Activation Of Muscle-specific Kinase Research Articles

ARGX-119 is an agonist antibody for human MuSK that reverses disease relapse in a mouse model of congenital myasthenic syndrome.

Muscle-specific kinase (MuSK) is essential for the formation, function, and preservation of neuromuscular synapses. Activation of MuSK by a MuSK agonist antibody may stabilize or improve the function of the neuromuscular junction (NMJ) in patients with disorders of the NMJ, such as congenital myasthenia (CM). Here, we generated and characterized ARGX-119, a first-in-class humanized agonist monoclonal antibody specific for MuSK, that is being developed for treatment of patients with neuromuscular diseases. We performed in vitro ligand-binding assays to show that ARGX-119 binds with high affinity to the Frizzled-like domain of human, nonhuman primate, rat, and mouse MuSK, without off-target binding, making it suitable for clinical development. Within the Fc region, ARGX-119 harbors L234A and L235A mutations to diminish potential immune-activating effector functions. Its mode of action is to activate MuSK, without interfering with its natural ligand neural Agrin, and cluster acetylcholine receptors in a dose-dependent manner, thereby stabilizing neuromuscular function. In a mouse model of DOK7 CM, ARGX-119 prevented early postnatal lethality and reversed disease relapse in adult Dok7 CM mice by restoring neuromuscular function and reducing muscle weakness and fatigability in a dose-dependent manner. Pharmacokinetic studies in nonhuman primates, rats, and mice revealed a nonlinear PK behavior of ARGX-119, indicative of target-mediated drug disposition and in vivo target engagement. On the basis of this proof-of-concept study, ARGX-119 has the potential to alleviate neuromuscular diseases hallmarked by impaired neuromuscular synaptic function, warranting further clinical development.

Internalization of Muscle-Specific Kinase Is Increased by Agrin and Independent of Kinase-Activity, Lrp4 and Dynamin.

Muscle-specific kinase (MuSK) is a receptor tyrosine kinase absolutely required for neuromuscular junction formation. MuSK is activated by binding of motor neuron-derived Agrin to low-density lipoprotein receptor related protein 4 (Lrp4), which forms a complex with MuSK. MuSK activation and downstream signaling are critical events during the development of the neuromuscular junction. Receptor tyrosine kinases are commonly internalized upon ligand binding and crosstalk between endocytosis and signaling has been implicated. To extend our knowledge about endocytosis of synaptic proteins and its role during postsynaptic differentiation at the neuromuscular junction, we studied the stability and internalization of Lrp4, MuSK and acetylcholine receptors (AChRs) in response to Agrin. We provide evidence that MuSK but not Lrp4 internalization is increased by Agrin stimulation. MuSK kinase-activity is not sufficient to induce MuSK internalization and the absence of Lrp4 has no effect on MuSK endocytosis. Moreover, MuSK internalization and signaling are unaffected by the inhibition of Dynamin suggesting that MuSK endocytosis uses a non-conventional pathway and is not required for MuSK-dependent downstream signaling.

Activation of Muscle-Specific Kinase (MuSK) Reduces Neuromuscular Defects in the Delta7 Mouse Model of Spinal Muscular Atrophy (SMA).

Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.

Mechanism of disease and therapeutic rescue of Dok7 congenital myasthenia

Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.

Muscle specific kinase (MuSK) activation preserves neuromuscular junctions in the diaphragm but is not sufficient to provide a functional benefit in the SOD1G93A mouse model of ALS

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons, is characterized by rapid decline of motor function and ultimately respiratory failure. As motor neuron death occurs late in the disease, therapeutics that prevent the initial disassembly of the neuromuscular junction may offer optimal functional benefit and delay disease progression. To test this hypothesis, we treated the SOD1G93A mouse model of ALS with an agonist antibody to muscle specific kinase (MuSK), a receptor tyrosine kinase required for the formation and maintenance of the neuromuscular junction. Chronic MuSK antibody treatment fully preserved innervation of the neuromuscular junction when compared with control-treated mice; however, no preservation of diaphragm function, motor neurons, or survival benefit was detected. These data show that anatomical preservation of neuromuscular junctions in the diaphragm via MuSK activation does not correlate with functional benefit in SOD1G93A mice, suggesting caution in employing MuSK activation as a therapeutic strategy for ALS patients.

MuSK Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop

The neuromuscular junction (NMJ) forms when a motor neuron contacts a muscle fibre. A reciprocal exchange of signals initiates a cascade of signalling events that result in pre- and postsynaptic differentiation. At the centre of these signalling events stands muscle specific kinase (MuSK). MuSK activation, kinase activity and subsequent downstream signalling are crucial for NMJ formation as well as maintenance. Therefore MuSK kinase activity is tightly regulated to ensure proper NMJ development. We have identified a novel serine phosphorylation site at position 751 in MuSK that is increasingly phosphorylated upon agrin stimulation. S751 is also phosphorylated in muscle tissue and its phosphorylation depends on MuSK kinase activity. A phosphomimetic mutant of S751 increases MuSK kinase activity in response to non-saturating agrin concentrations . In addition, basal MuSK and AChR phosphorylation as well as AChR cluster size are increased. We believe that the phosphorylation of S751 provides a novel mechanism to relief the autoinhibition of the MuSK activation loop. Such a lower autoinhibition could foster or stabilize MuSK kinase activation, especially during stages when no or low level of agrin are present. Phosphorylation of S751 might therefore represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMJ maintenance.

Muscle-specific kinase (MuSK) autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction.

Muscle-specific kinase (MuSK) autoantibodies from myasthenia gravis patients can block the activation of MuSK in vitro and/or reduce the postsynaptic localization of MuSK. Here we use a mouse model to examine the effects of MuSK autoantibodies upon some key components of the postsynaptic MuSK pathway and upon the regulation of junctional ACh receptor (AChR) numbers. Mice became weak after 14 daily injections of anti-MuSK-positive patient IgG. The intensity and area of AChR staining at the motor endplate was markedly reduced. Pulse-labelling of AChRs revealed an accelerated loss of pre-existing AChRs from postsynaptic AChR clusters without a compensatory increase in incorporation of (newly synthesized) replacement AChRs. Large, postsynaptic AChR clusters were replaced by a constellation of tiny AChR microaggregates. Puncta of AChR staining also appeared in the cytoplasm beneath the endplate. Endplate staining for MuSK, activated Src, rapsyn and AChR were all reduced in intensity. In the tibialis anterior muscle there was also evidence that phosphorylation of the AChR β-subunit-Y390 was reduced at endplates. In contrast, endplate staining for β-dystroglycan (through which rapsyn couples AChR to the synaptic basement membrane) remained intense. The results suggest that anti-MuSK IgG suppresses the endplate density of MuSK, thereby down-regulating MuSK signalling activity and the retention of junctional AChRs locally within the postsynaptic membrane scaffold.

Endosomal trafficking of the receptor tyrosine kinase MuSK proceeds via clathrin‐dependent pathways, Arf6 and actin

Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is the key player during the formation of the neuromuscular junction. Signal transduction events downstream of MuSK activation induce both pre-and postsynaptic differentiation, which, most prominently, includes the clustering of acetylcholine receptors at synaptic sites. More recently, regulated MuSK endocytosis and degradation have been implicated as crucial events for MuSK signalling activity, implicating a cross-talk between signalling and endocytosis. In the present study, we use a live imaging approach to study MuSK endocytosis. We find that MuSK is internalized via a clathrin-, dynamin-dependent pathway. MuSK is transported to Rab7-positive endosomes for degradation and recycled via Rab4-and Rab11-positive vesicles. MuSK activation by Dok7 mildly affects the localization of MuSK on the cell surface but has no effect on the rate of MuSK internalization. Interestingly, MuSK colocalizes with actin and Arf6 at the cell surface and during endosomal trafficking. Disruption of the actin cytoskeleton or the proper function of Arf6 concentrates MuSK in cell protrusions. Moreover, inhibition of Arf6 or cytoskeletal rearrangements impairs acetylcholine receptor clustering and phosphorylation. These results suggest that MuSK uses both classical and nonclassical endosomal pathways that involve a variety of different components of the endosomal machinery.Structured digital abstractMuSK and Arf6 colocalize by fluorescence microscopy (View Interaction: 1, 2)MuSK and Rab4 colocalize by fluorescence microscopy (View interaction)MuSK and Rab11 colocalize by fluorescence microscopy (View interaction)MuSK and Rab7 colocalize by fluorescence microscopy (View interaction)

Antibodies against Muscle-Specific Kinase Impair Both Presynaptic and Postsynaptic Functions in a Murine Model of Myasthenia Gravis

Antibodies against acetylcholine receptors (AChRs) cause pathogenicity in myasthenia gravis (MG) patients through complement pathway-mediated destruction of postsynaptic membranes at neuromuscular junctions (NMJs). However, antibodies against muscle-specific kinase (MuSK), which constitute a major subclass of antibodies found in MG patients, do not activate the complement pathway. To investigate the pathophysiology of MuSK-MG and establish an experimental autoimmune MG (EAMG) model, we injected MuSK protein into mice deficient in complement component five (C5). MuSK-injected mice simultaneously developed severe muscle weakness, accompanied by an electromyographic pattern such as is typically observed in MG patients. In addition, we observed morphological and functional defects in the NMJs of EAMG mice, demonstrating that complement activation is not necessary for the onset of MuSK-MG. Furthermore, MuSK-injected mice exhibited acetylcholinesterase (AChE) inhibitor-evoked cholinergic hypersensitivity, as is observed in MuSK-MG patients, and a decrease in both AChE and the AChE-anchoring protein collagen Q at postsynaptic membranes. These findings suggest that MuSK is indispensable for the maintenance of NMJ structure and function, and that disruption of MuSK activity by autoantibodies causes MG. This mouse model of EAMG could be used to develop appropriate medications for the treatment of MuSK-MG in humans.

Mechanism of Acetylcholine Receptor Cluster Formation Induced by DC Electric Field

BackgroundThe formation of acetylcholine receptor (AChR) cluster is a key event during the development of the neuromuscular junction. It is induced through the activation of muscle-specific kinase (MuSK) by the heparan-sulfate proteoglycan agrin released from the motor axon. On the other hand, DC electric field, a non-neuronal stimulus, is also highly effective in causing AChRs to cluster along the cathode-facing edge of muscle cells.Methodology/Principal FindingsTo understand its molecular mechanism, quantum dots (QDs) were used to follow the movement of AChRs as they became clustered under the influence of electric field. From analyses of trajectories of AChR movement in the membrane, it was concluded that diffuse receptors underwent Brownian motion until they were immobilized at sites of cluster formation. This supports the diffusion-mediated trapping model in explaining AChR clustering under the influence of this stimulus. Disrupting F-actin cytoskeleton assembly and interfering with rapsyn-AChR interaction suppressed this phenomenon, suggesting that these are integral components of the trapping mechanism induced by the electric field. Consistent with the idea that signaling pathways are activated by this stimulus, the localization of tyrosine-phosphorylated forms of AChR β-subunit and Src was observed at cathodal AChR clusters. Furthermore, disrupting MuSK activity through the expression of a kinase-dead form of this enzyme abolished electric field-induced AChR clustering.ConclusionsThese results suggest that DC electric field as a physical stimulus elicits molecular reactions in muscle cells in the form of cathodal MuSK activation in a ligand-free manner to trigger a signaling pathway that leads to cytoskeletal assembly and AChR clustering.

3PS2.1 Inherited Ataxia Update

acting on enzyme stability at presynaptic level. Transmission is compromised by desensitation and depolarization block at AChR at physiologic rate of stimulation in synaptic defects. Agrin stabilize laminin network being important for acetylcholinesterase (AChE) function, AChR clustering, and muscle-specific kinase (MuSK) autophosphorylation. Mutations in AChR impair ion channel gating, reducing number of AChR and cause slow channel, fast channel and AChR deficiency syndrome. AChR channel gating is impaired based probably on conformational transition by changing isomerisation between open and closed conformation. Dok7 mutations result in impaired activation of MuSK and rapsyn to concentrate AChR on junctional folds which cause small simplified and unstable neuromuscular junctions affecting both presynaptic and postsynaptic structures. MuSK mutations result in deficiency of presynaptic differentiation and postsynaptic development. Conclusions: There is no correlation of genotype and phenotype and there is no possibility to predict the severity of CMS according to known mutation. Molecular genetics is essential in differential diagnosis and understanding of the patomechanisms inducing CMS and its effective therapy.

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.

Crystal Structure of the Frizzled-Like Cysteine-Rich Domain of the Receptor Tyrosine Kinase MuSK

Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 Å resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ.

A Mammalian Homolog of Drosophila Tumorous Imaginal Discs, Tid1, Mediates Agrin Signaling at the Neuromuscular Junction

Motoneuron-derived agrin clusters nicotinic acetylcholine receptors (AChRs) in mammalian muscle cells. We used two-hybrid screens to identify a protein, tumorous imaginal discs (Tid1), that binds to the cytoplasmic domain of muscle-specific kinase (MuSK), a major component of the agrin receptor. Like MuSK, Tid1 colocalizes with AChRs at developing, adult, and denervated motor endplates. Knockdown of Tid1 by short hairpin RNA (shRNA) in skeletal muscle fibers dispersed synaptic AChR clusters and impaired neuromuscular transmission. In cultured myotubes, Tid1 knockdown inhibited AChR clustering, as well as agrin-induced activation of the Rac and Rho small GTPases and tyrosine phosphorylation of the AChR, without affecting MuSK activation. Tid1 knockdown also decreased Dok-7-induced clustering of AChRs. Overexpression of the N-terminal half of Tid1 induced agrin- and MuSK-independent phosphorylation and clustering of AChRs. These results demonstrate that Tid1 is an essential component of the agrin signaling pathway, crucial for synaptic development.

Rapsyn carboxyl terminal domains mediate muscle specific kinase–induced phosphorylation of the muscle acetylcholine receptor

At the developing vertebrate neuromuscular junction, postsynaptic localization of the acetylcholine receptor (AChR) is regulated by agrin signaling via the muscle specific kinase (MuSK) and requires an intracellular scaffolding protein called rapsyn. In addition to its structural role, rapsyn is also necessary for agrin-induced tyrosine phosphorylation of the AChR, which regulates some aspects of receptor localization. Here, we have investigated the molecular mechanism by which rapsyn mediates AChR phosphorylation at the rodent neuromuscular junction. In a heterologous COS cell system, we show that MuSK and rapsyn induced phosphorylation of β subunit tyrosine 390 (Y390) and δ subunit Y393, as in muscle cells. Mutation of β Y390 or δ Y393 did not inhibit MuSK/rapsyn-induced phosphorylation of the other subunit in COS cells, and mutation of β Y390 did not inhibit agrin-induced phosphorylation of the δ subunit in Sol8 muscle cells; thus, their phosphorylation occurs independently, downstream of MuSK activation. In COS cells, we further show that MuSK-induced phosphorylation of the β subunit was mediated by rapsyn, as MuSK plus rapsyn increased β Y390 phosphorylation more than rapsyn alone and MuSK alone had no effect. Intriguingly, MuSK also induced tyrosine phosphorylation of rapsyn itself. We then used deletion mutants to map the rapsyn domains responsible for activation of cytoplasmic tyrosine kinases that phosphorylate the AChR subunits. We found that rapsyn C-terminal domains (amino acids 212–412) are both necessary and sufficient for activation of tyrosine kinases and induction of cellular tyrosine phosphorylation. Moreover, deletion of the rapsyn RING domain (365–412) abolished MuSK-induced tyrosine phosphorylation of the AChR β subunit. Together, these findings suggest that rapsyn facilitates AChR phosphorylation by activating or localizing tyrosine kinases via its C-terminal domains.

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.

L‐type calcium channels mediate acetylcholine receptor aggregation on cultured muscle

Agrin activation of muscle specific kinase (MuSK) initiates postsynaptic development on skeletal muscle that includes the aggregation of acetylcholine receptors (AChRs; Glass et al. [1996]: Cell 85: 513-523; Gautam et al. [1996]: Cell 85: 525-535). Although the agrin/MuSK signaling pathway remains largely unknown, changes in intracellular calcium levels are required for agrin-induced AChR aggregation (Megeath and Fallon [1998]: J Neurosci 18: 672-678). Here, we show that L-type calcium channels (L-CaChs) are required for full agrin-induced aggregation of AChRs and sufficient to induce agrin-independent AChR aggregation. Blockade of L-CaChs in muscle cultures inhibited agrin-induced AChR aggregation but not tyrosine phosphorylation of MuSK or AChR beta subunits. Activation of L-CaChs in the absence of agrin induced AChR aggregation but not tyrosine phosphorylation of MuSK or AChR beta subunits. Agrin responsiveness was significantly reduced in primary muscle cultures from the muscular dysgenesis mouse, a natural mutant, which does not express the L-CaCh. Our results establish a novel role for L-CaChs as important sources of the intracellular calcium necessary for the aggregation of AChRs.

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.

Crystal Structure of the Agrin-responsive Immunoglobulin-like Domains 1 and 2 of the Receptor Tyrosine Kinase MuSK

Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed exclusively in skeletal muscle, where it is required for formation of the neuromuscular junction. MuSK is activated by agrin, a neuron-derived heparan sulfate proteoglycan. Here, we report the crystal structure of the agrin-responsive first and second immunoglobulin-like domains (Ig1 and Ig2) of the MuSK ectodomain at 2.2 Å resolution. The structure reveals that MuSK Ig1 and Ig2 are Ig-like domains of the I-set subfamily, which are configured in a linear, semi-rigid arrangement. In addition to the canonical internal disulfide bridge, Ig1 contains a second, solvent-exposed disulfide bridge, which our biochemical data indicate is critical for proper folding of Ig1 and processing of MuSK. Two Ig1-2 molecules form a non-crystallographic dimer that is mediated by a unique hydrophobic patch on the surface of Ig1. Biochemical analyses of MuSK mutants introduced into MuSK −/− myotubes demonstrate that residues in this hydrophobic patch are critical for agrin-induced MuSK activation.

Expression of MuSK in In Vitro-Innervated Human Muscle

Unlike rodent or avian muscle, which forms clusters of acetylcholine receptors (AChRs) on its surface, exhibits cross striations, and contracts spontaneously even if cultured in the absence of the nerve, human muscle must be innervated to reach such differentiation level under in vitro conditions (Kobayashi and Askanas, 1985; Mars et al., 2001). Because it is known that AChR clustering and other aspects of neuromuscular junction (NMJ) formation necessitate the activation of muscle-specific kinase (MuSK), one explanation of this inability of human muscle is that it has no MuSK or that it cannot be activated in the absence of the nerve. To test this hypothesis we analyzed cultured human muscle for the expression of MuSK at two stages of differentiation: postfusion myotube and innervated, contracting myotube. Analyses were carried out at the mRNA level, as no reliable anti-MuSK antibodies are available for the immunocytochemical demonstration of MuSK in cultured human muscle. The presence of MuSK, however, can be tested indirectly, as it can be activated in the absence of the nerve simply by growing muscle culture on laminin coating (Kummer et al., 2004). In the second part of our study, we therefore tested human myotubes for the presence and activation of MuSK by exposing them to laminin coating and by analyzing them afterwards for the areas of postsynaptic differentiation typical for NMJ formation.