatment. Agrin also caused a transient activation of the AChR-associated MuSK, as demonstrated by MuSK phosphorylation. In agrin-treated myotubes, MuSK phosphorylation increased with the same time course as phosphorylation of the beta subunit of the AChR, but declined more quickly. Although both herbimycin and staurosporine blocked agrin-induced AChR phosphorylation, only herbimycin inhibited the phosphorylation of MuSK. These results suggest that although agrin increases the amount of activated MuSK that is associated with the AChR, MuSK is not directly responsible for AChR phosphorylation but acts through other kinases.     

MAGI-1c: a synaptic MAGUK interacting with muSK at the vertebrate neuromuscular junction

The muscle-specific receptor tyrosine kinase (MuSK) forms part of a receptor complex, activated by nerve-derived agrin, that orchestrates the differentiation of the neuromuscular junction (NMJ). The molecular events linking MuSK activation with postsynaptic differentiation are not fully understood. In an attempt to identify partners and/or effectors of MuSK, cross-linking and immunopurification experiments were performed in purified postsynaptic membranes from the Torpedo electrocyte, a model system for the NMJ. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis was conducted on both cross-link products, and on the major peptide coimmunopurified with MuSK; this analysis identified a polypeptide corresponding to the COOH-terminal fragment of membrane-associated guanylate kinase (MAGUK) with inverted domain organization (MAGI)-1c. A bona fide MAGI-1c (150 kD) was detected by Western blotting in the postsynaptic membrane of Torpedo electrocytes, and in a high molecular mass cross-link product of MuSK. Immunofluorescence experiments showed that MAGI-1c is localized specifically at the adult rat NMJ, but is absent from agrin-induced acetylcholine receptor clusters in myotubes in vitro. In the central nervous system, MAGUKs play a primary role as scaffolding proteins that organize cytoskeletal signaling complexes at excitatory synapses. Our data suggest that a protein from the MAGUK family is involved in the MuSK signaling pathway at the vertebrate NMJ.     

Dual role for calcium in agrin signaling and acetylcholine receptor clustering

Agrin is a motoneuron‐derived factor that initiates neuromuscular synapse formation; however, the signaling pathway underlying postsynaptic differentiation is not yet understood. We have investigated the role of calcium in agrin signaling through the MuSK receptor tyrosine kinase and in the intracellular signaling cascade that leads to AChR phosphorylation and clustering. We find that agrin‐ and neuramindase‐induced MuSK activation in cultured myotubes is completely blocked by removal of extracellular calcium, but only slightly reduced by clamping of intracellular calcium transients with BAPTA. Following agrin's activation of MuSK, we find that the downstream tyrosine phosphorylation of the AChR β‐subunit was inhibited by BAPTA but not by a slower acting chelator, EGTA. Similarly, agrin‐induced clustering of the AChR was blocked by BAPTA but not EGTA. These findings indicate that extracellular calcium is required for the formation of a MuSK signaling complex, and that intracellular calcium regulates phosphorylation and clustering of the AChR in the postsynaptic membrane. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 69–79, 2002     

Molecular regulation of postsynaptic differentiation at the neuromuscular junction

The neuromuscular junction (NMJ) is a synapse that develops between a motor neuron and a muscle fiber. A defining feature of NMJ development in vertebrates is the re-distribution of muscle acetylcholine (ACh) receptors (AChRs) following innervation, which generates high-density AChR clusters at the postsynaptic membrane and disperses aneural AChR clusters formed in muscle before innervation. This process in vivo requires MuSK, a muscle-specific receptor tyrosine kinase that triggers AChR re-distribution when activated; rapsyn, a muscle protein that binds and clusters AChRs; agrin, a nerve-secreted heparan-sulfate proteoglycan that activates MuSK; and ACh, a neurotransmitter that stimulates muscle and also disperses aneural AChR clusters. Moreover, in cultured muscle cells, several additional muscle- and nerve-derived molecules induce, mediate or participate in AChR clustering and dispersal. In this review we discuss how regulation of AChR re-distribution by multiple factors ensures aggregation of AChRs exclusively at NMJs.