Agrin Signaling Research Articles

Research on the distal repair effect of Jiawei Buyang Huanwu Decoction on sciatic nerve injury through agrin signaling pathway

ObjectiveTo study the effects and potential control mechanisms of the agrin signaling pathway on the repair of distal sciatic nerve damage. Methods50 Sprague-Dawleyrats were randomly allocated into the control group (SCI group, n = 25) and the treatment group (Treat group, n = 25). In both groups, the sciatic nerve damage model was created. Following surgery, the SCI group and the Treat group received intragastric administrations of normal saline and Jiawei Buyang Huanwu Decoction, respectively. The dose for both groups was 2mL/(kg·d) for 12 consecutive weeks. At 4, 8, and 12 weeks post-surgery, the wet-to-weight ratio of the gastrocnemius muscle in the two groups was assessed; and hematoxylin-eosin staining and Masson staining were used to detect pathological alterations in the sciatic nerve and collagen. The expression of agrin, Dag1, Dmd, and Lamb2 proteins in the agrin pathway, as well as ERK, MEK, and their corresponding phosphorylated proteins, p-ERK and p-MEK, were all detected by Western blot in the gastrocnemius muscle. ResultsThe wet weight of the gastrocnemius muscle in the Treat group increased considerably (p < 0.05) after 4, 8, and 12 weeks following the operation compared to the SCI group, and it continued to rise over time. The expression level of Dag1, Dmd, and Lamb2 protein in agrin pathway, as well as p-ERK and p-MEK protein were all significantly lower than they were in the SCI group (p < 0.05). Additionally, Jiawei Buyang Huanwu Decoction greatly reduced the amount of nerve scars at the sciatic nerve injury and significantly healed sciatic neuropathy according to the results of pathological staining. ConclusionJiawei Buyang Huanwu Decoction can inhibit the expression of agrin, Dag1, Dmd, and Lamb2, as well as p-ERK and p-MEK. It can effectively promote the distal repair of sciatic nerve injury probably through argin pathway.

DOK7 Promotes NMJ Regeneration After Nerve Injury.

Motor function recovery from injury requires the regeneration of not only muscle fibers, but also the neuromuscular junction-the synapse between motor nerve terminals and muscle fibers. However, unlike muscle regeneration which has been extensively studied, little is known about the molecular mechanisms of NMJ regeneration. Recognizing the critical role of agrin-LRP4-MuSK signaling in NMJ formation and maintenance, we investigated whether increasing MuSK activity promotes NMJ regeneration. To this end, we evaluated the effect of DOK7, a protein that stimulates MuSK, on NMJ regeneration. Reinnervation, AChR cluster density, and endplate area were improved, and fragmentation was reduced in the AAV9-DOK7-GFP-injected muscles compared with muscles injected with AAV9-GFP. These results demonstrated expedited NMJ regeneration associated with increased DOK7 expression and support the hypothesis that increasing agrin signaling benefits motor function recovery after injury. Our findings propose a potentially new therapeutic strategy for functional recovery after muscle and nerve injury, i.e., promoting NMJ regeneration by increasing agrin signaling.

A link between agrin signalling and Cav3.2 at the neuromuscular junction in spinal muscular atrophy

SMN protein deficiency causes motoneuron disease spinal muscular atrophy (SMA). SMN-based therapies improve patient motor symptoms to variable degrees. An early hallmark of SMA is the perturbation of the neuromuscular junction (NMJ), a synapse between a motoneuron and muscle cell. NMJ formation depends on acetylcholine receptor (AChR) clustering triggered by agrin and its co-receptors lipoprotein receptor-related protein 4 (LRP4) and transmembrane muscle-specific kinase (MuSK) signalling pathway. We have previously shown that flunarizine improves NMJs in SMA model mice, but the mechanisms remain elusive. We show here that flunarizine promotes AChR clustering in cell-autonomous, dose- and agrin-dependent manners in C2C12 myotubes. This is associated with an increase in protein levels of LRP4, integrin-beta-1 and alpha-dystroglycan, three agrin co-receptors. Furthermore, flunarizine enhances MuSK interaction with integrin-beta-1 and phosphotyrosines. Moreover, the drug acts on the expression and splicing of Agrn and Cacna1h genes in a muscle-specific manner. We reveal that the Cacna1h encoded protein Cav3.2 closely associates in vitro with the agrin co-receptor LRP4. In vivo, it is enriched nearby NMJs during neonatal development and the drug increases this immunolabelling in SMA muscles. Thus, flunarizine modulates key players of the NMJ and identifies Cav3.2 as a new protein involved in the NMJ biology.

Deconstruction of Neurotrypsin Reveals a Multi-factorially Regulated Activity Affecting Myotube Formation and Neuronal Excitability

Neurotrypsin (NT) is a highly specific nervous system multi-domain serine protease best known for its selective processing of the potent synaptic organizer agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. These biological effects are likely to stem from NT-based regulation of agrin signaling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, due to the scarce molecular detail regarding NT activity and NT-agrin interactions. We developed a strategy to reliably produce and purify a catalytically competent engineered variant of NT called “NT-mini” and a library of C-terminal agrin fragments, with which we performed a thorough biochemical and biophysical characterization of NT enzyme functionality. We studied the regulatory effects of calcium ions and heparin, identified NT’s heparin-binding domain, and discovered how zinc ions induce modulation of enzymatic activity. Additionally, we investigated myotube differentiation and hippocampal neuron excitability, evidencing a dose-dependent increase in neuronal activity alongside a negative impact on myoblast fusion when using the active NT enzyme. Collectively, our results provide in vitro and cellular foundations to unravel the molecular underpinnings and biological significance of NT-agrin interactions.

Increasing LRP4 diminishes neuromuscular deficits in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease characterized by progressive wasting of skeletal muscles. The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, critical for the control of muscle contraction. The NMJ decline is observed in DMD patients, but the mechanism is unclear. LRP4 serves as a receptor for agrin, a proteoglycan secreted by motor neurons to induce NMJ, and plays a critical role in NMJ formation and maintenance. Interestingly, we found that protein levels of LRP4 were reduced both in muscles of the DMD patients and DMD model mdx mice. We explored whether increasing LRP4 is beneficial for DMD and crossed muscle-specific LRP4 transgenic mice with mdx mice (mdx; HSA-LRP4). The LRP4 transgene increased muscle strength, together with improved neuromuscular transmission in mdx mice. Furthermore, we found the LRP4 expression mitigated NMJ fragments and denervation in mdx mice. Mechanically, we showed that overexpression of LRP4 increased the activity of MuSK and expression of dystrophin-associated glycoprotein complex proteins in the mdx mice. Overall, our findings suggest that increasing LRP4 improves both function and structure of NMJ in the mdx mice and Agrin signaling might serve as a new therapeutic strategy in DMD.

APC2CDH1 negatively regulates agrin signaling by promoting the ubiquitination and proteolytic degradation of DOK7.

Neuromuscular junctions (NMJs) are peripheral synapses between motoneurons and skeletal muscle fibers that are critical for the control of muscle contraction. Dysfunction of these synapses has been implicated in congenital myasthenic syndrome (CMS). In vertebrates, agrin-LRP4-MuSK signaling plays a critical role in acetylcholine receptor (AChR) clustering and NMJ formation. The adaptor protein DOK7 is the downstream substrate of MuSK and also a cytoplasmic activator of MuSK. The role of DOK7 in the promotion of AChR clustering and the mechanisms involved have been well studied; however, the negative regulation of DOK7 after MuSK activation remains unknown. Anaphase-promoting complex 2 (APC2), the core subunit of APC/C E3 ligase complex, was originally believed to regulate cell-cycle transitions. Here, we show that APC2 is enriched at post-synapse of NMJs in postmitotic myotubes. In response to agrin stimulation, APC2 negatively regulates AChR clustering by promoting the ubiquitination of DOK7 at lysine 243 for its proteolytic degradation, which relies on MuSK kinase activity and the phosphorylation of tyrosine 106 in DOK7. Thus, this study provides a mechanism whereby agrin signaling is negatively regulated as part of vertebrate NMJ homeostasis.

Engineering 3D skeletal muscle primed for neuromuscular regeneration following volumetric muscle loss

Volumetric muscle loss (VML) overwhelms the native regenerative capabilities of skeletal muscle and has few effective treatments to regain lost muscle mass and function. Tissue engineered muscle constructs designed to promote neuromuscular regeneration are a promising therapeutic avenue. To date, there has been no engineered muscle construct for VML treatment that has incorporated a pharmacologic agent to promote neuromuscular regeneration. Here, we have modified electrospun fibrin microfiber bundles, which have demonstrated muscle regenerative potential, with the heparan sulfate proteoglycan, agrin, to stimulate innervation post-VML. Myoblasts cultured on microfiber bundles with either soluble or chemically tethered agrin demonstrated statistically significant increased clustering of acetylcholine receptors (AChRs) with soluble agrin displaying AChR clusters throughout the myofiber bundles, and tethered agrin displaying AChR clusters only at 10 μm from the substrate surface. Following implantation into murine VML defects for 4 weeks, constructs pre-treated with soluble or tethered agrin resulted in statistically significant increased neuromuscular junctions, regenerating myofibers, vascular infiltration, neural infiltration, and nuclear yes-associated protein (YAP) expression within the defect site compared to the control without agrin. The agrin-tethered microfiber bundles provided sustained agrin signaling within the regenerating site during the 4-week post-implantation periods and further augmented the density of regenerating myofibers in regenerated tissue with statistical significance compared to constructs with soluble agrin. These data demonstrate the neuromuscular regenerative potential of engineered muscle constructs pre-treated to induce AChR clustering with locally delivered agrin at the site of VML regeneration.

Src Family Kinases Play a Role in the Functional Clustering of Central Postsynaptic Nicotinic Acetylcholine Receptors

The mechanism of postsynaptic neurotransmitter receptor clustering has been best described at the neuromuscular junction (NMJ), where packets of nicotinic acetylcholine receptors (nAChRs) in muscle fibre are redistributed to the synaptic site upon motor neuron innervation. This process of receptor localisation is facilitated by agrin signalling, and the stability of the resulting nAChR clusters depends upon the activation of Src family kinases (SFKs). In contrast to the NMJ however, the cellular signalling mechanisms orchestrating the clustering of nAChRs in the central nervous system (CNS) remain poorly defined. Furthermore, our understanding of the role of SFKs in the CNS is also limited. Here we provide evidence that SFK activation is required for synapse formation between pairs of identified neurons isolated from the CNS of Lymnaea stagnalis. Furthermore, we suggest that SFKs are involved in the functional redistribution of nAChRs to the synaptic contact sites in isolated axons. To the best of our knowledge, this study is the first to demonstrate a role for SFKs in the clustering of nAChRs in central neurons, suggesting that the mechanisms of receptor clustering between the peripheral and central nervous system are likely conserved.

A mechanism in agrin signaling revealed by a prevalent Rapsyn mutation in congenital myasthenic syndrome.

Neuromuscular junction is a synapse between motoneurons and skeletal muscles, where acetylcholine receptors (AChRs) are concentrated to control muscle contraction. Studies of this synapse have contributed to our understanding of synapse assembly and pathological mechanisms of neuromuscular disorders. Nevertheless, underlying mechanisms of NMJ formation was not well understood. To this end, we took a novel approach - studying mutant genes implicated in congenital myasthenic syndrome (CMS). We showed that knock-in mice carrying N88K, a prevalent CMS mutation of Rapsyn (Rapsn), died soon after birth with profound NMJ deficits. Rapsn is an adapter protein that bridges AChRs to the cytoskeleton and possesses E3 ligase activity. In investigating how N88K impairs the NMJ, we uncovered a novel signaling pathway by which Agrin-LRP4-MuSK induces tyrosine phosphorylation of Rapsn, which is required for its self-association and E3 ligase activity. Our results also provide insight into pathological mechanisms of CMS.

Schwann cell-specific PTEN and EGFR dysfunctions affect neuromuscular junction development by impairing Agrin signaling and autophagy

The neuromuscular junction (NMJ) is formed by motor nerve terminals, post-junctional muscle membranes, and terminal Schwann cells (SCs). The formation of NMJ requires complex and dynamic molecular interactions. Nerve- and muscle-derived molecules have been well characterized but the mechanistic involvement of SC in NMJ development remains poorly understood. SC-specific phosphatase and tensin homolog (Pten) inactivation and epidermal growth factor receptor (EGFR) overexpression (Dhh-Cre; Cnp-EGFR; Ptenflox/flox or DET) mice were used and NMJ malformation was observed in these mice. Acetylcholine receptors (AChRs) were distorted and varicose presynaptic nerve terminals appeared in the tibialis anterior (TA) muscle of DET mice. Agrin signaling related to NMJ development, was downregulated in TA muscle. Both RAS/MEK/ERK and PI3K/AKT/mTOR signaling pathways were activated in the sciatic nerves of DET mice. In addition, autophagy was downregulated in these sciatic nerves. Interestingly, the use of Torin 2, an mTOR inhibitor, rescued the phenotype. The downregulated-autophagy might account for Agrin signaling abnormity, which induced NMJ malformation. Taken together, our results indicate that SCs-specific Pten and EGFR cooperation are essential for NMJ development.

Lipoprotein receptor‐related protein 4 autoantibodies in myasthenia gravis: Where are we and where are we going?

AbstractMyasthenia gravis (MG) is an autoimmune disorder of the neuromuscular junction that leads to fatigue of skeletal muscles and fluctuating weakness. The pathogenesis of MG is sufficiently diverse to include autoimmune mechanisms. In most patients with MG, autoantibodies against acetylcholine receptor can be detected. In a smaller proportion of patients without anti‐acetylcholine receptor antibodies, autoantibodies to muscle‐specific kinase and low‐density lipoprotein receptor‐related protein 4 (LRP4) are present. LRP4 is critical for neuromuscular junction formation and maintenance. A multimolecular complex is formed among agrin, LRP4 and muscle‐specific kinase that is critical for agrin signaling. The presence of anti‐LRP4 antibodies is associated with mild symptoms of MG, while thymomas are rarely observed in association with MG and LRP4 antibodies. We review recent findings on the pathogenesis, clinical features and treatment of LRP4 MG.

Agrin inhibits canonical WNT signalling through LRP4

Purpose: Osteoarthritis (OA) is a leading cause of disability for which there is no cure. The identification of molecules supporting cartilage homeostasis and regeneration is therefore a major pursuit in musculoskeletal medicine. Agrin is a heparan sulfate proteoglycan which, through binding to low-density lipoprotein receptor-related protein 4 (LRP4), is required for neuromuscular synapse formation. We recently identified Agrin as an essential molecule in articular cartilage homeostasis, uniquely requiring both α-dystroglycan and LRP4 to support SOX9 expression and chondrocyte differentiation in vitro and in vivo. Despite identifying α-dystroglycan and LRP4 as necessary for the chondrogenic effects of Agrin in chondrocytes, the signalling pathway(s) by which this occurs remains unclear. LRP4 signalling is known to negatively regulate WNT signalling. Therefore, we tested whether Agrin is required for the WNT-inhibitory properties of LRP4 and whether this is the mechanism by which Agrin signals in chondrocytes. Methods: Mammalian Agrin or GFP was overexpressed in bovine primary chondrocytes and cultured in micromass for 5 days in the presence or absence of 100ng/ml WNT3a. Chondrogenic potential was assessed by qPCR for SOX9 mRNA expression. Agrin or GFP stably expressing COS7 cells were transfected with the SUPER8TOPFLASH reporter plasmid in combination with Vehicle or LRP4 plasmids. COS7 monolayer cultures were treated with vehicle or increasing doses of WNT3a (50, 100, 200ng/ml). WNT signalling activation was determined by luciferase expression. Results: LRP4 overexpression was sufficient to induce SOX9 upregulation in chondrocytes, however this effect was abolished following Agrin knockdown demonstrating the requirement of Agrin for the chondrogenic effect of LRP4 signalling. In addition, downregulation of SOX9 mRNA expression induced by WNT3a treatment was rescued by Agrin overexpression in micromass cultures. Using the WNT reporter assay, activation of canonical WNT signalling was inhibited in COS7 cells transduced with human Agrin compared to GFP-transduced control COS7 cells; and this effect was exacerbated by overexpression of LRP4. Conclusions: Agrin inhibits canonical WNT signalling through LRP4 and enhance chondrocyte differentiation. However, it remains unclear how Agrin-LRP4 signalling achieves its specificity in promoting chondrogenesis compared to other LRP4 ligands.

An oncogenic role of Agrin in regulating focal adhesion integrity in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths globally. The identity and role of cell surface molecules driving complex biological events leading to HCC progression are poorly understood, hence representing major lacunae in HCC therapies. Here, combining SILAC quantitative proteomics and biochemical approaches, we uncover a critical oncogenic role of Agrin, which is overexpressed and secreted in HCC. Agrin enhances cellular proliferation, migration and oncogenic signalling. Mechanistically, Agrin’s extracellular matrix sensor activity provides oncogenic cues to regulate Arp2/3-dependent ruffling, invadopodia formation and epithelial–mesenchymal transition through sustained focal adhesion integrity that drives liver tumorigenesis. Furthermore, Agrin signalling through Lrp4-muscle-specific tyrosine kinase (MuSK) forms a critical oncogenic axis. Importantly, antibodies targeting Agrin reduced oncogenic signalling and tumour growth in vivo. Together, we demonstrate that Agrin is frequently upregulated and important for oncogenic property of HCC, and is an attractive target for antibody therapy.

Plastic Additives Decrease Agrin-Induced Acetylcholine Receptor Clusters and Myotube Formation in C2C12 Skeletal Muscle Cell Culture

Common additives in plastics such as bisphenol A (BPA) or phthalates like di-(2-ethylhexyl) phthalate (DEHP) are environmental estrogens that have been shown to be endocrine disruptors in some experimental animal models. This project used the C2C12 cell culture model to examine how exposure to BPA or DEHP affects two aspects of skeletal muscle development, the fusion of myoblasts into myotubes and agrin-induced clustering of acetylcholine receptors (AChRs). During myotube formation AChRs cluster spontaneously. Treatment with motor neuron derived agrin increases the frequency of AChR clusters through an agrin signaling pathway that also clusters other postsynaptic components of the neuromuscular synapse. For this project C2C12 cells were exposed to BPA or DEHP while myoblasts fused into myotubes. After exposure to 10 μM BPA or 100 μM DEHP the frequency of agrin-induced AChR clusters decreased. In addition, myotube formation decreased as a higher percentage of nuclei remained in myoblasts. Furthermore, BPA or DEHP reduced the amount of the myogenic regulatory factor myogenin. This suggests that BPA and DEHP decrease AChR clustering by reducing myogenin. Moreover, plastic additives like BPA and DEHP may pose a risk for skeletal muscle development in humans.

LRP4 is critical for neuromuscular junction maintenance.

The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires neuronal agrin that acts by binding to LRP4 to stimulate MuSK. Mutations have been identified in agrin, MuSK, and LRP4 in patients with congenital myasthenic syndrome, and patients with myasthenia gravis develop antibodies against agrin, LRP4, and MuSK. However, it remains unclear whether the agrin signaling pathway is critical for NMJ maintenance because null mutation of any of the three genes is perinatal lethal. In this study, we generated imKO mice, a mutant strain whose LRP4 gene can be deleted in muscles by doxycycline (Dox) treatment. Ablation of the LRP4 gene in adult muscle enabled studies of its role in NMJ maintenance. We demonstrate that Dox treatment of P30 mice reduced muscle strength and compound muscle action potentials. AChR clusters became fragmented with diminished junctional folds and synaptic vesicles. The amplitude and frequency of miniature endplate potentials were reduced, indicating impaired neuromuscular transmission and providing cellular mechanisms of adult LRP4 deficiency. We showed that LRP4 ablation led to the loss of synaptic agrin and the 90 kDa fragments, which occurred ahead of other prejunctional and postjunctional components, suggesting that LRP4 may regulate the stability of synaptic agrin. These observations demonstrate that LRP4 is essential for maintaining the structural and functional integrity of the NMJ and that loss of muscle LRP4 in adulthood alone is sufficient to cause myasthenic symptoms.

AChR Clustering is Increased by Neuregulin

In skeletal muscle development, agrin is released by the approaching motor neuron prior to innervation and the formation of the neuromuscular synapse. Agrin induces the clustering of existing acetylcholine receptors (AChRs) at the site of the future neuromuscular synapse through an agrin signaling pathway. Neuregulin is also released by the approaching motor neuron and induces the production of additional AChRs, especially at the nuclei near the future neuromuscular synapse. Our lab investigates myogenesis and neuromuscular synapse formation using the C2C12 myotube cell culture model. C2C12 myoblasts proliferate in growth medium, and then fuse into myotubes when switched to low‐serum differentiation medium (DM) for 72 hours. Myotubes are stimulated to cluster AChRs by the addition of agrin for the last 16 hours in DM. Our hypothesis was that neuregulin would increase AChR clustering in C2C12 myotubes as previously demonstrated in another cell line. Concentrations of agrin or neuregulin as low as 10 ng/ml was sufficient to increase AChR clustering. We conclude that AChR clustering in C2C12 myotubes is increased either by utilizing existing AChRs or the production of additional AChRs.Grant Funding Source: None

Role of extracellular matrix proteins and their receptors in the development of the vertebrate neuromuscular junction

The vertebrate neuromuscular junction (NMJ) remains the best-studied model for understanding the mechanisms involved in synaptogenesis, due to its relatively large size, its simplicity of patterning, and its unparalleled experimental accessibility. During neuromuscular development, each skeletal myofiber secretes and deposits around its extracellular surface an assemblage of extracellular matrix (ECM) proteins that ultimately form a basal lamina. This is also the case at the NMJ, where the motor nerve contributes additional factors. Before most of the current molecular components were known, it was clear that the synaptic ECM of adult skeletal muscles was unique in composition and contained factors sufficient to induce the differentiation of both pre- and postsynaptic membranes. Biochemical, genetic, and microscopy studies have confirmed that agrin, laminin (221, 421, and 521), collagen IV (α3-α6), collagen XIII, perlecan, and the ColQ-bound form of acetylcholinesterase are all synaptic ECM proteins with important roles in neuromuscular development. The roles of their many potential receptors and/or binding proteins have been more difficult to assess at the genetic level due to the complexity of membrane interactions with these large proteins, but roles for MuSK-LRP4 in agrin signaling and for integrins, dystroglycan, and voltage-gated calcium channels in laminin-dependent phenotypes have been identified. Synaptic ECM proteins and their receptors are involved in almost all aspects of synaptic development, including synaptic initiation, topography, ultrastructure, maturation, stability, and transmission.

A valid mouse model of AGRIN-associated congenital myasthenic syndrome

Congenital myasthenic syndromes (CMS) are inherited diseases affecting the neuromuscular junction (NMJ). Mutations in AGRIN (AGRN) and other genes in the AGRIN signaling pathway cause CMS, and gene targeting studies in mice confirm the importance of this pathway for NMJ formation. However, these mouse mutations are complete loss-of-function alleles that result in an embryonic failure of NMJ formation, and homozygous mice do not survive postpartum. Therefore, mouse models of AGRIN-related CMS that would allow preclinical testing or studies of postnatal disease progression are lacking. Using chemical mutagenesis in mice, we identified a point mutation in Agrn that results in a partial loss-of-function allele, creating a valid model of CMS. The mutation changes phenylalanine 1061 to serine in the SEA domain of AGRIN, a poorly characterized motif shared by other extracellular proteoglycans. NMJs in homozygous mice progressively degrade postnataly. Severity differs with genetic background, in different muscles, and in different regions within a muscle in a pattern matching mouse models of motor neuron disease. Mutant NMJs have decreased acetylcholine receptor density and an increased subsynaptic reticulum, evident by electron microscopy. Synapses eventually denervate and the muscles atrophy. Molecularly, several factors contribute to the partial loss of AGRIN's function. The mutant protein is found at NMJs, but is processed differently than wild-type, with decreased glycosylation, changes in sensitivity to the protease neurotrypsin and other proteolysis, and less efficient externalization and secretion. Therefore, the Agrn point mutation is a model for CMS caused by Agrn mutations and potentially other related neuromuscular diseases.

Modulation of Agrin-induced Acetylcholine Receptor Clustering by Extracellular Signal-regulated Kinases 1 and 2 in Cultured Myotubes

Agrin released by motoneurons induces and/or maintains acetylcholine receptor (AChR) clustering and other aspects of postsynaptic differentiation at the vertebrate neuromuscular junction. Agrin acts by binding and activating a receptor complex containing LDL receptor protein 4 (Lrp4) and muscle-specific kinase (MuSK). Two critical downstream components of this signaling cascade, Dox-7 and rapsyn, have been identified. However, additional intracellular essential elements remain unknown. Prior observations by others and us suggested antagonistic interactions between agrin and neuregulin-1 (Nrg-1) signaling in cultured myotubes and developing muscle fibers in vivo. A hallmark of Nrg-1 signaling in skeletal muscle cells is the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2 are also activated in most cells by phorbol 12-myristate 13-acetate, a classical inhibitor of agrin-induced AChR clustering in myotubes. Here, it was investigated whether agrin activates ERK1/2 directly and whether such activation modulates agrin-induced AChR clustering. Agrin induced a rapid but transient activation of ERK1/2 in myotubes that was Lrp4/MuSK-dependent. However, blocking this ERK1/2 activation did not prevent but potentiated AChR clustering induced by agrin. ERK1/2 activation was dispensable for Nrg-1-mediated inhibition of the AChR clustering activity of agrin, but was indispensable for such activity by phorbol 12-myristate 13-acetate. Together, these results suggest agrin-induced activation of ERK1/2 is a negative modulator of agrin signaling in skeletal muscle cells.

How are ion pumps and agrin signaling integrated?

Na(+),K(+)-ATPase (NKA) has a fundamental role in ion transport across the plasma membrane of animal cells and uses approximately 50% of brain energy consumption. Recent work has uncovered additional roles for NKA in signal transduction. How might such different functions of the sodium-potassium pump be connected and regulated? We envision an integrated model of ion pumping and signaling, considering in particular the recently discovered regulation of the sodium-potassium pump by agrin, a protein that is cleaved specifically by neurotrypsin at the synapse. Based on the recently solved structure of NKA and sequence analysis, we propose a molecular model for the agrin-NKA interaction, in which agrin displaces the NKA β-subunit and exploits the ouabain-binding pocket.