The juxtamembrane region of MuSK has a critical role in agrin-mediated signaling

MuSK is a receptor tyrosine kinase expressed selectively in skeletal muscle and localized to neuromuscular synapses. Agrin activates MuSK and stimulates phosphorylation and clustering of acetylcholine receptors (AChRs) at synaptic sites. We expressed wild-type or mutant MuSK in MuSK(-/-) myotubes and identified tyrosine residues in the MuSK cytoplasmic domain that are necessary for agrin-stimulated phosphorylation and clustering of AChRs. The activation loop tyrosines and the single juxtamembrane tyrosine were found to be essential for agrin-stimulated phosphorylation and clustering of AChRs. Further, we show that the juxtamembrane tyrosine, contained within an NPXY motif, is phosphorylated in vivo by agrin stimulation. We constructed chimeras containing extracellular and transmembrane domains from MuSK and cytoplasmic sequences from TrkA and found that inclusion of 13 amino acids from the MuSK juxtamembrane region, including the NPXY motif, is sufficient to convert a phosphorylated but inactive MuSK-TrkA chimera into a phosphorylated active chimera. These data suggest that phosphorylation of the MuSK NPXY site leads to recruitment of a phosphotyrosine-binding domain-containing protein that functions to stimulate phosphorylation and clustering of AChRs.     

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.     

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.     

Polyomavirus middle-T antigen associates with the kinase domain of Src-related tyrosine kinases.

Middle-T antigen of mouse polyomavirus, an oncogenic DNA virus, associates with and activates the cellular tyrosine kinases c-Src, c-Yes, and Fyn. This interaction is essential for polyomavirus-mediated transformation of cells in culture and tumor formation in animals. To determine the domain of c-Src directing association with middle-T, mutant c-Src proteins lacking the amino-terminal unique domain and the myristylation signal, the SH2 domain, the SH3 domain, or all three of these domains were coexpressed with middle-T in NIH 3T3 cells. All mutants were found to associate with middle-T, demonstrating that the kinase domain of c-Src, including the carboxy-terminal regulatory tail, is sufficient for association with middle-T. Moreover, we found that Hck, another member of the Src kinase family, does not bind middle-T, while chimeric kinases consisting of the amino-terminal domains of c-Src fused to the kinase domain of Hck or the amino-terminal domains of Hck fused to the kinase domain of c-Src associated with middle-T. Hck mutated at its carboxy-terminal regulatory residue, tyrosine 501, was also found to associate with middle-T. These results suggest that in Hck, the postulated intramolecular interaction between the carboxy-terminal regulatory tyrosine and the SH2 domain prevents association with middle-T. This intramolecular interaction apparently also limits the ability of c-Src to associate with middle-T, since removal of the SH2 or SH3 domain increases the efficiency with which middle-T binds c-Src.     

MuSK glycosylation restrains MuSK activation and acetylcholine receptor clustering

MuSK, a muscle-specific receptor tyrosine kinase that is activated by agrin, has a critical role in neuromuscular synapse formation. In cultured myotubes, agrin stimulates the rapid phosphorylation of MuSK, leading to MuSK activation and tyrosine phosphorylation and clustering of acetylcholine receptors. Agrin, however, fails to stimulate tyrosine phosphorylation of MuSK that is force-expressed in myoblasts and fibroblasts, indicating that myotubes contain an additional activity that is required for agrin to stimulate MuSK. Certain glycosyltransferases are expressed selectively at synaptic sites in skeletal muscle, raising the possibility that carbohydrate modifications of MuSK, catalyzed by glycosyltransferases expressed selectively in myotubes, may be essential for agrin to bind and activate MuSK. We identifed two N-linked glycosylation sites in MuSK, and we expressed MuSK mutants lacking one or both N-linked sites into MuSK mutant myotubes to determine whether N-linked carbohydrate modifications of MuSK have a role in MuSK activation. We found that N-linked glycosylation restrains ligand-independent tyrosine phosphorylation of MuSK and downstream signaling but is not necessary for agrin to stimulate MuSK.     

A putative ariadne-like E3 ubiquitin ligase (PAUL) that interacts with the muscle-specific kinase (MuSK)

Formation of the postsynaptic membrane at the skeletal neuromuscular junction (NMJ) requires activation of the muscle-specific receptor tyrosine kinase (MuSK). Few intracellular mediators or modulators of MuSK actions are known. E3 ubiquitin ligases may serve this role, because activities of several receptor tyrosine kinases, G-protein-coupled receptors and channels are modulated by ubiquitination. Here, we report identification of a putative Ariadne-like ubiquitin ligase (PAUL) that binds to the cytoplasmic domain of MuSK. PAUL is expressed in numerous tissues of developing and adult mice, and is present at NMJs in muscle fibers but is not confined to them.     

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.     

Crystal structure of the MuSK tyrosine kinase: insights into receptor autoregulation

Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed selectively in skeletal muscle. During neuromuscular synapse formation, agrin released from motor neurons stimulates MuSK autophosphorylation in the kinase activation loop and in the juxtamembrane region, leading to clustering of acetylcholine receptors. We have determined the crystal structure of the cytoplasmic domain of unphosphorylated MuSK at 2.05 A resolution. The structure reveals an autoinhibited kinase domain in which the activation loop obstructs ATP and substrate binding. Steady-state kinetic analysis demonstrates that autophosphorylation results in a 200-fold increase in k(cat) and a 10-fold decrease in the K(m) for ATP. These studies provide a molecular basis for understanding the regulation of MuSK catalytic activity and suggest that an additional in vivo component may contribute to regulation via the juxtamembrane region.     

COOH-terminal collagen Q (COLQ) mutants causing human deficiency of endplate acetylcholinesterase impair the interaction of ColQ with proteins of the basal lamina

Collagen Q (ColQ) is a key multidomain functional protein of the neuromuscular junction (NMJ), crucial for anchoring acetylcholinesterase (AChE) to the basal lamina (BL) and accumulating AChE at the NMJ. The attachment of AChE to the BL is primarily accomplished by the binding of the ColQ collagen domain to the heparan sulfate proteoglycan perlecan and the COOH-terminus to the muscle-specific receptor tyrosine kinase (MuSK), which in turn plays a fundamental role in the development and maintenance of the NMJ. Yet, the precise mechanism by which ColQ anchors AChE at the NMJ remains unknown. We identified five novel mutations at the COOH-terminus of ColQ in seven patients from five families affected with endplate (EP) AChE deficiency. We found that the mutations do not affect the assembly of ColQ with AChE to form asymmetric forms of AChE or impair the interaction of ColQ with perlecan. By contrast, all mutations impair in varied degree the interaction of ColQ with MuSK as well as basement membrane extract (BME) that have no detectable MuSK. Our data confirm that the interaction of ColQ to perlecan and MuSK is crucial for anchoring AChE to the NMJ. In addition, the identified COOH-terminal mutants not only reduce the interaction of ColQ with MuSK, but also diminish the interaction of ColQ with BME. These findings suggest that the impaired attachment of COOH-terminal mutants causing EP AChE deficiency is in part independent of MuSK, and that the COOH-terminus of ColQ may interact with other proteins at the BL.     

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.     

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.     

Identification and characterization of a novel splice variant of MuSK

MuSK is a receptor tyrosine kinase that initiates the formation of neuromuscular junctions in response to agrin. Little is known about the ligand-induced activation and kinase-dependent signalling that leads to the clustering of acetylcholine receptors. The ectodomain of these molecule is composed of four Ig-like domains. We describe here the isolation of a novel MuSK splice variant that lacks the third Ig-like domain in its ectodomain. The corresponding RNA is the result of alternative splicing which eliminates two exons. There is 10 times less mRNA for this shorter form than for the long form of MuSK and both forms are regulated coordinately. They decrease strongly after birth and are elevated in denervated muscle. Gene transfer by muscle injection of MuSK DNA into individual muscle fibers demonstrates that kinase-induced acetylcholine receptor clustering caused by overexpression of the two kinases does not depend on the presence of the third Ig-like domain.     

Evolutionary Divergence in the Catalytic Activity of the CAM-1, ROR1 and ROR2 Kinase Domains

Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.     

Agrin binds to the N-terminal region of Lrp4 protein and stimulates association between Lrp4 and the first immunoglobulin-like domain in muscle-specific kinase (MuSK)

Neuromuscular synapse formation depends upon coordinated interactions between motor neurons and muscle fibers, leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. Synapse formation begins as motor axons approach muscles that are prepatterned in the prospective synaptic region in a manner that depends upon Lrp4, a member of the LDL receptor family, and muscle-specific kinase (MuSK), a receptor tyrosine kinase. Motor axons supply Agrin, which binds Lrp4 and stimulates further MuSK phosphorylation, stabilizing nascent synapses. How Agrin binds Lrp4 and stimulates MuSK kinase activity is poorly understood. Here, we demonstrate that Agrin binds to the N-terminal region of Lrp4, including a subset of the LDLa repeats and the first of four β-propeller domains, which promotes association between Lrp4 and MuSK and stimulates MuSK kinase activity. In addition, we show that Agrin stimulates the formation of a functional complex between Lrp4 and MuSK on the surface of myotubes in the absence of the transmembrane and intracellular domains of Lrp4. Further, we demonstrate that the first Ig-like domain in MuSK, which shares homology with the NGF-binding region in Tropomyosin Receptor Kinase (TrKA), is required for MuSK to bind Lrp4. These findings suggest that Lrp4 is a cis-acting ligand for MuSK, whereas Agrin functions as an allosteric and paracrine regulator to promote association between Lrp4 and MuSK.     

Direct transmembrane clustering and cytoplasmic dimerization of focal adhesion kinase initiates its tyrosine phosphorylation

We investigated mechanisms for inducing focal adhesion kinase (FAK) tyrosine phosphorylation and their ability to trigger MAP kinase signaling using transmembrane chimeras that localize FAK and its mutants to the plasma membrane. We tested whether tyrosine phosphorylation was triggered by FAK transmembrane aggregation using antibodies against the chimeric extracellular domain. Experimental clustering of chimeras containing integrin beta cytoplasmic domains or FAK induced FAK tyrosine phosphorylation and trans-phosphorylation of endogenous FAK, as well as strong ERK activation. Next, we examined whether lower-order molecular proximity, namely dimerization, could regulate FAK tyrosine phosphorylation. We found that even relatively low-affinity FAK dimerization (K(d)=3.9 x 10(-5) M), in either of two different orientations, could induce FAK tyrosine phosphorylation. However, this cytoplasmic FAK dimerization could not induce MAP kinase activation or trans-phosphorylation of endogenous FAK. We conclude that dimerization of FAK is sufficient to induce its tyrosine phosphorylation, but that higher-order molecular proximity (clustering) at the cell membrane is apparently needed for additional biochemical events. This study identifies a proximity mechanism for regulating the initiation of FAK-mediated biochemical signaling.     

Implication of geranylgeranyltransferase I in synapse formation

Agrin activates the transmembrane tyrosine kinase MuSK to mediate acetylcholine receptor (AChR) clustering at the neuromuscular junction (NMJ). However, the intracellular signaling mechanism downstream of MuSK is poorly characterized. This study provides evidence that geranylgeranyltransferase I (GGT) is an important signaling component in the Agrin/MuSK pathway. Agrin causes a rapid increase in tyrosine phosphorylation of the alpha(G/F) subunit of GGT and in GGT activity. Inhibition of GGT activity or expression prevents muscle cells from forming AChR clusters in response to Agrin and attenuates the formation of neuromuscular synapses in spinal neuron-muscle cocultures. Importantly, transgenic mice expressing an alpha(G/F) mutant demonstrate NMJ defects with wider endplate bands and smaller AChR plaques. These results support the notion that prenylation is necessary for AChR clustering and the NMJ formation and/or maintenance, revealing an active role of GGT in Agrin/MuSK signaling.     

The neurotrophin receptor, gp75, forms a complex with the receptor tyrosine kinase TrkA

The high-affinity NGF receptor is thought to be a complex of two receptors , gp75 and the tyrosine kinase TrkA, but direct biochemical evidence for such an association had been lacking. In this report, we demonstrate the existence of such a gp75-TrkA complex by a copatching technique. Gp75 on the surface of intact cells is patched with an anti- gp75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with and anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression of wild-type and mutated NGF receptors. TrkA and gp75 copatch in both the absence and presence of NGF. The association is specific, since gp75 does not copatch with other tyrosine kinase receptors, including TrkB, platelet-derived growth factor receptor- beta, and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with gp75. A chimeric receptor with TrkA transmembrane and intracellular domains show partial copatching with gp75. Deletion of the intracellular domain of gp75 decreases but does not eliminate copatching. A point mutation which inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with gp75. Hence, although interactions between the gp75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role.     

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.