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.     

Laminin-1 redistributes postsynaptic proteins and requires rapsyn,tyrosine phosphorylation,and Src and Fyn to stably cluster acetylcholine receptors

Clustering of acetylcholine receptors (AChRs) is a critical step in neuromuscular synaptogenesis, and is induced by agrin and laminin which are thought to act through different signaling mechanisms. We addressed whether laminin redistributes postsynaptic proteins and requires key elements of the agrin signaling pathway to cause AChR aggregation. In myotubes, laminin-1 rearranged dystroglycans and syntrophins into a laminin-like network, whereas inducing AChR-containing clusters of dystrobrevin, utrophin, and, to a marginal degree, MuSK. Laminin-1 also caused extensive coclustering of rapsyn and phosphotyrosine with AChRs, but none of these clusters were observed in rapsyn -/- myotubes. In parallel with clustering, laminin-1 induced tyrosine phosphorylation of AChR beta and delta subunits. Staurosporine and herbimycin, inhibitors of tyrosine kinases, prevented laminin-induced AChR phosphorylation and AChR and phosphotyrosine clustering, and caused rapid dispersal of clusters previously induced by laminin-1. Finally, laminin-1 caused normal aggregation of AChRs and phosphotyrosine in myotubes lacking both Src and Fyn kinases, but these clusters dispersed rapidly after laminin withdrawal. Thus, laminin-1 redistributes postsynaptic proteins and, like agrin, requires tyrosine kinases for AChR phosphorylation and clustering, and rapsyn for AChR cluster formation, whereas cluster stabilization depends on Src and Fyn. Therefore, the laminin and agrin signaling pathways overlap intracellularly, which may be important for neuromuscular synapse formation.     

Recycling of acetylcholine receptors at ectopic postsynaptic clusters induced by exogenous agrin in living rats

During the development of the neuromuscular junction, motor axons induce the clustering of acetylcholine receptors (AChRs) and increase their metabolic stability in the muscle membrane. Here, we asked whether the synaptic organizer agrin might regulate the metabolic stability and density of AChRs by promoting the recycling of internalized AChRs, which would otherwise be destined for degradation, into synaptic sites. We show that at nerve-free AChR clusters induced by agrin in extrasynaptic membrane, internalized AChRs are driven back into the ectopic synaptic clusters where they intermingle with pre-existing and new receptors. The extent of AChR recycling depended on the strength of the agrin stimulus, but not on the development of junctional folds, another hallmark of mature postsynaptic membranes. In chronically denervated muscles, in which both AChR stability and recycling are significantly decreased by muscle inactivity, agrin maintained the amount of recycled AChRs at agrin-induced clusters at a level similar to that at denervated original endplates. In contrast, AChRs did not recycle at agrin-induced clusters in C2C12 or primary myotubes. Thus, in muscles in vivo, but not in cultured myotubes, neural agrin promotes the recycling of AChRs and thereby increases their metabolic stability.     

Nicotine decreases agrin signaling and acetylcholine receptor clustering in C2C12 myotube culture

The clustering of acetylcholine receptors (AChRs) in skeletal muscle fibers is a critical event in neuromuscular synaptogenesis. AChRs in concert with other molecules form postsynaptic scaffolds in response to agrin released from motor neurons as motor neurons near skeletal muscle fibers in development. Agrin drives an intracellular signaling pathway that precedes AChR clustering and includes the tyrosine phosphorylation of AChRs. In C2C12 myotube culture, agrin application stimulates the agrin signaling pathway and AChR clustering. Previous studies have determined that the frequency of spontaneous AChR clustering is decreased and AChRs are partially inactivated when bound by the acetylcholine agonist nicotine. We hypothesized that nicotine interferes with AChR clustering and consequent postsynaptic scaffold formation. In the present study, C2C12 myoblasts were cultured with growth medium to stimulate proliferation and then differentiation medium to stimulate fusion into myotubes. They were bathed in a physiologically relevant concentration of nicotine and then subject to agrin treatment after myotube formation. Our results demonstrate that nicotine decreases agrin-induced tyrosine phosphorylation of AChRs and decreases the frequency of spontaneous as well as agrin-induced AChR clustering. We conclude that nicotine interferes with postsynaptic scaffold formation by preventing the tyrosine phosphorylation of AChRs, an agrin signaling event that precedes AChR clustering.     

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.     

Neuregulin inhibits acetylcholine receptor aggregation in myotubes

The high local concentration of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction results from their aggregation by the agrin/MuSK signaling pathway and their synthetic up-regulation by the neuregulin/ErbB pathway. Here, we show a novel role for the neuregulin/ErbB pathway, the inhibition of AChR aggregation on the muscle surface. Treatment of C2C12 myotubes with the neuregulin epidermal growth factor domain decreased the number of both spontaneous and agrin-induced AChR clusters, in part by increasing the rate of cluster disassembly. Upon cluster disassembly, AChRs were internalized into caveolae (as identified by caveolin-3). Time-lapse microscopy revealed that individual AChR clusters fragmented into puncta, and application of neuregulin accelerated the rate at which AChR clusters decreased in area without affecting the density of AChRs remaining in individual clusters (as measured by the fluorescence intensity/unit area). We propose that this novel action of neuregulin regulates synaptic competition at the developing neuromuscular junction.     

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.     

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.     

Acetylcholine receptors are required for agrin-induced clustering of postsynaptic proteins.

We have investigated the role of acetylcholine receptors (AChRs) in an early step of postsynaptic assembly at the neuromuscular synapse, the clustering of postsynaptic proteins induced by nerve-released agrin. To achieve this, we used two variants of C2 myotubes virtually lacking AChRs and C2 cells in which surface AChRs were down-regulated by AChR antibodies. In all cases, agrin caused normal clustering of the agrin receptor component MuSK, alpha-dystrobrevin and utrophin, but failed to aggregate AChRs, alpha- and beta-dystroglycan, syntrophin isoforms and rapsyn, an AChR-anchoring protein necessary for postsynaptic assembly and AChR clustering. In C2 variants, the stability of rapsyn was decreased, whereas in antibody-treated cells, rapsyn efficiently co-localized with remaining AChRs in microaggregates. Upon ectopic injection into myofibers in vivo, rapsyn did not form clusters in the absence of AChRs. These results show that AChRs and rapsyn are interdependent components of a pre-assembled protein complex that is required for agrin-induced clustering of a full set of postsynaptic proteins, thus providing evidence for an active role of AChRs in postsynaptic assembly.     

Regulation of ACh receptor clustering by the tyrosine phosphatase Shp2

At the vertebrate neuromuscular junction (NMJ), postsynaptic aggregation of muscle acetylcholine receptors (AChRs) depends on the activation of MuSK, a muscle-specific tyrosine kinase that is stimulated by neural agrin and regulated by muscle-intrinsic tyrosine kinases and phosphatases. We recently reported that Shp2, a tyrosine phosphatase containing src homology two domains, suppressed MuSK-dependent AChR clustering in cultured myotubes, but how this effect of Shp2 is controlled has remained unclear. In this study, biochemical assays showed that agrin-treatment of C2 mouse myotubes enhanced the tyrosine phosphorylation of signal regulatory protein alpha1 (SIRPalpha1), a known activator of Shp2, and promoted SIRPalpha1's interaction with Shp2. Moreover, in situ experiments revealed that treatment of myotubes with the Shp2-selective inhibitor NSC-87877 increased spontaneous and agrin-induced AChR clustering, and that AChR clustering was also enhanced in myotubes ectopically expressing inactive (dominant-negative) Shp2; in contrast, AChR clustering was reduced in myotubes expressing constitutively active Shp2. Significantly, expression of truncated (nonShp2-binding) and full-length (Shp2-binding) forms of SIRPalpha1 in myotubes also increased and decreased AChR clustering, respectively, and coexpression of truncated SIRPalpha1 with active Shp2 and full-length SIRPalpha1 with inactive Shp2 reversed the actions of the exogenous Shp2 proteins on AChR clustering. These results suggest that SIRPalpha1 is a novel downstream target of MuSK that activates Shp2, which, in turn, suppresses AChR clustering. We propose that an inhibitory loop involving both tyrosine kinases and phosphatases sets the level of agrin/MuSK signaling and constrains it spatially to help generate high-density AChR clusters selectively at NMJs.     

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.     

Agrin-induced activation of acetylcholine receptor-bound Src family kinases requires Rapsyn and correlates with acetylcholine receptor clustering

During neuromuscular synaptogenesis, neurally released agrin induces aggregation and tyrosine phosphorylation of acetylcholine receptors (AChRs) by acting through both the receptor tyrosine kinase MuSK (muscle-specific kinase) and the AChR-associated protein, rapsyn. To elucidate this signaling mechanism, we examined tyrosine phosphorylation of AChR-associated proteins, particularly addressing whether agrin activates Src family kinases bound to the AChR. In C2 myotubes, agrin induced tyrosine phosphorylation of these kinases, of AChR-bound MuSK, and of the AChR beta and delta subunits, as observed in phosphotyrosine immunoblotting experiments. Kinase assays revealed that the activity of AChR-associated Src kinases was increased by agrin, whereas phosphorylation of the total cellular kinase pool was unaffected. In both rapsyn-deficient myotubes and staurosporine-treated C2 myotubes, where AChRs are not clustered, agrin activated MuSK but did not cause either Src family or AChR phosphorylation. In S27 mutant myotubes, which fail to aggregate AChRs, no agrin-induced phosphorylation of AChR-bound Src kinases, MuSK, or AChRs was observed. These results demonstrate first that agrin leads to phosphorylation and activation of AChR-associated Src-related kinases, which requires rapsyn, occurs downstream of MuSK, and causes AChR phosphorylation. Second, this activation intimately correlates with AChR clustering, suggesting that these kinases may play a role in agrin-induced AChR aggregation by forming an AChR-bound signaling cascade.     

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.     

Conjugation of LG domains of agrins and perlecan to polymerizing laminin-2 promotes acetylcholine receptor clustering

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.     

The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

Background

Postsynaptic enrichment of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction (NMJ) depends on the activation of the muscle receptor tyrosine MuSK by neural agrin. Agrin-stimulation of MuSK is known to initiate an intracellular signaling cascade that leads to the clustering of AChRs in an actin polymerization-dependent manner, but the molecular steps which link MuSK activation to AChR aggregation remain incompletely defined.

Methodology/Principal Findings

In this study we used biochemical, cell biological and molecular assays to investigate a possible role in AChR clustering of cortactin, a protein which is a tyrosine kinase substrate and a regulator of F-actin assembly and which has also been previously localized at AChR clustering sites. We report that cortactin was co-enriched at AChR clusters in situ with its target the Arp2/3 complex, which is a key stimulator of actin polymerization in cells. Cortactin was further preferentially tyrosine phosphorylated at AChR clustering sites and treatment of myotubes with agrin significantly enhanced the tyrosine phosphorylation of cortactin. Importantly, forced expression in myotubes of a tyrosine phosphorylation-defective cortactin mutant (but not wild-type cortactin) suppressed agrin-dependent AChR clustering, as did the reduction of endogenous cortactin levels using RNA interference, and introduction of the mutant cortactin into muscle cells potently inhibited synaptic AChR aggregation in response to innervation.

Conclusion

Our results suggest a novel function of phosphorylation-dependent cortactin signaling downstream from agrin/MuSK in facilitating AChR clustering at the developing NMJ.     

A single pulse of agrin triggers a pathway that acts to cluster acetylcholine receptors

Agrin triggers signaling mechanisms of high temporal and spatial specificity to achieve phosphorylation, clustering, and stabilization of postsynaptic acetylcholine receptors (AChRs). Agrin transiently activates the kinase MuSK; MuSK activation has largely vanished when AChR clusters appear. Thus, a tyrosine kinase cascade acts downstream from MuSK, as illustrated by the agrin-evoked long-lasting activation of Src family kinases (SFKs) and their requirement for AChR cluster stabilization. We have investigated this cascade and report that pharmacological inhibition of SFKs reduces early but not later agrin-induced phosphorylation of MuSK and AChRs, while inhibition of Abl kinases reduces late phosphorylation. Interestingly, SFK inhibition applied selectively during agrin-induced AChR cluster formation caused rapid cluster dispersal later upon agrin withdrawal. We also report that a single 5-min agrin pulse, followed by extensive washing, triggered long-lasting MuSK and AChR phosphorylation and efficient AChR clustering. Following the pulse, MuSK phosphorylation increased and, beyond a certain level, caused maximal clustering. These data reveal novel temporal aspects of tyrosine kinase action in agrin signaling. First, during AChR cluster formation, SFKs initiate early phosphorylation and an AChR stabilization program that acts much later. Second, a kinase mechanism rapidly activated by agrin acts thereafter autonomously in agrin's absence to further increase MuSK phosphorylation and cluster AChRs.     

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.     

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     

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 beta-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.