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Rapsyn may function as a link between the acetylcholine receptor and the agrin-binding dystrophin-associated glycoprotein complex

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Abstract

The 43 kDa AChR-associated protein rapsyn is required for the clustering of nicotinic acetylcholine receptors (AChRs) at the developing neuromuscular junction, but the functions of other postsynaptic proteins colocalized with the AChR are less clear. Here we use a fibroblast expression system to investigate the role of the dystrophin-glycoprotein complex (DGC) in AChR clustering. The agrin-binding component of the DGC, dystroglycan, is found evenly distributed across the cell surface when expressed in fibroblasts. However, dystroglycan colocalizes with AChR-rapsyn clusters when these proteins are coexpressed. Furthermore, dystroglycan colocalizes with rapsyn clusters even in the absence of AChR, indicating that rapsyn can cluster dystroglycan and AChR independently. Immunofluorescence staining using a polyclonal antibody to utrophin reveals a lack of staining of clusters, suggesting that the immunoreactive species, like the AChR, does not mediate the observed rapsyndystroglycan interaction. Rapsyn may therefore be a molecular link connecting the AChR to the DGC. At the neuromuscular synapse, rapsyn-mediated linkage of the AChR to the cytoskeleton-anchored DGC may underlie AChR cluster stabilization.

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... RAPSN encodes the 43 kDa receptor-associated scaffold protein of the synapse or rapsyn, which is essential for the postsynaptic specialization of the NMJ . Rapsyn is enriched at the postsynaptic membrane, acting as a linker between the AChRs and the cytoskeleton via the dystrophinassociated glycoprotein complex (Apel et al., 1995;Moransard et al., 2003). Early cross-linking studies showed that rapsyn is located in close proximity to the AChR-β subunit (Burden et al., 1983). ...
... However, the lack of a crystallographic structure means that the detailed composition of the AChRsrapsyn network is not well understood (Zuber and Unwin, 2013). Rapsyn is composed of an N-terminal myristoylation moiety (N-Myr) necessary for submembrane localization; seven tetratricopeptide (TRP) domains responsible for rapsyn selfassociation (Ramarao et al., 2001;Lee et al., 2008); a coiled-coil domain that binds to the cytoplasmic loops of AChRs , and a RING-H2 domain that links rapsyn to the cytoskeleton through its interaction with the dystroglycan complex ( Figure 3D; Apel et al., 1995). RAPSN mutations identified in humans are found along the length of the gene and the common p.N88K is located within the TRP domains . 1) is a large proteoglycan (>200 KDa) with multiple domains that binds to laminins through the N-terminal domain, and to LRP4 and α-dystroglycan via C-terminus. ...
... Dashes are used to mark the different exons. (D) Rapsyn (RefSeq NP_005046.2) is composed of a N-terminal myristoylation moiety (N-Myr) necessary for submembrane localization; seven tetratricopeptide (TRP) domains responsible for rapsyn self-association and MuSK binding (Ramarao et al., 2001;Lee et al., 2008); a coiled-coil domain that binds to the cytoplasmic loops of AChRs , and a RING-H2 domain that interacts with the dystroglycan complex and links with the cytoskeleton (Apel et al., 1995). ...
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The neuromuscular junction (NMJ) is a highly specialized synapse between a motor neuron nerve terminal and its muscle fiber that are responsible for converting electrical impulses generated by the motor neuron into electrical activity in the muscle fibers. On arrival of the motor nerve action potential, calcium enters the presynaptic terminal, which leads to the release of the neurotransmitter acetylcholine (ACh). ACh crosses the synaptic gap and binds to ACh receptors (AChRs) tightly clustered on the surface of the muscle fiber; this leads to the endplate potential which initiates the muscle action potential that results in muscle contraction. This is a simplified version of the events in neuromuscular transmission that take place within milliseconds, and are dependent on a tiny but highly structured NMJ. Much of this review is devoted to describing in more detail the development, maturation, maintenance and regeneration of the NMJ, but first we describe briefly the most important molecules involved and the conditions that affect their numbers and function. Most important clinically worldwide, are myasthenia gravis (MG), the Lambert-Eaton myasthenic syndrome (LEMS) and congenital myasthenic syndromes (CMS), each of which causes specific molecular defects. In addition, we mention the neurotoxins from bacteria, snakes and many other species that interfere with neuromuscular transmission and cause potentially fatal diseases, but have also provided useful probes for investigating neuromuscular transmission. There are also changes in NMJ structure and function in motor neuron disease, spinal muscle atrophy and sarcopenia that are likely to be secondary but might provide treatment targets. The NMJ is one of the best studied and most disease-prone synapses in the nervous system and it is amenable to in vivo and ex vivo investigation and to systemic therapies that can help restore normal function.
... The ability of syntrophin to link NaChs to dystrophin and the DAPC is reminiscent of the AChRassociated protein rapsyn. Cotransfection experiments have demonstrated that rapsyn can cluster AChRs (Froehner et al., 1990;Phillips et al., 1991;Apel et al., 1995) and ␣-and ␤-dystroglycan (Apel et al., 1995(Apel et al., , 1997, extracellular and transmembrane proteins, respectively, of the DAPC (Ervasti and C ampbell, 1991). Thus, rapsyn may f unction as a link between AChRs and the DAPC. ...
... The ability of syntrophin to link NaChs to dystrophin and the DAPC is reminiscent of the AChRassociated protein rapsyn. Cotransfection experiments have demonstrated that rapsyn can cluster AChRs (Froehner et al., 1990;Phillips et al., 1991;Apel et al., 1995) and ␣-and ␤-dystroglycan (Apel et al., 1995(Apel et al., , 1997, extracellular and transmembrane proteins, respectively, of the DAPC (Ervasti and C ampbell, 1991). Thus, rapsyn may f unction as a link between AChRs and the DAPC. ...
... There are two routes by which laminin, acting through DG, may aggregate MuSK along with AChRs to achieve a density high enough to lead to MuSK dimerization and activation. First, DG interacts intracellularly with rapsyn [4,11] which self assembles and aggregates AChRs as well as DG [30]. Rapsyn interacts, in turn, with AChRs and AChRs with MuSk ( [3,4,30,31], Fuhrer, [88]). ...
... First, DG interacts intracellularly with rapsyn [4,11] which self assembles and aggregates AChRs as well as DG [30]. Rapsyn interacts, in turn, with AChRs and AChRs with MuSk ( [3,4,30,31], Fuhrer, [88]). Thus binding of DG to laminin, which has the ability to polymerize in the ECM, could cause aggregation of DG in the plasma membrane and its associated proteins. ...
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Previous work has shown that myotubes cultured on laminin-coated substrates form complex aggregates of synaptic proteins that are similar in shape and composition to neuromuscular junctions (NMJs). Here we show that laminin instructs the location of complex aggregates which form only on the lower surface when laminin is coated onto culture dishes but over the entire cell when laminin is added in solution. Silencing of myotubes by agents that block electrical activity (tetrodotoxin, verapamil) or by inhibitors of calmodulin dependent kinase (CaMKII) render the myotube permissive for the formation of complex aggregates. Treatment with laminin alone will facilitate the formation of complex aggregates hours later when myotubes are made permissive by inhibiting CaMKII. The AChR agonist carbachol disperses pre formed aggregates suggesting that non-permissiveness may involve active dispersal of AChRs. The permissive period requires ongoing protein synthesis. The latter may reflect a requirement for rapsyn, which turns over rapidly, and is necessary for aggregation. Consistent with this geldanamycin, an agent that increases rapsyn turnover disrupts complex aggregates. Agrin is well known to induce small clusters of AChRs but does not induce complex aggregates even though aggregate formation requires MuSK, a receptor tyrosine kinase activated by agrin. Dystroglycan (DG) is the major laminin receptor mediating complex aggregate formation with some contribution from β1 integrins. In addition, there is a pool of CaMKII associated with DG. We discuss how these permissive and instructive mechanisms bear on NMJ formation in vivo.
... The first Ig-like domain is required for agrin responsiveness . MuSK mutants without the fourth Ig-like domain don't co-cluster with rapsyn, suggesting that this domain may be involved in interacting with rapsyn (indirectly via a hypothetic protein RATL, for rapsyn associated transmembrane linker) (Apel et al., 1995). Other noticeable domains include a conserved cysteine rich domain (CRD) known as C6-box, which shows homology to frizzled, a wnt receptor (Masiakowski and Yancopoulos, 1998;Saldanha et al., 1998) and a kringle domain of unknown function that is present in MuSK of torpedo, chick and Xenopus but absent from mammalian homologues (Jennings et al., 1993;Fu et al., 1999;Ip et al., 2000). ...
... Co-expression of rapsyn with AChRs in heterologous cells leads to aggregation of the receptors, which are diffusely distributed when expressed on their own (Froehner et al., 1990;Phillips et al., 1991a). When coexpressed in QT-6 fibroblasts, rapsyn is also able to aggregate MuSK and dystroglycan and to activate MuSK (Apel et al., 1995;Gillespie et al., 1996;Apel et al., 1997), suggesting that rapsyn is a general mediator of protein clustering by forming intracellular aggregates and by recruiting other proteins to the MuSK scaffold. ...
Article
Muscle specific tyrosine kinase (MuSK) has been shown to be expressed in muscle fiber in which it mediates the formation of neuromuscular junctions. In this study we show that MuSK is expressed in the central nervous system (CNS), particularly in the brain and eye of rodents. In the retina MuSK was expressed in astrocytes between postnatal days 7 and 14, i.e. at the time when the eyes open. Interestingly, agrin an activator of MuSK in muscle cells was also detected in the CNS. We found that agrin was localized adjacent to MuSK-expressing astrocytes which in turn were detected close to the inner limiting membrane of the rodent retina. These findings raise an interesting possibility that, in addition to the known function in the formation of the neuromuscular junctions, MuSK may be involved in neural development. To get new insight into the functions of MuSK, a yeast-two-hybrid approach was undertaken to identify partners and/or effectors of MuSK. Two of the identified proteins interacting with the intracellular domain of MuSK were the beta subunit of the protein kinase CK2 (CK2 beta) and the carboxy-terminal part of ErbB2 interacting protein (Erbin). Further studies have shown that not only the regulatory beta subunit but also the catalytic beta subunit of CK2 interact with MuSK. Epitope-mapping studies define the area of Erbin comprising amino acid residues between 1175 and 1229 in the carboxy terminus of the protein is necessary for its interaction with MuSK
... a-dystrogl ycan binds to merosin in the extra-cellular matrix (Ervasti and Campbell, 1993), while &dystroglycan binds to the W W motif of dystrophin (Jung et al., 1995). In addition to this structural rote, the dystroglycans have also been postulated to be involved in the Rcniitrnent of proteins involved in signal transduction (Apel et al., 1995;Jung et al., 1995). In contrast to the dystroglycans, the sarcoglycans are poorly defineci, and little is known about their functioa The sarcoglycan complex is composed of a-, f3-, y-, 6and e-sarcoglycan, as well as a channel protein cailed sarcospan (Crosbie et al., 1997). ...
... In cardiology, a large number of genes (Shy et al., 2013), including SNTA1 (Wu et al., 2008), MOG1 (Kattygnarath et al., 2011), andGP1DL (Van Norstrand et al., 2007) have been shown to interact with Na v 1.5, and have been implicated in causing Brugada and long-QT syndromes. In neuromuscular disease, rapsyn, encoded by the RAPSN gene, is known to play a major role in the localisation of skeletal muscle acetylcholine receptors (Apel et al., 1995), and RAPSN mutations are associated with congenital myaesthenic syndrome (Ohno et al., 2002) and fatal akinesia deformation sequence (Michalk et al., 2009). ...
Article
The genetic channelopathies are a broad collection of diseases. Many ion channel genes demonstrate wide phenotypic pleiotropy, but nonetheless concerted efforts have been made to characterise genotype-phenotype relationships. In this review we give an overview of the factors that influence genotype-phenotype relationships across this group of diseases as a whole, using specific individual channelopathies as examples. We suggest reasons for the limitations observed in these relationships. We discuss the role of ion channel variation in polygenic disease and highlight research that has contributed to unravelling the complex aetiological nature of these conditions. We focus specifically on the quest for modifying genes in inherited channelopathies, using the voltage-gated sodium channels as an example. Epilepsy related to genetic channelopathy is one area in which precision medicine is showing promise. We will discuss the successes and limitations of precision medicine in these conditions.
... Agrin activates a small GTPase Cdc42 that, in turn, regulates an Arp2/3 complex to promote actin polymerization through the activation of Wiskott-Aldrich Syndrome protein (WASp) [27]. The postsynaptic stabilization of AChR clusters is achieved by the rapsynmediated linkage of the AChR to the cytoskeleton-anchored dystrophin-glycoprotein complex [28]. Apart from serving as a stable cytoskeletal scaffold for the docking and anchoring of structural and signaling molecules, our previous studies have provided definitive evidence to show that another dynamic pool of F-actin, regulated by actin depolymerizing factor (ADF)/cofilin, actively facilitates the site-directed delivery of AChRs to the nascent postsynaptic sites by modulating dynamic actin turnover locally. ...
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Purpose of Review At present, the most common diagnostic measures for the autoimmune neuromuscular disease myasthenia gravis (MG) are radioimmunoprecipitation assay (RIPA), enzyme-linked immunosorbent assay (ELISA), and cell-based assay (CBA). Considering the pitfalls of these diagnostic assays, this review describes the advantages of using Xenopus tissue cultures for MG diagnosis and research. Recent Findings Our recent study described a novel CBA involving Xenopus tissue cultures for MG serological diagnosis. Moreover, this CBA can potentially be applied to elucidate the pathogenic mechanisms underlying acetylcholine receptor endocytosis and degradation and to develop and validate potential therapeutic strategies for MG. Summary Although most CBAs are relatively labor intensive, Xenopus CBA is a promising tool for the initial clinical serological diagnosis and for the pathological research of MG. The future studies will be devoted to gain a better understanding of the etiology of MG and to provide a therapeutic intervention for this disease.
... Similarly, nAChR packing density in purified Torpedo synaptic membranes also correlates with rapsyn abundance [80]. Second, rapsyn anchors the nAChR to several components of the postsynaptic cytoskeleton and also to transmembrane scaffolding proteins [35,57,[59][60][61][62]91]. Again, this is likely enhanced by higher stoichiometries of rapsyn binding, which could allow for more links to the postsynaptic scaffold ( Figure 2C). ...
Article
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Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission at neuromuscular and autonomic ganglionic synapses in the peripheral nervous system. The postsynaptic localization of muscle ((α1)2β1γδ) and neuronal ((α3β4)2β4) nicotinic receptors at these synapses is mediated by interactions between the nAChR intracellular domains and cytoplasmic scaffolding proteins. Recent high resolution structures and functional studies provide new insights into the molecular determinants that mediate these interactions. Surprisingly, they reveal that the muscle nAChR binds 1–3 rapsyn scaffolding molecules, which dimerize and thereby form an interconnected lattice between receptors. Moreover, rapsyn binds two distinct sites on the nAChR subunit cytoplasmic loops; the MA-helix on one or more subunits and a motif specific to the β subunit. Binding at the latter site is regulated by agrin-induced phosphorylation of βY390, and increases the stoichiometry of rapsyn/AChR complexes. Similarly, the neuronal nAChR may be localized at ganglionic synapses by phosphorylation-dependent interactions with 14-3-3 adaptor proteins which bind specific motifs in each of the α3 subunit cytoplasmic loops. Thus, postsynaptic localization of nAChRs is mediated by regulated interactions with multiple scaffolding molecules, and the stoichiometry of these complexes likely helps regulate the number, density, and stability of receptors at the synapse.
... Rapsyn links the receptor to the cytoskeleton-anchored dystrophin-associated glycoprotein complex (Apel et al, 1995). Clustering of the nicotinic receptors at the neuromuscular junction is dependent on the presence of rapsyn (Phillips et al, 1991;Phillips et al, 1993;Scodand et al, 1993). ...
... More recently, we have found that rapsyn and α-dbn are also reduced in α-syn -/muscles (Martinez-Pena y . The presence of fewer rapsyn molecules at the α-syn -/-NMJ could be a consequence of the loss of utrophin, as rapsyn is known to associate the DGC and nAChRs through utrophin (Apel et al., 1995;Cartaud et al., 1998). ...
Article
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The effectiveness of synaptic transmission at most mammalian synapses depends largely on the maintenance of a high density of postsynaptic receptors. In a mature synapse, this density is highly dynamic and can be regulated by several factors including synaptic activity, post-translational modifications of receptors, and scaffold proteins. In my thesis work, I focused on the regulation of AChR clustering, which is the hallmark of a neuromuscular junction, a well characterized cholinergic synapse between the motor neuron and the skeletal muscle. Among several pathways, I first focused on the role of alpha-syntrophin (syn), a member of the dystrophin glycoprotein complex (DGC), in the development and modulation of nAChR dynamics of the mouse NMJ. Using syn knock-out mice, I showed that syn is not required for synapse formation, but it is essential for synapse maturation. Particularly, I demonstrated that during the maturation of synapses, the integrity of the postsynaptic apparatus is altered, the turnover rate of AChRs increases significantly, and the number/density of AChRs is impaired. The synaptic alterations observed in this mouse mutant were explained by the loss of tyrosine phosphorylated alpha-dystrobrevin (dbn). Interestingly, when GFP-dbn1 was electroporated into sternomastoid muscles of syn mutant, most of synaptic abnormalities were partially restored. In the second part of my thesis work, I investigated the role of serine/threonine kinases, particularly PKC and PKA on the regulation of nAChR trafficking. We found that PKC accelerates nAChR removal and inhibits recycling at the NMJ, while PKA has the opposite effect. Finally, I begin to address the role of the Wnt/beta-catenin pathway in the adult NMJ, and we show that beta-catenin interacts with the DGC in mature synapses, via rapsyn. Taken together, these results provide new insights into the cellular and molecular underlying signaling of the regulation of nAChR trafficking and dynamics.
... The causative RAPSN gene encodes a 43-KD (Kilodalton) protein that connects AChR at the neuromuscular junction, thereby stabilizing AChR clustering [8]. Mutations in this gene are associated with AChR deficiency [7]. ...
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We highlight the importance of exome sequencing in solving a clinical case of a child who died at 14 months after a series of respiratory crises. He was the half-brother of a girl diagnosed at 7 years with the early-onset seizure variant of Rett syndrome due to CDKL5 mutation. We performed a test for CDKL5 in the boy, which came back negative. Driven by the mother's compelling need for a diagnosis, we moved forward performing whole exome sequencing analysis. Surprisingly, two missense mutations in compound heterozygosity were identified in the RAPSN gene encoding a receptor-associated protein with a key role in clustering and anchoring nicotinic acetylcholine receptors at synaptic sites. This gene is responsible for a congenital form of myasthenic syndrome, a disease potentially treatable with cholinesterase inhibitors. Therefore, an earlier diagnosis in this boy would have led to a better clinical management and prognosis. Our study supports the key role of exome sequencing in achieving a definite diagnosis in severe perinatal diseases, an essential step especially when a specific therapy is available.
... 34,57 The C-terminal cytoplasmic domain of ␤-dystroglycan was shown to bind the WW, EF-hand (EF), and ZZ domains of dystrophin and utrophin, AChRs, and the scaffold protein rapsyn, which clusters AChRs. 36,37,[58][59][60] Additionally, ␤-dystroglycan was shown to interact with TKS5, a podosome-associated protein that can bind N-WASP, which is involved in actin organization. 61 ␤-Dystroglycan and caveolin-3 were also reported to competitively recruit dystrophin to the sarcolemma. ...
Article
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The proper function of skeletal muscles relies on their ability to process signals derived from motor neurons, transmit stimuli along the muscle fibers, contract, and regenerate efficiently after injury. The dystrophin–glycoprotein complex (DGC; also called the dystrophin-associated protein complex) plays a central role in all of these processes. It acts as a transmembrane platform that anchors the extracellular matrix (ECM) to the intracellular cytoskeleton and makes muscle fibers more resistant to injury. The DGC also contributes to the transmission of contraction-evoked force from the sarcomere to the ECM. The dysfunction of DGC-associated proteins can lead to myopathies, including Duchenne's muscular dystrophy, manifested by progressive muscle damage and impairments in regeneration. The DGC also plays a pivotal role in the organization of neuromuscular junctions (NMJs), where it stabilizes postsynaptic machinery, including receptors for the neurotransmitter acetylcholine (AChRs). Here, we focus on the role of the DGC complex in NMJ and skeletal muscle physiology and discuss the novel components that are associated with the complex.
... In analogy to dystrophin, the dystrophin-related protein utrophin, which is located mostly at the neuromuscular junction [60], also interacts with dystrophin-associated glycoproteins [61]. A key utrophin-associated protein is represented by α-dystroglycan, which functions as an extracellular receptor for the large proteoglycan agrin at the postsynaptic membrane of the neuromuscular junction [62][63][64]. Since agrin is essential for the development and specialization of the neuromuscular junction and aggregation of the nicotinic acetylcholine receptor complex during synaptogenesis [65], the utrophin/dystrophinglycoprotein complex at the sarcolemma/neuromuscular junction plays an essential role in neurotransmission [66]. ...
Article
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The development of advanced mass spectrometric methodology has decisively enhanced the analytical capabilities for studies into the composition and dynamics of multi-subunit protein complexes and their associated components. Large-scale complexome profiling is an approach that combines the systematic isolation and enrichment of protein assemblies with sophisticated mass spectrometry-based identification methods. In skeletal muscles, the membrane cytoskeletal protein dystrophin of 427kDa forms tight interactions with a variety of sarcolemmal, cytosolic and extracellular proteins, which in turn associate with key components of the extracellular matrix and the intracellular cytoskeleton. A major function of this enormous assembly of proteins, including dystroglycans, sarcoglycans, syntrophins, dystrobrevins, sarcospan, laminin and cortical actin, is postulated to stabilize muscle fibres during the physical tensions of continuous excitation-contraction-relaxation cycles. This article reviews the evidence from recent proteomic studies that have focused on the characterization of the dystrophin-glycoprotein complex and its central role in the establishment of the cytoskeleton-sarcolemma-matrisome axis. Proteomic findings suggest a close linkage of the core dystrophin complex with a variety of protein species, including tubulin, vimentin, desmin, annexin, proteoglycans and collagens. Since the almost complete absence of dystrophin is the underlying cause for X-linked muscular dystrophy, a more detailed understanding of the composition, structure and plasticity of the dystrophin complexome may have considerable biomedical implications.
... The accumulation of molecules of the DGC at the postsynaptic membrane of the NMJ and their co-distribution with AChR clusters in vitro suggested a role of the DGC in synaptogenesis (Yang et al., 1993). Furthermore, Rapsyn and β-DG have been shown to interact in the postsynaptic membrane (Apel et al., 1995;Cartaud et al., 1998). ...
... However, components of the synaptic basal lamina, such as AChE and laminin-␤2, still accumulate at the synapse. Interestingly, MuSK is still aggregated underneath the nerve terminal (8), and Chrna1 mRNA is highly expressed in the fundamental myonuclei (152). These experiments show that rapsyn is essential for the tethering of AChRs to the postsynaptic apparatus but not for synaptic gene transcription; furthermore, they imply that at early postnatal stages even nonclustered AChRs at the neural contact site are sufficient to support some low-level muscle contraction. ...
Article
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The neuromuscular junction is the chemical synapse between motor neurons and skeletal muscle fibers. It is designed to reliably convert the action potential from the presynaptic motor neuron into the contraction of the postsynaptic muscle fiber. Diseases that affect the neuromuscular junction may cause failure of this conversion and result in loss of ambulation and respiration. The loss of motor input also causes muscle wasting as muscle mass is constantly adapted to contractile needs by the balancing of protein synthesis and protein degradation. Finally, neuromuscular activity and muscle mass have a major impact on metabolic properties of the organisms. This review discusses the mechanisms involved in the development and maintenance of the neuromuscular junction, the consequences of and the mechanisms involved in its dysfunction, and its role in maintaining muscle mass during aging. As life expectancy is increasing, loss of muscle mass during aging, called sarcopenia, has emerged as a field of high medical need. Interestingly, aging is also accompanied by structural changes at the neuromuscular junction, suggesting that the mechanisms involved in neuromuscular junction maintenance might be disturbed during aging. In addition, there is now evidence that behavioral paradigms and signaling pathways that are involved in longevity also affect neuromuscular junction stability and sarcopenia. Copyright © 2015 the American Physiological Society.
... The 43 kDa AchR-associated protein rapsyn is required for the clustering of AchR at the developing neuromuscular junction (Cartaud et al., 1998). The hypothesis is that rapsyn may be a molecular link connecting the AchR to the DAPC (Apel et al., 1995). Activation of AchRs leads to calcium influx via the ligand-gated ion channel (i.e.,AchRs) or indirectly via sodium influx-stimulated calcium from intracellular stores Allard et al.,1996;Cherednichenko et al., 2004 . ...
Article
Duchenne Muscular Dystrophy (DMD) is an X-linked progressive muscle disease which is caused by mutations in the dystrophin gene. Until now, there is no effective therapy for DMD. As the largest gene in human beings, it produces a 427-kDa cytoskeleton protein: Dystrophin. Dystrophin links actin and dystrophin associated protein complex (DAPC) in muscles. Currently, there are 3 hypotheses to explain the mechanisms of DMD. They suggest that the absence of dystrophin could lead to periodic muscle cell membrane ruptures, or affect the distribution and function of ion channels, or perturb signal transduction pathways. In Caenorhabditis elegans, there is only one homologue of mammalian dystrophin gene named dys-1, and the nematode protein DYS-1 presents 37% similar to the human one. The double mutant dys-1; hlh-1 exhibits a severe progressive muscle degeneration. The protein composition of the sarcomere has been studied and it has revealed a high degree of similarity with mammalian sarcomere. These allow C. elegans be a relevant animal model to study DMD.To understand why the lack of dystrophin induces muscle degeneration in mammals and worms, and to find new drugs that might help in reducing muscle degeneration, L. Ségalat and his coworkers performed several screens for drugs and genes suppressing muscle degeneration. An interesting gene pkc-2 came out and was considered as a possible regulator in the process of muscle degeneration in C. elegans. The protein that is encoded by this gene in C. elegans is an orthologous of the human gene Protein Kinase C Alpha (PKC), which belongs to the family of serine/threonine specific protein kinases. To study the function of pkc-2, we generated different recombinant constructs, analyzed the expression pattern of pkc-2 with immunocytochemistry, and performed yeast two-hybrid to search for PKC-2 binding partners. In addition, a neurotransmitter serotonin (5-HT) was found by drug screening to be an active blocker of striated muscle degeneration. As C. elegans lacking PKC-2 displays a severe blister phenotype in exogenous 5-HT, studying the correlation between PKC-2 and 5-HT therefore seems to be an opportunity to explore the reasons of muscle degeneration. A genetic screen with EMS (ethane methyl sulfonate) to search serotonin resistant mutant in strain pkc-2 (ok328) would help us study further about the role of pkc-2.In this thesis, different clones myo3::pkc-2 and pkc-2::gfp were made to inject into wild-type animals. The results revealed that pkc-2 expressed intensely in neurons and pharynx, but was not found in body-wall muscles. Mutants dys-1;hlh-1 fed with pkc-2 RNAi did not reduce muscle degeneration statistically comparing to triple mutant pkc-2;dys-1;hlh-1. This indicated that PKC-2 may be dominantly acting in neurons. A yeast two-hybrid screen identified the gene Y59A8A.3, which is a homologue to mammalian filamin A interacting protein 1 isoform 3, as a binding partner of PKC-2. Filamin A is a cytoskeleton protein, anchoring various trans-membrane proteins to the actin cytoskeleton and may also function as an important signaling scaffold. The result suggested that PKC-2 may therefore modulate filamin A activity through the filamin interacting protein 1. Genetic screen by EMS presented 8 candidates named cx253, cx254, cx259, cx263, cx267, cx268, cx270, cx276, which were mapped on chromosomes by SNP mapping using a polymorphic C. elegans strain, but time was too short to identify these genes formally. The experiment also offered possibilities of searching links between PKC-2 and serotonin pathways.In summary, this work studied the gene pkc-2 in order to reveal the function of PKC-2 and its involvement in muscle degeneration. The present results answered some questions about pkc-2, and needed further researches to elucidate the in vivo role of PKC-2 protein and its interaction with other proteins in the mechanism of muscle dystrophy in C. elegans.
... The ability of activated Rac/Cdc42 to induce reorganization of cortical actin by modulating the dynamics of actin polymerization is now well documented (for review see Hall, 1998). Moreover, surface AChR is thought to be attached to the actin cytoskeleton via a complex in which rapsyn links AChR to the actin-binding protein utrophin, and recent findings indicate that agrininduced AChR aggregation involves the clustering of these diffusely distributed complexes at sites of MuSK activation (Apel et al., 1995;Fuhrer et al., 1999). According to our proposed scheme shown in Fig. 6, agrin-induced activation of Rac/Cdc42 produces highly localized reorganization of cortical actin cytoskeleton, resulting in redistribu- Figure 6. ...
Article
During neuromuscular junction formation, agrin secreted from motor neurons causes muscle cell surface acetylcholine receptors (AChRs) to cluster at synaptic sites by mechanisms that are insufficiently understood. The Rho family of small guanosine triphosphatases (GTPases), including Rac and Cdc42, can mediate focal reorganization of the cell periphery in response to extracellular signals. Here, we investigated the role of Rac and Cdc42 in coupling agrin signaling to AChR clustering. We found that agrin causes marked muscle-specific activation of Rac and Cdc42 in differentiated myotubes, as detected by biochemical measurements. Moreover, this activation is crucial for AChR clustering, since the expression of dominant interfering mutants of either Rac or Cdc42 in myotubes blocks agrin-induced AChR clustering. In contrast, constitutively active Rac and Cdc42 mutants cause AChR to aggregate in the absence of agrin. By indicating that agrin-dependent activation of Rac and Cdc42 constitutes a critical step in the signaling pathway leading to AChR clustering, these findings suggest a novel role for these Rho-GTPases: the coupling of neuronal signaling to a key step in neuromuscular synaptogenesis.
... In cardiology, a large number of genes (Shy et al., 2013), including SNTA1 (Wu et al., 2008), MOG1 (Kattygnarath et al., 2011), andGP1DL (Van Norstrand et al., 2007) have been shown to interact with Na v 1.5, and have been implicated in causing Brugada and long-QT syndromes. In neuromuscular disease, rapsyn, encoded by the RAPSN gene, is known to play a major role in the localisation of skeletal muscle acetylcholine receptors (Apel et al., 1995), and RAPSN mutations are associated with congenital myaesthenic syndrome (Ohno et al., 2002) and fatal akinesia deformation sequence (Michalk et al., 2009). ...
Article
The genetic channelopathies are a broad collection of diseases. Many ion channel genes demonstrate wide phenotypic pleiotropy, but nonetheless concerted efforts have been made to characterise genotype-phenotype relationships. In this review we give an overview of the factors that influence genotype-phenotype relationships across this group of diseases as a whole, using specific individual channelopathies as examples. We suggest reasons for the limitations observed in these relationships. We discuss the role of ion channel variation in polygenic disease and highlight research that has contributed to unravelling the complex aetiological nature of these conditions. We focus specifically on the quest for modifying genes in inherited channelopathies, using the voltage-gated sodium channels as an example. Epilepsy related to genetic channelopathy is one area in which precision medicine is showing promise. We will discuss the successes and limitations of precision medicine in these conditions. This article is part of the Special Issue entitled ‘Channelopathies.’
... This domain is highly conserved among vertebrates including zebrafish, mouse, and human ( Figures 1A and 1B). In vitro data suggest that it could bind to b-dystroglycan (Lim and Campbell, 1998), a transmembrane protein that interacts with dystrophin and utrophin in the subplasmalemmal cytoskeleton (Apel et al., 1995;Bartoli et al., 2001). Mutation of b-dystroglycan impairs muscle development, but its effect on NMJ formation appeared to be mild (Cô té et al., 1999;Jacobson et al., 2001), suggesting additional functions of the RING domain. ...
Article
Neurotransmission is ensured by a high concentration of neurotransmitter receptors at the postsynaptic membrane. This is mediated by scaffold proteins that bridge the receptors with cytoskeleton. One such protein is rapsyn (receptor-associated protein at synapse), which is essential for acetylcholine receptor (AChR) clustering and NMJ (neuromuscular junction) formation. We show that the RING domain of rapsyn contains E3 ligase activity. Mutation of the RING domain that abolishes the enzyme activity inhibits rapsyn- as well as agrin-induced AChR clustering in heterologous and muscle cells. Further biological and genetic studies support a working model where rapsyn, a classic scaffold protein, serves as an E3 ligase to induce AChR clustering and NMJ formation, possibly by regulation of AChR neddylation. This study identifies a previously unappreciated enzymatic function of rapsyn and a role of neddylation in synapse formation, and reveals a potential target of therapeutic intervention for relevant neurological disorders.
... "~,~~ Clusters of AChR are connected to the cytoskeleton via the dystrophin-glycoprotein complex (DGC). 56 The DGC contains a-and P-dystroglycan, adhalin, agrin, syntrophin, utrophin (dystrophin-related protein), and additional 2 5 , 3 5 , and 87-kDa proteins. The 87-kDa protein may be the dystrophin-related phosphoprotein dy~trobrevin.~~ ...
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The mammalian neuromuscular junction is a chemical synapse that uses acetylcholine as transmitter. Acetylcholine (ACh) is stored in the nerve terminal and is released from small membrane-bound vesicles that fuse with the nerve terminal membrane after depolarization of the nerve terminal. Calcium entry via P/Q-type calcium channels is necessary for transmitter release. The postsynaptic membrane of the muscle fiber has several unique properties that facilitate neuromuscular transmission. The postsynaptic membrane area is greatly expanded by synaptic folds. Acetylcholine receptors (AChR) are concentrated at the peaks of synaptic folds and sodium channels are concentrated in the troughs of the synaptic folds. The safety factor for neuromuscular transmission results from a combination of factors: the amount of ACh released, the high concentration of AChR, and the high concentration of sodium channels. AChR are firmly anchored in the membrane by a complex of proteins. The autoimmune attack in myasthenia gravis (MG) directed at acetylcholine receptors results in loss of postsynaptic membrane, AChR, and sodium channels. This article describes the different types of neuromuscular junctions on extraocular muscle fibers and their possible relevance to the susceptibility of extraocular muscle to MG.
... Noticeably, agrin-LRP4-MuSK signaling is critical (DeChiara et al., 1996;Gautam et al., 1996;Hesser et al., 2006;Weatherbee et al., 2006;Chevessier et al., 2008;Kim et al., 2008;Zhang et al., 2008;Samuel et al., 2012;Zong et al., 2012;Barik et al., 2014). The intracellular protein rapsyn (receptor-associated protein at the synapse) is also necessary, likely by serving as an anchor for AChRs (Burden et al., 1983;Walker et al., 1984;LaRochelle and Froehner, 1986;Apel et al., 1995;Gautam et al., 1995) and/or as an E3 ligase to regulate proteins by posttranslational modification including neddylation (Li et al., 2016). Moreover, rapsyn turns over with a half-life of several hours in muscle cells and its stability requires Hsp90b . ...
Article
During aging, skeletal muscles become atrophic and lose contractile force. Aging can also impact the neuromuscular junction (NMJ), a synapse that transmits signals from motoneurons to muscle fibers to control muscle contraction. However, in contrast to muscle aging that has been studied extensively, less is known about the molecular mechanisms of NMJ aging although its structure and function are impaired in aged animals. To this end, we performed RNA-seq analysis to identify genes whose expression in synapse-rich region is altered. Gene Ontology analysis highlighted genes relating to nuclear structure or function. In particular, lamin A/C, an intermediate filament protein critical for the interphase nuclear architecture, was reduced. Remarkably, mutation of lamin A/C in muscles or motoneurons had no effect on NMJ formation in either sex of mice, but the muscle mutation caused progressive denervation, AChR cluster fragmentation, and neuromuscular dysfunction. Interestingly, rapsyn, a protein critical to AChR clustering, was reduced in mutant muscle cells; and expressing rapsyn in muscles attenuated NMJ deficits of HSA-Lmna-/- mice. These results reveal a role of lamin A/C in NMJ maintenance and suggest that nuclear dysfunction or deficiency may contribute to NMJ deficits in aged muscles.Significance statement:This study provides evidence that lamin A/C, a scaffolding component of the nuclear envelope, is critical to maintaining the NMJ in mice. Its muscle-specific mutation led to progressive NMJ degeneration in vivo We showed that the mutation reduced the level of rapsyn, a protein necessary for AChR clustering; and expression of rapsyn in muscles attenuated NMJ deficits of HSA-Lmna-/- mice. These results reveal a role of lamin A/C in NMJ maintenance and suggest that nuclear dysfunction or deficiency may contribute to NMJ deficits in aged muscles.
... [29][30][31][32][33] This signaling induces cytosolic proteins Dok-7 and rapsyn to accumulate AChR and postsynaptic specialization. [34][35][36][37] In MG, transmembrane proteins, AChRs, and MuSK become targets of autoantibodies. The roles of MuSK are described in detail next. ...
Article
Neuromuscular junctions (NMJs) form between nerve terminals of spinal cord motor neurons and skeletal muscles, and perisynaptic Schwann cells and kranocytes cap NMJs. One muscle fiber has one NMJ, which is innervated by one motor nerve terminal. NMJs are excitatory synapses that use P/Q-type voltage-gated calcium channels to release the neurotransmitter acetylcholine. Acetylcholine receptors accumulate at the postsynaptic specialization called the end plate on the muscle fiber membrane, the sarcolemma. Proteins essential for the organization of end plates include agrin secreted from nerve terminals, Lrp4 and MuSK receptors for agrin, and Dok-7 and rapsyn cytosolic proteins in the muscle.
... RAPSN encodes the 43 kDa receptor-associated scaffold protein of the synapse [102], which is essential for stabilisation of AChR clusters at the muscle endplate [103,104]. Rapsyn is enriched at postsynaptic membranes and acts as a linker between the AChRs and the cytoskeleton via the dystrophin-associated glycoprotein complex [105]. The detailed organisation of the AChRs-rapsyn network is not fully understood since the crystallographic structure has not been solved [106]. ...
Article
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Congenital myasthenic syndromes (CMS) are genetic disorders characterised by impaired neuromuscular transmission. This review provides an overview on CMS and highlights recent advances in the field, including novel CMS causative genes and improved therapeutic strategies. CMS due to mutations in SLC5A7 and SLC18A3, impairing the synthesis and recycling of acetylcholine, have recently been described. In addition, a novel group of CMS due to mutations in SNAP25B, SYT2, VAMP1, and UNC13A1 encoding molecules implicated in synaptic vesicles exocytosis has been characterised. The increasing number of presynaptic CMS exhibiting CNS manifestations along with neuromuscular weakness demonstrate that the myasthenia can be only a small part of a much more extensive disease phenotype. Moreover, the spectrum of glycosylation abnormalities has been increased with the report that GMPPB mutations can cause CMS, thus bridging myasthenic disorders with dystroglycanopathies. Finally, the discovery of COL13A1 mutations and laminin α5 deficiency has helped to draw attention to the role of extracellular matrix proteins for the formation and maintenance of muscle endplates. The benefit of β2-adrenergic agonists alone or combined with pyridostigmine or 3,4-Dyaminopiridine is increasingly being reported for different subtypes of CMS including AChR-deficiency and glycosylation abnormalities, thus expanding the therapeutic repertoire available.
... Self-association of these rapsyn struts appears to draw AChRs together to form microaggregates (Phillips et al. 1993;Ramarao & Cohen, 1998;Ramarao et al. 2001;Zubera & Unwin, 2013). In contrast, the formation of large, contiguous AChR clusters may depend upon the binding of the carboxyl-terminal RING-H2 domain of rapsyn to β-DG (Apel et al. 1995;Bezakova & Bloch, 1998;Cartaud et al. 1998;Bartoli et al. 2001). Through β-DG, rapsyn may couple AChR microaggregates into a much larger postsynaptic membrane lattice that includes synaptic basement membrane proteins (Sugiyama et al. 1997;Montanaro et al. 1998;Cote et al. 1999;Grady et al. 2000;Jacobson et al. 2001;Pilgram et al. 2010). ...
Article
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.This article is protected by copyright. All rights reserved
... It codes for a postsynaptic protein (rapsyn) that links the AChR to the agrin-binding dystrophin-associated glycoprotein complex and stabilizes the AChR at the neuromuscular junction. 13 Homozygosity or compound heterozygosity for mutations results in AChR deficiency and FADS or CMS. Michalk et al reported the case of a brother and a sister in a nonconsanguineous Pakistani family. ...
Article
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Pena–Shokeir syndrome (PSS) type 1, also known as fetal akinesia deformation sequence, is a rare genetic syndrome that almost always results in intrauterine or early neonatal death. It is characterized by markedly decreased fetal movements, intrauterine growth restriction, joint contractures, short umbilical cord, and features of pulmonary hypoplasia. Antenatal diagnosis can be difficult. Ultrasound features are varied and may overlap with those of Trisomy 18. The poor prognosis of PSS is due to pulmonary hypoplasia, which is an important feature that distinguishes PSS from arthrogryposis multiplex congenital without pulmonary hypoplasia, which has a better prognosis. If diagnosed in the antenatal period, a late termination of pregnancy can be considered following ethical discussion (if the law allows). In most cases, a diagnosis is only made in the neonatal period. Parents of a baby affected with PSS require detailed counseling that includes information on the imprecise recurrence risks and a plan for subsequent pregnancies.
... It could induce AChR clusters in heterologous cells (Froehner et al., 1990;Li et al., 2016;Phillips et al., 1991); Rapsn null mutant mice die soon after birth without AChR clusters (Gautam et al., 1995), indicating a critical role in NMJ formation. Being a classic adapter protein, Rapsn is thought to bridge the AChR to the cytoskeleton (Apel et al., 1995;Bartoli et al., 2001;Chen et al., 2016;Lee et al., 2009;Maimone and Merlie, 1993;Miyazawa et al., 1999;Ramarao et al., 2001;Ramarao and Cohen, 1998). We showed recently that Rapsn possesses E3 ligase activity (Li et al., 2016). ...
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14 Neuromuscular junction is a synapse between motoneurons and skeletal 15 muscles, where acetylcholine receptors (AChRs) are concentrated to control 16 muscle contraction. Studies of this synapse have contributed to our 17 understanding of synapse assembly and pathological mechanisms of 18 neuromuscular disorders. Nevertheless, underlying mechanisms of NMJ 19 formation was not well understood. To this end, we took a novel approach-20 studying mutant genes implicated in congenital myasthenic syndrome (CMS). 21 We showed that knock-in mice carrying N88K, a prevalent CMS mutation of 22 Rapsyn (Rapsn), died soon after birth with profound NMJ deficits. Rapsn is an 23 adapter protein that bridges AChRs to the cytoskeleton and possesses E3 24 ligase activity. In investigating how N88K impairs the NMJ, we uncovered a 25 novel signaling pathway by which Agrin-LRP4-MuSK induces tyrosine 26 phosphorylation of Rapsn, which is required for its self-association and E3 27 ligase activity. Our results also provide insight into pathological mechanisms of 28 CMS. 29 30 3
... Although it is critical for AChR clustering in vivo [26], exactly how it is involved in the process is still a mystery. Initially proposed as a purely structural anchoring link between AChR clusters and the underlying synaptic cytoskeleton [48], recent findings indicate that Rapsyn is also endowed with enzymatic activity, specifically neddylation [49], that seems important for AChR clustering. ...
Article
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The neuromuscular junction is the synapse between a motor neuron of the spinal cord and a skeletal muscle fiber in the periphery. Reciprocal interactions between these excitable cells, and between them and others cell types present within the muscle tissue, shape the development, homeostasis and plasticity of skeletal muscle. An important aim in the field is to understand the molecular mechanisms underlying these cellular interactions, which include identifying the nature of the signals and receptors involved but also of the downstream intracellular signaling cascades elicited by them. This review focuses on work that shows that skeletal muscle fiber-derived extracellular signal-regulated kinases 1 and 2 (ERK1/2), ubiquitous and prototypical intracellular mitogen-activated protein kinases, have modulatory roles in the maintenance of the neuromuscular synapse and in the acquisition and preservation of fiber type identity in skeletal muscle.
... Therefore, the integrin-GIRK interaction may link the channel to the cytoskeleton. Several other channels have links to the cytoskeleton (41)(42)(43)(44)(45)(46)(47), and previous studies show that the actin cytoskeleton helps to stabilize some inward rectifiers (48). Me-chanical stress, presumably transmitted via cytoskeletal elements, affects both the integrin-mediated transduction pathway (49) and GIRK activation (50). ...
Article
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Heteromultimeric G protein-activated inward rectifier K⁺ (GIRK) channels, abundant in heart and brain, help to determine the cellular membrane potential as well as the frequency and duration of electrical impulses. The sequence arginine-glycine-aspartate (RGD), located extracellularly between the first membrane-spanning region and the pore, is conserved among all identified GIRK subunits but is not found in the extracellular domain of any other cloned K⁺ channels. Many integrins, which, like channels, are integral membrane proteins, recognize this RGD sequence on other proteins, usually in the extracellular matrix. We therefore asked whether GIRK activity might be regulated by direct interaction with integrin. Here, we present evidence that mutation of the RGD site to RGE, particularly on the GIRK4 subunit, decreases or abolishes GIRK current. Furthermore, wild-type channels can be co-immunoprecipitated with integrin. The total cellular amount of expressed mutant GIRK channel protein is the same as the wild-type protein; however, the amount of mutant channel protein that localizes to the plasma membrane is decreased relative to wild-type, most likely accounting for the diminished GIRK current detected. GIRK channels appear to bind directly to integrin and to require this interaction for proper GIRK channel membrane localization and function.
... The dystrophin-glycoprotein complex containing four integral membrane glycoproteins is also linked to the ion channel to form more complex structures in neuromuscular junctions (26). This complex is associated with a peripheral membrane glycoprotein ␣-dystroglycan (27), which in the neuromuscular junction is a receptor for agrin, an extracellular matrix protein that promotes clustering of the acetylcholine receptor (28)(29)(30). Multisubunit receptors of the immune system, including the T cell antigen receptor, the B cell antigen receptor, and the high affinity receptor for immunoglobulin E, form associations with the cytoskeletal matrix upon cell activation (reviewed in Ref. 31). ...
Article
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Microfilaments associate with the microvillar membrane of 13762 ascites mammary adenocarcinoma cells via a large transmembrane complex (TMC) comprising the major glycoproteins TMC-gp120, -110, -80, -65, and -55, the receptor kinase p185neu, and the cytoplasmic proteins actin and p58gag, linking the receptor with microfilaments in a signal transduction particle. Immunoblot screening with polyclonal antisera to TMC glycoproteins showed selective epithelial expression in normal rat tissues and epithelially derived tumor cells. The TMC glycoproteins were isolated by solubilization of microfilament core preparations in SDS, dilution, and separation on a concanavalin A-agarose affinity column. The large p185neu-containing complex was reconstituted from the column eluate after displacement of SDS with nonionic detergent, demonstrated by gel filtration and co-immunoprecipitation of the glycoproteins with anti-gp55 or anti-p185neu. Exhaustive biotinylation of the glycoproteins gave a stoichiometry of gp120:gp110:gp80:gp65:gp55 of approximately 1:1:1:0.5:1. Overlay blots with biotinylated actin and in vitro translated, [³⁵S]methionine-labeled p58gag, respectively, showed specific interactions of actin with gp55 and gp120 and of p58gag with gp65 and gp55. These results provide evidence for a specific complex of microfilament-associated glycoproteins containing p185neu and p58gag and suggest a role for the complex in signal transduction scaffolding.
... It has also been shown to interact with components of the utrophin complex. Coexpression studies in QT-6 fibroblasts, show that dystroglycan targets rapsyn-induced clusters (Apel et al. 1995). Moreover, utrophin and syntrophin are dramatically reduced at synaptic sites in knockout mice for the rapsyn gene (Gautam et al. 1995). ...
Thesis
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Duchenne and Becker muscular dystrophies (DMD/BMD) are caused by mutations in the dystrophin gene. DMD is also associated with a variable degree of mental impairment. Several dystrophin transcripts are expressed in the brain including a novel transcript (P-type dystrophin) expressed specifically in Purkinje cells; its expression is controlled by an alternative promoter. This study shows that the P-type mRNA is also expressed in skeletal and cardiac muscle but not in smooth muscle. Its first exon encodes a specific, short amino terminus that is highly conserved in mammals and to a lesser extent in chicken. The nucleotide sequence of the P-type first exon and putative promoter region is also conserved. In mice, the 5'-end of the P-type transcript was found to be structurally diverse arising from alternative splicing events at the 5'-UTR. This may occur separately or in combination with insertion of a part of intron I resulting in premature termination of translation. There are multiple transcription initiation sites, the predominant one being conserved in human and mouse. Moreover, alternative usage of ATG codons may result in alternative N-termini in rodents or short upstream open reading frames in other species. Several regulatory elements are conserved in different species. The TATA box found in human sequence is not conserved and is outside the region that directed CAT reporter gene expression in differentiated myotubes in culture.
... A 43 kDa protein called rapsyn is associated with the receptor in stoichiometric amounts (Frail et al. 1988). It is required for clustering of nAChRs at the neuromuscular junction and may play a role in attaching the receptor to other cytoskeletally-anchored components such as dystroglycan, the agrin-binding component of the dystrophin-glycoprotein complex (DOC) (Apel et al. 1995). Hucho et al (1996). ...
Thesis
Nicotinic acetylcholine receptors (nAChRs) are pentameric multi-subunit proteins that form ligand-gated ion channels in the central nervous systems of vertebrates and invertebrates and at the neuromuscular junction of vertebrate species. In insects, they play a major role in excitatory synaptic transmission and are the primary target site for neonicotinoid insecticides such as imidacloprid. Despite their commercial importance as insecticide targets and the recent cloning of nAChR subunit genes from a range of insect species, the insect nAChR is not well characterized at the molecular level. This study investigates the molecular diversity of nAChR subunit genes of two important agricultural pests, the peach-potato aphid Myzus persicae and the tobacco whitefly Bemisia tabaci. Five genes have already been cloned as full-length cDNAs from M. persicae and one from B. tabaci. Heterologous expression of M. persicae subunit genes in Drosophila S2 cells has revealed some evidence for co-assembly of certain a/p subunit combinations, although functional reconstitution has so far been dependant on co-expression of insect a subunits with a vertebrate β subunit. This suggests that one or more key subunits required for functional reconstitution of the native receptor have yet to be cloned. Heterologous expression of insect nAChR subunits has demonstrated that the binding affinity and agonist potency of imidacloprid is influenced markedly by nAChR subunit composition and only certain M. persicae a/rat β2 complexes display high affinity binding of 3H-imidacloprid, indicating selectivity of this ligand for different a subunits. In this study additional novel nAChR subunit genes have been cloned from both M. persicae and B. tabaci, utilising the recently sequenced D. melanogaster genome to help target a degenerate PCR approach. The contribution of individual amino acids in M. persicae a subunits to imidacloprid binding has been examined by site-directed mutagenesis, heterologous expression and radioligand binding studies.
Article
Mutations in the dystrophin-glycoprotein complex cause muscle degeneration and dysfunctions in the central nervous system, including an impaired synaptic transmission in the outer plexiform layer (OPL) of the retina. To investigate the basis for this ocular phenotype, we analyzed the distribution of β-dystroglycan, a central member of the dystrophin-glycoprotein complex, in the chick retina by using the 43DAG/8D5 monoclonal antibody. This antibody reacted specifically with chick β-dystroglycan, as indicated by its staining of the neuromuscular junction, and its reactivity with a single 43-kilodalton band in Western blots. In the retina, β-dystroglycan was highly concentrated in the OPL and at the vitreal border of the retina, around the inner limiting membrane. Mechanically isolated and flat-mounted inner limiting membranes were stained by the anti-β-dystroglycan antibody, and this immunoreactivity could be extracted by detergent, indicating that β-dystroglycan is associated with membranous structures bound to the basal lamina. Consistently, electron microscopy showed a concentration of β-dystroglycan in the endfeet of Müller glial cells exclusively in the region of direct contact to the inner limiting membrane. In the OPL, β-dystroglycan immunoreactivity was concentrated in the distal extensions of rod and cone terminals protruding into the outer plexiform layer. There, β-dystroglycan codistributed with the α1B subunit of the N-type voltage-gated calcium channel. By contrast to previous reports, we did not detect β-dystroglycan directly associated with the synaptic regions of conventional or ribbon synapses of the retina. These results show that in the retina β-dystroglycan is exclusively expressed by photoreceptors and glial cells and that β-dystroglycan is highly concentrated in subcellular regions of glial cell endfeet and photoreceptor terminals. Moreover, the colocalization of β-dystroglycan with N-type calcium channels in the outer plexiform layer indicates that both proteins might be part of a macromolecular complex. J. Comp. Neurol. 389:668–678, 1997. © 1997 Wiley-Liss, Inc.
Article
At the neuromuscular junction (NMJ), the postsynaptic localization of muscle acetylcholine receptor (AChR) is regulated by neural signals and occurs via several processes including metabolic stabilization of the receptor. However, the molecular mechanisms that influence receptor stability remain poorly defined. Here, we show that neural agrin and the tyrosine phosphatase inhibitor, pervanadate slow the degradation of surface receptor in cultured muscle cells. Their action is mediated by tyrosine phosphorylation of the AChR β subunit, as agrin and pervandate had no effect on receptor half‐life in AChR‐β3F/3F muscle cells, which have targeted mutations of the β subunit cytoplasmic tyrosines. Moreover, in wild type AChR‐β3Y muscle cells, we found a linear relationship between average receptor half‐life and the percentage of AChR with phosphorylated β subunit, with half‐lives of 12.7 and 23 h for nonphosphorylated and phosphorylated receptor, respectively. Surprisingly, pervanadate increased receptor half‐life in AChR‐β3Y myotubes in the absence of clustering, and agrin failed to increase receptor half‐life in AChR‐β3F/3F myotubes even in the presence of clustering. The metabolic stabilization of the AChR was mediated specifically by phosphorylation of βY390 as mutation of this residue abolished β subunit phosphorylation but did not affect δ subunit phosphorylation. Receptor stabilization also led to higher receptor levels, as agrin increased surface AChR by 30% in AChR‐β3Y but not AChR‐β3F/3F myotubes. Together, these findings identify an unexpected role for agrin‐induced phosphorylation of βY390 in downregulating AChR turnover. This likely stabilizes AChR at developing synapses, and contributes to the extended half‐life of AChR at adult NMJs. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 399–410, 2013
Article
In the central nervous system, mechanisms of postsynaptic clustering of neurotransmitter receptors are best understood at glycinergic inhibitory synapses. The glycine receptor is a pentameric protein composed of three α and two β transmembrane proteins: the ligand-binding α subunit and a structural β subunit. At glycinergic synapses, these receptors form clusters at the postsynaptic membrane directly opposite to the presynaptic release sites. Anchoring of glycine receptors in the neuronal membrane depends on a peripheral membrane protein named gephyrin. Gephyrin binds to the glycine receptor via the β subunit and links the receptor to the subsynaptic cytoskeleton. Gephyrin may be involved in the anchoring of other neurotransmitter receptors such as the γ-aminobutyric acid (GABAA) receptor.
Article
The extracellular matrix is a well organized structure with profound effects on the development and the integrity of adherent tissues. Agrin is a component of many matrices, such as the basement membrane of kidney, blood capillaries and the muscle fiber basal lamina, where it is highly concentrated at the neuromuscular junction. During synapse formation agrin is believed to promote differentiation of the postsynaptic muscle fibers and the presynaptic motor neuron. This complex process is, at least in part, based on specific interactions of agrin with other matrix molecules and with membrane-associated or integral membrane proteins of the abutting cells. This review summarizes studies concerning the integration of agrin with other molecules and highlights possible functions of agrin in the synaptic basal lamina and in other matrices.
Article
This chapter discusses the regulation of membrane-protein organization at the neuromuscular junction. Agrin is an extracellular-matrix protein released by the motor neuron at the time of synapse formation. It resides in the synaptic cleft of the neuromuscular junction (NMJ), where it forms part of the stable basal lamina. In this location, agrin induces the formation of Ach receptor (AchR) clusters under the nerve-muscle synapse. The underlying molecular mechanisms of agrin's actions are likely to involve tethering AChR to the cytoskeleton. Several lines of evidence support this hypothesis. First, agrin induces cluster formation by redistributing AChR already present on the muscle-cell membrane and has no effect on AchR-subunit synthesis. Second, AChR clusters are more resistant to detergent extraction than unclustered receptors. Third, many spectrin-like molecules, including syntrophin, utrophin, p-spectrin, the 87-kDa protein, and rapsyn, are specifically co-localized with AChRs at the NMJ. These molecules are likely to serve as a link between AChRs and the actin cytoskeleton. Agrin binds to α-dystroglycan (DG), a glycoprotein complex (GC) member. The GC is linked to the cytoskeleton by binding dystrophin or utrophin, spectrin-like proteins known to bind F-actin. These data provide a model in which agrin, by binding to a-DG, traps the AChRs as they diffuse into the agrin-receptorcytoskeleton complex.
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In mouse sympathetic superior cervical ganglion (SCG), cortical cytoskeletal proteins such as dystrophin (Dys) and beta 1 Sigma 2 spectrin colocalize with beta-dystoglycan (beta-DG), a transmembrane dystrophin-associated protein, and the acetylcholine receptor (AChR) at the postsynaptic specialization. The function of the dystrophin-dystloglycan complex in the organization of the neuronal cholinergic postsynaptic apparatus was studied following changes in the immunoreactivity of these proteins during the disassembly and subsequent reassembly of the postsynaptic specializations induced by axotomy of the ganglionic neurons. After axotomy, a decrease in the number of inh aganglionic synapses was observed (t1/2 8 h 45'), preceded by a rapid decline of postsynaptic specializations immunopositive for beta-DG, Dys, and alpha 3 AChR subunit alpha 3AChR (t1/2 3 h 45', 4 h 30' and 6 h, respectively). In contrast, the percentage of postsynaptic densities immunopositive for beta 1 Sigma 2 spectrin remained unaltered. When the axotomized neurons began to regenerate their axons, the number of intraganglionic synapses increased,as did that of postsynaptic specializations immunopositive for beta-DG, Dys, and alpha 3AChR. The latter number increased more slowly than that of Dys and beta-DG. These observations suggest that in SCG neurons, the dystrophin-dystroglycan complex might play a role in the assembly-disassembly of the postsynaptic apparatus, and is probably involved in the stabilization of AChR clusters.
Article
Introduction: Nonimmune hydrops fetalis (NIHF) has varied etiology. We assessed the etiological spectrum and evaluated the utility of fetal whole exome sequencing (fWES) for the diagnosis of NIHF. Methods: In this prospective cohort study, we evaluated antenatally diagnosed fetuses with NIHF between July 2018 and December 2019 according to the routine diagnostic algorithm. Fetuses that remained undiagnosed after routine NIHF workup were subjected to fetal chromosomal microarray and/or WES. Pregnancies were followed up for clinical outcomes. Results: Of the 45 fetuses, consanguinity and recurrent hydrops fetalis were observed in 13.3% (6/45) and 28.8% (13/45), respectively. Overall, an etiological diagnosis was possible in 75.5% (34/45) of fetuses, while the cause remained unknown in 24.4% (11/45). A genetic etiology was identified in 46.6% (21/45): aneuploidy and monogenic disorders in 28.8% (13/45) and 17.8% (8/45), respectively. fWES on 19 fetuses detected disease-causing variants in 42.1% (8/19). Nine novel variants were detected in RAPSN, ASCC1, NEB, PKD1L1, GUSB, and PIEZO1. Only 8.8% (4/45) of the cohort survived without morbidity. Conclusions: This study describes the etiological spectrum and the disease-causing variants in an Indian cohort of hydropic fetuses.
Chapter
Nicotinic acetylcholine receptors (AChRs) from skeletal muscle and fish electric organs have the best characterized structural and functional properties of any neurotransmitter-gated ion channels. Subunit sequence homologies among families of receptors in the gene superfamily validate muscle AChRs as a model for the general structural features of glycine, γ-aminobutyric acid (GABA)-A, and serotonin 5-HT3 receptors that comprise this superfamily. The most elegant evidence for the structural homologies in this superfamily are experiments showing that it is possible to change the selectivity of the ion channel of a neuronal AChR from cations to anions by changing only three channel-lining amino acids to those characteristic of glycine and GABAA receptors (GALZI et al. 1992), and experiments showing that it is possible to form a chimera consisting of the extracellular domain of a neuronal AChR subunit and the remainder of a 5-HT3 receptor subunit and produce a receptor with the channel properties of a 5-HT3 receptor which is activated by ACh (EISELE et al. 1993). Actually, there are significant sequence differences between the subunits of the receptors in the superfamily, so in general it is not trivial to mix parts of them. Muscle AChRs are especially relevant as models for neuronal AChRs, to which they are most closely related by sequence homology.
Article
The cloning of the dystrophin gene has led to major advances in the understanding of the molecular genetic basis of Duchenne, Becker, and other muscular dystrophies associated with mutations in genes encoding members of the dystrophin-associated glycoprotein complex. The recent introduction of pharmaceutical agents such as prednisone has shown great promise in delaying the progression of Duchenne muscular dystrophy but there remains a need to develop more longterm therapeutic interventions. Knowledge of the nature of the dystrophin gene and the glycoprotein complex has led many researchers to think that somatic gene replacement represents the most promising approach to treatment. The potential use of this strategy has been shown in the mdx mouse model of Duchenne muscular dystrophy, where germ line gene transfer of either a full-length or a smaller Becker-type dystrophin minigene prevents necrosis and restores normal muscle function.
Article
Fast and accurate synaptic transmission requires high-density accumulation of neurotransmitter receptors in the postsynaptic membrane. During development of the neuromuscular junction, clustering of acetylcholine receptors (AChR) is one of the first signs of postsynaptic specialization and is induced by nerve-released agrin. Recent studies have revealed that different mechanisms regulate assembly vs stabilization of AChR clusters and of the postsynaptic apparatus. MUSK, a receptor tyrosine kinase and component of the agrin receptor, and rapsyn, an AChR-associated anchoring protein, play crucial roles in the postsynaptic assembly. Once formed, AChR clusters and the postsynaptic membrane are stabilized by components of the dystrophin/utrophin glycoprotein complex, some of which also direct aspects of synaptic maturation such as formation of postjunctional folds. Nicotinic receptors are also expressed across the peripheral and central nervous system (PNS/CNS). These receptors are localized not only at the pre- but also at the postsynaptic sites where they carry out major synaptic transmission. In neurons, they are found as clusters at synaptic or extrasynaptic sites, suggesting that different mechanisms might underlie this specific localization of nicotinic receptors. This review summarizes the current knowledge about formation and stabilization of the postsynaptic apparatus at the neuromuscular junction and extends this to explore the synaptic structures of interneuronal cholinergic synapses.
Article
Agrin induces both phosphorylation and aggregation of nicotinic acetylcholine receptors (AChRs) when added to myotubes in culture, apparently by binding to a specific receptor on the myotube surface. One such agrin receptor is alpha‐dystroglycan, although binding to alpha‐dystroglycan appears not to mediate AChR aggregation. To determine whether agrin‐induced AChR phosphorylation is mediated by alpha‐dystroglycan or by a different agrin receptor, fragments of recombinant agrin that differ in affinity for alpha‐dystroglycan were examined for their ability to induce AChR phosphorylation and aggregation in mouse C2 myotubes. The carboxy‐terminal 95 kDa agrin fragment agrin‐c95(A0B0), which binds to alpha‐dystroglycan with high affinity, failed to induce AChR phosphorylation and aggregation. In contrast, agrin‐c95(A4B8) which binds less strongly to alpha‐dystroglycan, induced both phosphorylation and aggregation, as did a small 21 kDa fragment of agrin, agrin‐c21(B8), that completely lacks the binding domain for alpha‐dystroglycan. We conclude that agrin‐induced AChR phosphorylation and aggregation are triggered by an agrin receptor that is distinct from alpha‐dystroglycan.
Article
Der Aufbau eines korrekt funktionierenden Nervensystems hängt entscheidend von der präzisen Vernetzung verschiedener Zellgruppen ab. Synaptogenese ist hierbei ein zentraler Punkt und das Verständnis davon, wie synaptische Kontakte gebildet und erhalten werden, von grossem Interesse. Als Modellsystem hat sich die neuromuskuläre Endplatte sehr bewährt, da diese leicht zugänglich und relativ gross ist. Diese Dissertation erforschte den Einfluss von Src-Familien-Kinasen auf die Stabilität dieser Synapse. Es wurde gezeigt, dass ein Ungleichgewicht in der Src- Aktivität einen dramatischen Einfluss auf die Stabilität postsynaptischer Strukturen bewirkt - in vivo in elektroporierten Mausmuskeln ebenso wie in vitro in mutanten Muskelzellen, denen Src-Kinasen fehlen. Wechselwirkungen zwischen kritischen postsynaptischen Proteinen waren ebenso betroffen wie Proteinphosphorylierungen und das Zytoskelett, und Src-Kinasen erreichten ihre Wirkung zusammen mit speziellen Membrandomänen, sogenannten Lipid Rafts. Die Resultate liefern neue Erkenntnisse über die Synapsen-stabilisierenden Eigenschaften der Src-Kinasen, welche eventuell dazu beitragen könnten, verschiedene Muskelschwächen und Probleme in der synaptischen Transmission besser zu verstehen. The assembly of a proper functional nervous system is strongly dependent on the precise linkage of different cell types. Synaptogenesis is therefore a central issue and it is of great interest to understand how synaptic contacts are formed and maintained. The neuromuscular junction has been useful as a good model system since it is easily accessible and relatively big. This thesis investigated the influence of Src-family kinases on the stability of the neuromuscular synapse. It was shown that an unbalanced Src-activity can have a dramatic effect on the stability of postsynaptic structures - in vivo in electroporated mice muscles and in vitro in mutant muscle cells which lack Src-kinases.Interactions between critical postsynaptic proteins, protein phosphorylations and the cytoskeleton were all compromised. Src-kinases were associated with special membrane domains called lipid rafts. These results provide new insights in the synapse-stabilizing properties of Src-kinases, which might contribute to a better understanding of different muscle diseases and defects in the synaptic transmission.
Article
The acetylcholine receptor (AChR) is highly concentrated at the neuromuscular junction (NMJ), ensuring efficient signal transmission from motoneurons to muscle fibers. This requires the agrin-LRP4-MuSK signaling as well as rapsyn, a peripheral, intracellular protein that is enriched at the NMJ. Mutations of rapsyn have been associated with NMJ diseases including congenital myasthenia syndromes. Rapsyn is a prototype of synaptic adaptor proteins that is thought to bind and anchor neurotransmitter receptors to the postsynaptic membrane. In accord, it interacts with the AChR and a plethora of proteins that associate or regulate the cytoskeleton. Rapsyn also interacts with signaling molecules. Recent studies show that it possesses E3 ligase activity that is required for NMJ formation, revealing a novel function of this classic adaptor protein. Identifying rapsyn as a signaling molecule provides a handle in studies of mechanisms of NMJ formation, maintenance, aging and disorders.
Thesis
Utrophin is a large protein, which accummulates at the neuromuscular and myotendinous junctions in the adult skeletal muscle, and is widely expressed in several non-muscle tissues. Evidence from a variety of sources suggests that a successful strategy for treatment of Duchenne muscular dystrophy will be to increase the expression of utrophin in muscle. In order to facilitate this much needs to be learnt about utrophin gene regulation, in particular about alternative isoforms, their promoters and their role in muscle and non-muscle tissues. My project involved searching for novel transcripts transcribed from the utrophin gene. Two novel transcripts of utrophin, Up71 and Up140, with unique first exons and promoters located in intron 62 and intron 44, respectively have been identified in mRNA from brain by 5' RACE. 5' RACE using primers designed to amplify forms of the full-length utrophin failed to find alternative full-length forms in the brain or lung. The expression pattern for Up140 and Up71 was investigated using RT-PCR and Western blotting, which revealed that both Up140 and Up71 mRNA are expressed in a wide variety of both human and mouse tissues, including skeletal muscle. However there was little evidence that this mRNA was translated widely. However a novel 120 kDa polypeptide was specifically detected in kidney. Further characterisation of Up140 and Up71 mRNA revealed that they show transcript-specific differential splicing of exon 71 similar to that described for dystrophin isoforms. However no evidence for splicing of exon 78 of the utrophin gene was found, which was in contrast to dystrophin and may reflect subtle differences in the pattern of phosphorylation between the two proteins. Genomic sequences corresponding to Up71 and Up140 were isolated and the sequence of their proximal promoters explored. The possible functions for the short utrophin transcripts are described and their evolutionary significance explored.
Article
Four-and-a-half LIM domain protein 1 (FHL1) is a member of the FHL protein family that serves as a scaffold protein to maintain normal cellular structure and function. Its mutations have been implicated in multiple muscular diseases. These FHL1 related myopathies are characterized by symptoms such as progressive muscle loss, rigid or bent spine, even cardiac or respiratory failure in some patients, which implies pathological problems not only in muscles, but also in the nervous system. Moreover, decreased FHL1 protein level has been found in patients with FHL1 mutations, indicating the protein loss-of-function as a pathological cause of such diseases. These findings suggest the significance of understanding the systemic role of FHL1 in the homeostasis of nervous system and muscle. Here we reported that Fhl1 loss in C2C12 myotubes obscured acetylcholine receptor (AChR) clustering in addition to myotube fusion, which was associated with impaired MuSK phosphorylation. Mechanistically, myostatin-SMAD2/3 signaling was enhanced, whereas IGF-PI3K-AKT signaling was suppressed in Fhl1−/− C2C12 myotubes. Reversion of these molecular alterations rescued AChR clustering and differentiation deficits. These data outline a systemic regulation of AChR clustering and myotube fusion by FHL1, which may offer clues for mechanism study and development of therapeutic strategies to treat FHL1 related myopathies.
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Background Neuromuscular junctions (NMJs) are chemical synapses formed between motor neurons and skeletal muscle fibers and are essential for controlling muscle contraction. NMJ dysfunction causes motor disorders, muscle wasting, and even breathing difficulties. Increasing evidence suggests that many NMJ disorders are closely related to alterations in specific gene products that are highly concentrated in the synaptic region of the muscle. However, many of these proteins are still undiscovered. Thus, screening for NMJ-specific proteins is essential for studying NMJ and the pathogenesis of NMJ diseases. Results In this study, synaptic regions (SRs) and nonsynaptic regions (NSRs) of diaphragm samples from newborn (P0) and adult (3-month-old) mice were used for RNA-seq. A total of 92 and 182 genes were identified as differentially expressed between the SR and NSR in newborn and adult mice, respectively. Meanwhile, a total of 1563 genes were identified as differentially expressed between the newborn SR and adult SR. Gene Ontology (GO) enrichment analyses, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and gene set enrichment analysis (GSEA) of the DEGs were performed. Protein–protein interaction (PPI) networks were constructed using STRING and Cytoscape. Further analysis identified some novel proteins and pathways that may be important for NMJ development, maintenance and maturation. Specifically, Sv2b, Ptgir, Gabrb3, P2rx3, Dlgap1 and Rims1 may play roles in NMJ development. Hcn1 may localize to the muscle membrane to regulate NMJ maintenance. Trim63, Fbxo32 and several Asb family proteins may regulate muscle developmental-related processes. Conclusion Here, we present a complete dataset describing the spatiotemporal transcriptome changes in synaptic genes and important synaptic pathways. The neuronal projection-related pathway, ion channel activity and neuroactive ligand-receptor interaction pathway are important for NMJ development. The myelination and voltage-gated ion channel activity pathway may be important for NMJ maintenance. These data will facilitate the understanding of the molecular mechanisms underlying the development and maintenance of NMJ and the pathogenesis of NMJ disorders.
Article
The peripheral nerve provides the pathway for motor, sensory, and vegetative axons belonging to the peripheral nervous system. It transmits information between these neurons and their peripheral effectors in both directions (sensory receptors, skeletal muscles, and viscera). The afferences to the periphery correspond to the nerve motor content, whereas efferences from the periphery, in charge of delivering information to the central integrators, correspond to nerve-sensitive content. This information support depends on the intrinsic properties of the nerve itself. Peripheral nerve injuries are frequent and generate significant deficits. Their treatment sometimes leads to functional recovery but is mostly incomplete or unpredictable, despite the regular use of sophisticated repair techniques. The clinician must clearly understand the peripheral nervous system's responses to injury, which reveal surprising degenerating and spontaneous regenerating abilities. This potential recovery is a peripheral nervous system specificity and follows a relatively complex process. Peripheral neurons depend on glial cell structure and metabolism, inducing the global and dynamic response of the whole axon environment, even in cases of focal lesion, modulated by the initial type and mechanism of injury. Today's progress remains insufficient to improve functional prognosis significantly, but a better understanding of peripheral nerve regenerating processes obtained in cellular and molecular biology has opened the door to new medical and surgical advances.
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Dystrophin is associated with a complex of muscle membrane (sarcolemmal) glycoproteins that provide a linkage to the extracellular matrix protein, laminin. The absence of dystrophin leads to a dramatic reduction of the dystrophin-associated proteins (156DAG, 59DAP, 50DAG, 43DAG and 35DAG) in the sarcolemma of patients with Duchenne muscular dystrophy and mdx mice. Here we demonstrate that dystrophin-related protein (DRP, utrophin), an autosomal homologue of dystrophin, is associated with an identical or antigenically similar complex of sarcolemmal proteins and that DRP and the dystrophin/DRP-associated proteins colocalize to the neuromuscular junction in Duchenne muscular dystrophy and mdx muscle. The DRP and dystrophin/DRP-associated proteins are found throughout the sarcolemma in small-calibre skeletal muscles and cardiac muscle of adult mdx mice. Because these muscles show minimal pathological changes, our results could provide a basis for the upregulation of DRP as a potential therapeutic approach.
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Dystrophin was purified by immunoaffinity chromatography from detergent-solubilized Torpedo electric organ postsynaptic membranes using monoclonal antibodies. A major doublet of proteins at Mr 58,000 and minor proteins at Mr 87,000, Mr 45,000, and Mr 30,000 reproducibly copurified with dystrophin. The Mr 58,000 and Mr 87,000 proteins were identical to previously described peripheral membrane proteins (Mr 58,000 protein and 87,000 protein) whose muscle homologs are associated with the sarcolemma (Froehner, S. C., Murnane, A. A., Tobler, M., Peng, H. B., and Sealock, R. (1987) J. Cell Biol. 104, 1633-1646; Carr, C., Fischbach, G. D., and Cohen, J. B. (1989) J. Cell Biol. 109, 1753-1764). The copurification of dystrophin and Mr 58,000 protein was shown to be specific, since dystrophin was also captured with a monoclonal antibody against the Mr 58,000 protein but not by several control antibodies. The Mr 87,000 protein was a major component (along with the Mr 58,000 protein) in material purified on anti-58,000 columns, suggesting that the Mr 58,000 protein forms a distinct complex with the Mr 87,000 protein, as well as with dystrophin. Immunofluorescence staining of skeletal and cardiac muscle from the dystrophin-minus mdx mouse with the anti-58,000 antibody was confined to the sarcolemma as in normal muscle but was much reduced in intensity, even though immunoblotting demonstrated that the contents of Mr 58,000 protein in normal and mdx muscle were comparable. Thus, the Mr 58,000 protein appears to associate inefficiently with the sarcolemmal membrane in the absence of dystrophin. This deficiency may contribute to the membrane abnormalities that lead to muscle necrosis in dystrophic muscle.
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The postsynaptic membrane of the neuromuscular junction contains a myristoylated 43-kD protein (43k) that is closely associated with the cytoplasmic face of the nicotinic acetylcholine receptor (AChR)-rich plasma membrane. Previously, we described fibroblast cell lines expressing recombinant AChRs. Transfection of these cell lines with 43k was necessary and sufficient for reorganization of AChR into discrete 43k-rich plasma membrane domains (Phillips, W. D., C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we demonstrate the utility of this expression system for the study of 43k function by site-directed mutagenesis. Substitution of a termination codon for Asp254 produced a truncated (28-kD) protein that associated poorly with the cell membrane. The conversion of Gly2 to Ala2, to preclude NH2-terminal myristoylation, reduced the frequency with which 43k formed plasma membrane domains by threefold, but did not eliminate the aggregation of AChRs at these domains. Since both NH2 and COOH-termini seemed important for association of 43k with the plasma membrane, a deletion mutant was constructed in which the codon Gln15 was fused in-frame to Ile255 to create a 19-kD protein. This mutated protein formed 43k-rich plasma membrane domains at wild-type frequency, but the domains failed to aggregate AChRs, suggesting that the central part of the 43k polypeptide may be involved in AChR aggregation. Our results suggest that membrane association and AChR interactions are separable functions of the 43k molecule.
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An alpha subunit cDNA of the mouse nicotinic acetylcholine receptor under transcriptional control of the Rous Sarcoma virus long terminal repeat was transfected into and expressed in a quail fibroblast cell line. The biosynthesis and post-translational modification of the alpha subunit protein made in this heterologous system have been studied using immunoprecipitation and ligand binding assays. The polypeptide is present at high steady-state levels and inserted in the correct transmembrane orientation. However, in the absence of assembly with other subunits the alpha subunit is confined to an intracellular membrane compartment and is not transported to the plasma membrane. Twenty percent of the newly synthesized alpha subunit acquired high affinity alpha bungarotoxin binding in a time-dependent process within 20 min of translation. Sucrose gradient fractionation demonstrated that both the polypeptide and toxin binding forms of the alpha subunit have a sedimentation coefficient of 5 s suggesting the absence of stable homo-oligomers. Quantitative binding assays demonstrated that the apparent affinity and rate of association of alpha bungarotoxin to the unassembled alpha subunit are greater than for native receptor. On the other hand, the affinities for the small ligands D-tubocurarine and gallamine are 10(3) lower than for native receptor; no detectable binding was observed for decamethonium, hexamethonium, or carbamylcholine. Thus, the acetylcholine receptor alpha subunit, independent of other subunits of the receptor, acquires a mature conformation and high affinity alpha bungarotoxin binding when expressed in a quail fibroblast cell line.
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The nicotinic acetylcholine receptor and a receptor-associated protein of 43 kDa are the major proteins present in postsynaptic membranes isolated from Torpedo electric organ. Immunochemical analyses indicated that a protein sharing antigenic determinants with the receptor-associated protein is also present at receptor clusters of muscle cell lines and postsynaptic membranes of vertebrate neuromuscular junctions. We now provide definitive proof that a homolog of the 43-kDa protein exists in mammals. Complimentary DNA clones encoding the complete protein sequence have been isolated from the mouse muscle cell line, BC3H1. We heretofore refer to these proteins as nicotinic receptor-associated proteins at synapses or N-RAP-syns. The deduced sequence of mouse RAPsyn has 412 amino acids and a molecular mass of 46,392 daltons. The overall identity with Torpedo RAPsyn is 70%; some regions are extremely well conserved and are therefore postulated to be functionally important. Important domains, including the amino terminus and a cAMP-dependent protein kinase phosphorylation site, are conserved between species. Several structural features are consistent with the proposal that RAPsyn is a peripheral membrane protein that associates with membranes by virtue of covalently bound myristate. Although multiple mRNAs were previously identified in Torpedo electric organ, RNA blot analysis reveals a single polyadenylated RAPsyn mRNA of approximately equal to 2.0 kilobases in newborn and 4-week-old mouse muscle. Finally, genomic DNA blot analysis indicates that a single N-RAPsyn gene is present in the mouse genome.
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In the study of proteins that may participate in the events responsible for organization of macromolecules in the postsynaptic membrane, we have used a mAb to an Mr 58,000 protein (58K protein) found in purified acetylcholine receptor (AChR)-enriched membranes from Torpedo electrocytes. Immunogold labeling with the mAb shows that the 58K protein is located on the cytoplasmic side of Torpedo postsynaptic membranes and is most concentrated near the crests of the postjunctional folds, i.e., at sites of high AChR concentration. The mAb also recognizes a skeletal muscle protein with biochemical characteristics very similar to the electrocyte 58K protein. In immunofluorescence experiments on adult mammalian skeletal muscle, the 58K protein mAb labels endplates very intensely, but staining of extrasynaptic membrane is also seen. Endplate staining is not due entirely to membrane infoldings since a similar pattern is seen in neonatal rat diaphragm in which postjunctional folds are shallow and rudimentary, and in chicken muscle, which lacks folds entirely. Furthermore, clusters of AChR that occur spontaneously on cultured Xenopus myotomal cells and mouse muscle cells of the C2 line are also stained more intensely than the surrounding membrane with the 58K mAb. Denervation of adult rat diaphragm muscle for relatively long times causes a dramatic decrease in the endplate staining intensity. Thus, the concentration of this evolutionarily conserved protein at postsynaptic sites may be regulated by innervation or by muscle activity.
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A protein of Mr 43,000 (43-kDa protein) occurs on the postsynaptic membrane in close association with the acetylcholine receptor and comprises a major part of the postsynaptic cytoskeletal apparatus. We have devised an immunological assay for the 43-kDa protein to determine if it is confined to receptor-specific sites or if it, like general cytoskeletal proteins, has a more widespread tissue distribution. The assay utilizes monoclonal antibodies (Mab) to the 43-kDa protein that recognize two spatially separate epitopes. One Mab, attached to the well of a microtiter plate, binds the antigen which is then available to bind the biotin-derivatized second Mab. Bound second antibody is detected with either avidin-alkaline phosphatase or a more elaborate system using avidin, rabbit anti-avidin, and anti-rabbit IgG-alkaline phosphatase conjugate. A similar assay was developed for the receptor. The 43-kDa protein and the receptor are found in electric organ and, in 500-fold lower concentrations, in skeletal muscle but are not detectable in heart, liver, pancreas, or brain. In electric organ, the receptor and the 43-kDa protein are present in approximately equimolar concentrations. These results indicate that the 43-kDa protein is not a general membrane-associated cytoskeletal element and that its occurrence, and possibly also its function, is related to the acetylcholine receptor.
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A partial cDNA clone for the Duchenne's muscular dystrophy (DMD) locus was used to investigate the expression of this locus in human muscle in vitro. Hybridization to a 14-kilobase RNA transcript was demonstrated in both fetal and mature human skeletal muscle and four lines of human muscle cells in culture. The DMD transcript was not detected in cultured cells outside the muscle lineage. In cultured muscle cells, gene expression was evident only in myotubes both before and after innervation with mouse spinal cord. Primary cultures of human myoblasts did not show the presence of the DMD transcript prior to fusion to form myotubes. An in vitro model is potentially an excellent system in which to investigate factors controlling expression of the DMD gene in normal muscle and how this expression is altered in cultured DMD muscle.
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Forty monoclonal antibodies to acetylcholine receptor from the electric organs of Electrophorus electricus have been characterized by immunoglobulin isotype, affinity for receptor, and specificity for species, subunit, and determinants within subunits. Using these antibodies, nine immunogenic regions on the receptor molecule were distinguished. Most of these are species specific, and are located on various subunits of the acetylcholine receptor. The least species-specific region forms the "main immunogenic region" (MIR). Most monoclonal antibodies and most antibodies in conventional antisera are directed at this region. The MIR is located on the extracellular surface of the alpha subunits and is homologous to the MIR which we previously described on Torpedo californica receptor. An homologous MIR is also a characteristic feature of receptor from mammalian muscle. The possible immunological and structural significance of the MIR is discussed.
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The rotational diffusion of the acetylcholine (ACh) receptor in subsynaptic membrane fragments from Torpedo marmorata electric organ was investigated with a spin-labelled alpha-bungarotoxin. A toxin with two spin labels was first synthesized; the conventional electron spin resonance spectrum (e.s.r.) of this toxin bound to the receptor indicated: (1) a complete immobilization of the probes; and (2) a strong spin-spin interaction that was not, or barely, seen in solution. The modification of the degree of spin-spin interaction is taken as an indication of a toxin conformational change accompanying its binding to the ACh-receptor. To avoid spin-spin interaction a single-labelled toxin was made and used to follow the rotational diffusion of the receptor by saturation transfer e.s.r. (ST-e.s.r.). With native membranes a high immobilization of the ACh-receptor was noticed. Reduction of the membranes by dithiothreitol had little effect on this motion. Only extraction of the 43 000 protein(s) by pH 11 treatment was able to enhance the rotational diffusion of the ACh-receptor protein (rotational correlation time by ST-e.s.r. in the 0.5 - 1 X 10(-4) s range) and to allow its lateral diffusion in the plane of the membrane fragments (observed by electron microscopy after freeze-etching or negative staining).
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Highly purified cholinergic postsynaptic membranes from Torpedo electric tissue contain, in addition to the acetylcholine receptor (AcChoR), major proteins of Mr 43,000 and Mr approximately 90,000 and minor proteins that can be removed from the membranes by alkaline treatment. We have prepared an antiserum to these alkaline-extractable proteins that reacts with the Mr 43,000 protein but not with any of the other major membrane proteins, including the AcChoR subunits. Immunofluorescent staining of sections of Torpedo electric tissue shows that this antiserum binds to the innervated but not the uninnervated surface of the electrocytes. In rat diaphragm muscle, the antigens recognized by this antiserum are highly concentrated at the synapse. Synaptic staining of muscle is eliminated by prior incubation of the antiserum with the Mr 43,000 protein but not by incubation with affinity-purified AcChoR. This antiserum stains end plates of muscles denervated for 7 days. Antiserum to AcChoR binds to the subsynaptic membranes of electrocytes and muscle but does not react with the Mr 43,000 protein. Purified AcChoR blocks staining of synapses by anti-AcChoR but the Mr 43,000 protein does not. These results indicate that the Mr 43,000 protein is located in the innervated membrane of Torpedo electrocytes and that an immunologically similar component is highly concentrated in the postsynaptic membrane of mammalian muscle.
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After fixation with glutaraldehyde and impregnation with tannic acid, the membrane that underlies the nerve terminals in Torpedo marmorata electroplaque presents a typical asymmetric triple-layered structure with an unusual thickness; in addition, it is coated with electron-dense material on its inner, cytoplasmic face. Filamentous structures are frequently found attached to these "subsynaptic densities." The organization of the subsynaptic membrane is partly preserved after homogenization of the electric organ and purification of acetylcholine-receptor (AchR)-rich membrane fragments. In vitro treatment at pH 11 and 4 degrees C of these AchR-rich membranes releases an extrinsic protein of 43,000 mol wt and at the same time causes the complete disappearance of the cytoplasmic condensations. Freeze-etching of native membrane fragments discloses remnants of the ribbonlike organization of the AchR rosettes. This organization disappears ater alkaline treatment and is replaced by a network which is not observed after rapid freezing and, therefore, most likely results from the lateral redistribution of the AchR rosettes during condition of slow freezing. A dispersion of the AchR rosettes in the plane of the membrane also occurs after fusion of the pH 11-treated fragments with phospholipid vesicles. These results are interpreted in terms of a structural stabilization and immobilization of the AchR by the 43,000-Mr protein binding to the inner face of the subsynaptic membrane.
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A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.
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We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.
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The carboxy-terminal region of dystrophin has been suggested to be crucially important for its function to prevent muscle degeneration. We have previously shown that this region is the locus that interacts with the sarcolemmal glycoprotein complex, which mediates membrane anchoring of dystrophin, as well as with the cytoplasmic peripheral membrane protein, A0 and beta 1-syntrophin (Suzuki, A., M. Yoshida, K. Hayashi, Y. Mizuno, Y. Hagiwara, and E. Ozawa. 1994. Eur. J. Biochem. 220:283-292). In this work, by using the overlay assay technique developed previously, we further analyzed the dystrophin-syntrophin/A0 interaction. Two forms of mammalian syntrophin, alpha 1- and beta 1-syntrophin, were found to bind to very close but discrete regions on the dystrophin molecule. Their binding sites are located at the vicinity of the glycoprotein-binding site, and correspond to the amino acid residues encoded by exons 73-74 which are alternatively spliced out in some isoforms. This suggests that the function of syntrophin is tightly linked to the functional diversity among dystrophin isoforms. Pathologically, it is important that the binding site for alpha 1-syntrophin, which is predominantly expressed in skeletal muscle, coincides with the region whose deletion was suggested to result in a severe phenotype. In addition, A0, a minor component of dystrophin-associated proteins with a molecular mass of 94 kD which is immunochemically related to syntrophin, binds to the same site as beta 1-syntrophin. Finally, based on our accumulated evidence, we propose a revised model of the domain organization of dystrophin from the view point of protein-protein interactions.
Article
Four mouse monoclonal antibodies (mabs) were shown by immunoblotting procedures to recognize the major, basic, membrane-bound Mr 43,000 protein (43K protein) of acetylcholine receptor-rich postsynaptic membranes from Torpedo nobiliana . These mabs and a mab against an extracellular determinant on the acetylcholine receptor were used to localize the two proteins in electroplax (Torpedo californica) and on unsealed postsynaptic membrane fragments at the ultrastructural level. Bound mabs were revealed with a rabbit anti-mouse Ig serum and protein A-colloidal gold. The anti-43K mabs bound only to the cytoplasmic surface of the postsynaptic membrane. The distributions of the receptor and the 43K protein along the membrane were found to be coextensive. Distances between the membrane center and gold particles were very similar for anti-receptor and anti-43K mabs (29 +/- 7 nm and 26 to 29 +/- 7 to 10 nm, respectively). These results show that the 43K protein is a receptor-specific protein having a restricted spatial relationship to the membrane. They thus support models in which the 43K protein is associated with the cytoplasmic domains of the receptor molecule.
Article
After fixation with glutaraldehyde and impregnation with tannic acid, the membrane that underlies the nerve terminals in Torpedo marmorata electroplaque presents a typical asymmetric triple-layered structure with an unusual thickness; in addition, it is coated with electron-dense material on its inner, cytoplasmic face. Filamentous structures are frequently found attached to these "subsynaptic densities." The organization of the subsynaptic membrane is partly preserved after homogenization of the electric organ and purification of acetylcholine-receptor (AchR)-rich membrane fragments. In vitro treatment at pH 11 and 4 degrees C of these AchR-rich membranes releases an extrinsic protein of 43,000 mol wt and at the same time causes the complete disappearance of the cytoplasmic condensations. Freeze-etching of native membrane fragments discloses remnants of the ribbonlike organization of the AchR rosettes. This organization disappears ater alkaline treatment and is replaced by a network which is not observed after rapid freezing and, therefore, most likely results from the lateral redistribution of the AchR rosettes during condition of slow freezing. A dispersion of the AchR rosettes in the plane of the membrane also occurs after fusion of the pH 11-treated fragments with phospholipid vesicles. These results are interpreted in terms of a structural stabilization and immobilization of the AchR by the 43,000-Mr protein binding to the inner face of the subsynaptic membrane.
Article
To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43- and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87- and 270-kD proteins were cleaved by Ca++-activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.
Article
A 58-kD protein, identified in extracts of postsynaptic membrane from Torpedo electric organ, is enriched at sites where acetylcholine receptors (AChR) are concentrated in vertebrate muscle (Froehner, S. C., A. A. Murnane, M. Tobler, H. B. Peng, and R. Sealock. 1987. J. Cell Biol. 104:1633-1646). We have studied the 58-kD protein in AChR clusters isolated from cultured rat myotubes. Using immunofluorescence microscopy we show that the 58-kD protein is highly enriched at AChR clusters, but is also present in regions of the myotube membrane lacking AChR. Within clusters, the 58-kD protein codistributes with AChR, and is absent from adjacent membrane domains involved in myotube-substrate contact. Semiquantitative fluorescence measurements suggest that molecules of the 58-kD protein and AChR are present in approximately equal numbers. Differential extraction of peripheral membrane proteins from isolated AChR clusters suggests that the 58-kD protein is more tightly bound to cluster membrane than is actin or spectrin, but less tightly bound than the receptor-associated 43-kD protein. When AChR clusters are disrupted either in intact cells or after isolation, the 58-kD protein still codistributes with AChR. Clusters visualized by electron microscopy after immunogold labeling and quick-freeze, deep-etch replication show that, within AChR clusters, the 58-kD protein is sharply confined to AChR-rich domains, where it is present in a network of filaments lying on the cytoplasmic surface of the membrane. Additional actin filaments overlie, and are attached to, this network. Our results suggest that within AChR domains of clusters, the 58-kD protein lies between AChR and the receptor-associated 43-kD protein, and the membrane-skeletal proteins, beta-spectrin, and actin.
Article
After alkaline extraction, purified subsynaptic fragments isolated from Torpedo electric tissue exhibit on sodium dodecyl sulfate/polyacrylamide gel electrophoresis predominant peptides of apparent Mr 41,000, 50,000, and 65,000 (i.e., the peptides characteristic of the nicotinic receptor purified and isolated in detergent solutions). The peptide of Mr 43,000 that is also found in the isolated postsynaptic membranes is recovered in the supernatant after alkaline extraction. The alkaline-extracted membranes were functionally intact, as demonstrated by the following criteria. The kinetics of binding of [3H]acetylcholine in the presence and absence of 30 micron carbamoylcholine to occupy acetylcholine binding sites, [14C]-meproadifen [2-(diethylmethylaminoethyl)-2,2-diphenylvalerate iodide ] was bound with a dissociation constant, KD, of 0.3 +/- 0.1 micron to 0.3 +/- 0.1 site per [3H]alpha-toxin site. This binding was displaced by perhydrohistrionicotoxin. The carbamoylcholine-stimulated efflux of 22Na+ from the Torpedo vesicles were preserved after alkaline extraction. It is concluded that not only the acetylcholine binding site, but also the local anesthetic binding site, must be associated with the peptides of the cholinergic receptor itself and not that of Mr 43,000. Those peptides remaining after alkaline extraction are also sufficient for permeability control.
Article
Several continuous tissue culture cell lines were established from methylcholanthrene-induced fibrosarcomas of Japanese quail. The lines consist either of fibroblastic elements, round refractile cells or polygonal cells. They show transformed characteristics in agar colony formation and hexose uptake, and most are tumorigenic. Their cloning efficiency in plastic dishes is not increased over that of normal quail embryo fibroblasts. The quail tumor cell lines do not produce endogenous avian oncoviruses and fail to complement the Bryan high titer strain of Rous sarcoma virus; those tested lack the p27 protein of avian oncoviruses. Most of the cell lines are susceptible to subgroup A avian sarcoma viruses, but are relatively resistant to viruses of subgroups C, E and F as compared to normal quail embryo fibroblasts.
Article
1. The distribution of alpha-bungarotoxin binding sites on embryonic and neonatal rat skeletal muscle fibres was determined by autoradiography. Most of the bungarotoxin binding could be inhibited by curare. This observation, together with the spatial distribution of toxin-binding sites, indicates that the distribution of bound toxin reflects that of acetylcholine (ACh) receptors on these developing muscle cells.2. At 15 days of embryogenesis, muscle fibres showed an essentially uniform distribution of receptors. By 16 days, many fibres showed an accumulation of receptors in their mid-region. This accumulation was at the same location as histochemically demonstrated cholinesterase activity.3. At 16 days ACh receptors were distributed over the entire length of the fibres, with a gradient of increasing density as the accumulation was appoached. The density of toxin binding sites in the accumulation was greater than the general level on 15 day cells, suggesting that the high junctional density does not develop solely by the loss of extrajunctional receptors.4. The accumulations of ACh receptors became more pronounced and circumscribed with embryonic development, and after birth the extent of the localizations appeared to follow the size of the neuromuscular junction. The extrajunctional receptor density decreased with development, and by 1 week after birth was undetectable by the methods used.5. The results suggest that the high junctional receptor density found on adult, innervated skeletal muscle fibres develops after the formation of the neuromuscular junction.
Article
Agrin is thought to mediate the motor neuron-induced aggregation of synaptic proteins on the surface of muscle fibers at neuromuscular junctions. Recent experiments provide direct evidence in support of this hypothesis, reveal the nature of agrin immunoreactivity at sites other than neuromuscular junctions, and have resulted in findings that are consistent with the possibility that agrin plays a role in synaptogenesis throughout the nervous system.
Article
The developing neuromuscular junction has provided an important paradigm for studying synapse formation. An outstanding feature of neuromuscular differentiation is the aggregation of acetylcholine receptors (AChRs) at high density in the postsynaptic membrane. While AChR aggregation is generally believed to be induced by the nerve, the mechanisms underlying aggregation remain to be clarified. A 43-kD protein (43k) normally associated with the cytoplasmic aspect of AChR clusters has long been suspected of immobilizing AChRs by linking them to the cytoskeleton. In recent studies, the AChR clustering activity of 43k has, at last, been demonstrated by expressing recombinant AChR and 43k in non-muscle cells. Mutagenesis of 43k has revealed distinct domains within the primary structure which may be responsible for plasma membrane targeting and AChR binding. Other lines of study have provided clues as to how nerve-derived (extracellular) AChR-cluster inducing factors such as agrin might activate 43k-driven postsynaptic membrane specialization.
Article
The distributions of dystrophin, 'dystrophin-related protein' (DRP) and beta-spectrin were compared with that of acetylcholine receptors (AChRs) at rat nerve-muscle junctions (NMJs) using immunofluorescence techniques. In sections, monoclonal antibodies (MAbs) to dystrophin and beta-spectrin labelled the entire sarcolemma but were concentrated at the NMJs while those to DRP labelled only NMJs. In permeabilized muscle fibres, DRP was precisely co-localized with the AChRs, whereas the zone of high density labelling of dystrophin and beta-spectrin extended 0.3-0.4 microns beyond the AChRs. Within the NMJ, the labelling of DRP appeared as a series of interconnecting lines similar to that of AChRs. However, labelling of dystrophin and beta-spectrin was consistently more punctate. These data suggest DRP is more closely associated with AChRs than are dystrophin or beta-spectrin.
Article
Dystrophin-related protein (DRP) is an autosomal gene product with high homology to dystrophin. We have used highly specific antibodies to the unique C-terminal peptide sequences of DRP and dystrophin to examine the subcellular localization and biochemical properties of DRP in adult skeletal muscle. DRP is enriched in isolated sarcolemma from control and mdx mouse muscle, but is much less abundant than dystrophin. Immunofluorescence microscopy localized DRP almost exclusively to the neuromuscular junction region in rabbit and mouse skeletal muscle, as well as mdx mouse muscle and denervated mouse muscle. DRP is also present in normal size and abundance and localizes to the neuromuscular junction region in muscle from the dystrophic mouse model dy/dy. Thus, DRP is a junction-specific membrane cytoskeletal protein that may play an important role in the organization of the postsynaptic membrane of the neuromuscular junction.
Article
Nicotinic acetylcholine receptors (AChRs) are localized at high concentrations in the postsynaptic membrane of the neuromuscular junction. A peripheral membrane protein of Mr 43,000 (43K protein) is closely associated with AChRs and has been proposed to anchor receptors at postsynaptic sites. We have used the Xenopus oocyte expression system to test the idea that the 43K protein clusters AChRs. Mouse muscle AChRs expressed in oocytes after injection of RNA encoding receptor subunits are uniformly distributed in the surface membrane. Coinjection of AChR RNA and RNA encoding the mouse muscle 43K protein causes AChRs to form clusters of 0.5-1.5 microns diameter. AChR clustering is not a consequence of increased receptor expression in the surface membrane or nonspecific clustering of all membrane proteins. The 43K protein is colocalized with AChRs in clusters when the two proteins are expressed together and forms clusters of similar size even in the absence of AChRs. These results provide direct evidence that the 43K protein causes clustering of AChRs and suggest that regulation of 43K protein clustering may be a key step in neuromuscular synaptogenesis.
Article
Neurotransmitter receptors are generally clustered in the postsynaptic membrane. The mechanism of clustering was analyzed with fibroblast cell lines that were stably transfected with the four subunits for fetal (alpha, beta, gamma, delta) or adult (alpha, beta, epsilon, delta) type mouse muscle nicotinic acetylcholine receptors (AChRs). Immunofluorescent staining indicated that AChRs were dispersed on the surface of these cells. When transiently transfected with an expression construct encoding a 43-kilodalton protein that is normally concentrated under the postsynaptic membrane, AChRs expressed in these cells became aggregated in large cell-surface clusters, colocalized with the 43-kilodalton protein. This suggests that 43-kilodalton protein can induce AChR clustering and that cluster induction involves direct contact between AChR and 43-kilodalton protein.
Article
Dystrophin Related Protein is the recently identified protein product of a large autosomal transcript, showing significant similarity to dystrophin at the carboxyl terminus. Dystrophin related protein and dystrophin share a similar abundance and molecular weight, however, they differ both in their tissue distribution and expression in Duchenne/Becker muscular dystrophy. Here we define the immunolocalization of dystrophin related protein to neuromuscular and myotendinous junctions, along with peripheral nerves and vasculature of skeletal muscle. Groups of regenerating muscle fibres as well as embryonic and neonatal muscle express far greater amounts of dystrophin related protein compared with adult mdx mice. These findings may explain the paradoxical labelling seen using dystrophin antibodies in Duchenne patients and dystrophin deficient mdx mice. Finally, no abnormalities of dystrophin related protein expression were detected in three patients with Duchenne-like autosomal recessive muscular dystrophy.
Article
The stoichiometry, cellular location, glycosylation, and hydrophobic properties of the components in the dystrophin-glycoprotein complex were examined. The 156, 59, 50, 43, and 35 kd dystrophin-associated proteins each possess unique antigenic determinants, enrich quantitatively with dystrophin, and were localized to the skeletal muscle sarcolemma. The 156, 50, 43, and 35 kd dystrophin-associated proteins contained Asn-linked oligosaccharides. The 156 kd dystrophin-associated glycoprotein contained terminally sialylated Ser/Thr-linked oligosaccharides. Dystrophin, the 156 kd, and the 59 kd dystrophin-associated proteins were found to be peripheral membrane proteins, while the 50 kd, 43 kd, and 35 kd dystrophin-associated glycoproteins and the 25 kd dystrophin-associated protein were confirmed as integral membrane proteins. These results demonstrate that dystrophin and its 59 kd associated protein are cytoskeletal elements that are tightly linked to a 156 kd extracellular glycoprotein by way of a complex of transmembrane proteins.
Article
A 58-kD protein, identified in extracts of postsynaptic membrane from Torpedo electric organ, is enriched at sites where acetylcholine receptors (AChR) are concentrated in vertebrate muscle (Froehner, S. C., A. A. Murnane, M. Tobler, H. B. Peng, and R. Sealock. 1987. J. Cell Biol. 104:1633-1646). We have studied the 58-kD protein in AChR clusters isolated from cultured rat myotubes. Using immunofluorescence microscopy we show that the 58-kD protein is highly enriched at AChR clusters, but is also present in regions of the myotube membrane lacking AChR. Within clusters, the 58-kD protein codistributes with AChR, and is absent from adjacent membrane domains involved in myotube-substrate contact. Semiquantitative fluorescence measurements suggest that molecules of the 58-kD protein and AChR are present in approximately equal numbers. Differential extraction of peripheral membrane proteins from isolated AChR clusters suggests that the 58-kD protein is more tightly bound to cluster membrane than is actin or spectrin, but less tightly bound than the receptor-associated 43-kD protein. When AChR clusters are disrupted either in intact cells or after isolation, the 58-kD protein still codistributes with AChR. Clusters visualized by electron microscopy after immunogold labeling and quick-freeze, deep-etch replication show that, within AChR clusters, the 58-kD protein is sharply confined to AChR-rich domains, where it is present in a network of filaments lying on the cytoplasmic surface of the membrane. Additional actin filaments overlie, and are attached to, this network. Our results suggest that within AChR domains of clusters, the 58-kD protein lies between AChR and the receptor-associated 43-kD protein, and the membrane-skeletal proteins, beta-spectrin, and actin.
Article
mAbs specific for protein components of the surface membrane of rabbit skeletal muscle have been used as markers in the isolation and characterization of skeletal muscle sarcolemma membranes. Highly purified sarcolemma membranes from rabbit skeletal muscle were isolated from a crude surface membrane preparation by wheat germ agglutination. Immunoblot analysis of subcellular fractions from skeletal muscle revealed that dystrophin and its associated glycoproteins of 156 and 50 kD are greatly enriched in purified sarcolemma vesicles. The purified sarcolemma was also enriched in novel sarcolemma markers (SL45, SL/TS230) and Na+/K(+)-ATPase, whereas t-tubule markers (alpha 1 and alpha 2 subunits of dihydropyridine receptor, TS28) and sarcoplasmic reticulum markers (Ca2(+)-ATPase, ryanodine receptor) were greatly diminished in this preparation. Analysis of isolated sarcolemma by SDS-PAGE and densitometric scanning demonstrated that dystrophin made up 2% of the total protein in the rabbit sarcolemma preparation. Therefore, our results demonstrate that although dystrophin is a minor muscle protein it is a major constituent of the sarcolemma membrane in skeletal muscle. Thus the absence of dystrophin in Duchenne muscular dystrophy may result in a major disruption of the cytoskeletal network underlying the sarcolemma in dystrophic muscle.
Article
We found six groups of proteins, A0-A5, besides dystrophin itself in a dystrophin preparation obtained by the reported method [Campbell, K.P. & Kahl, S.D.(1989) Nature 338, 259-262] with some modifications. Their molecular weights were 94, 62, 52, 43, 36, and 24 kDa, respectively. Their molar ratios to dystrophin were 0.14, 2.2, 0.88, 0.90, 1.7, and 0.34, respectively. Each of A1, A3, and A4 was split into several bands. But each group of bands except A3 seemed to behave like the same kind of protein. The doublet of A3 was subdivided into A3a and A3b in the decreasing order of molecular weight. All the A-proteins except A2 were cross-linked with dystrophin molecule by a cross-linker, bis(sulfosuccinimidyl)suberate, suggesting them to be dystrophin-associated proteins. When dystrophin preparation was treated with KI, which is known to break membrane cytoskeletal interactions, as described by Campbell and Kahl, A2, A3, and A4 were absorbed by wheat germ lectin (WGL) Sepharose, but the dystrophin molecule and A1 were not absorbed. On the other hand, A2 and A3b reacted with biotinyl WGL but A3a and A4 did not in blotting analysis. This apparent discrepancy can be explained if we postulate that A3a and/or A4 would associate with A2 and/or A3b. On the basis of these results including stoichiometric considerations, we are of the opinion that the complex of A2.A4 among various possible ones is the most important to anchor dystrophin to sarcolemma. In this A2.A4 complex, A4 but not A2 is directly associated with dystrophin.
Article
To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43- and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87- and 270-kD proteins were cleaved by Ca++-activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.
Article
Duchenne muscular dystrophy (DMD), a sex-linked degenerative disorder of the muscle, is one of the most common lethal genetic diseases in man. It affects about one male in 3,500, with an estimated one-third of cases being caused by new mutations. A less severe disease, Becker's muscular dystrophy (BMD), maps to the same chromosomal locus and is most probably an allelic form of DMD. Both diseases are sometimes associated with various degrees of mental retardation; the molecular basis of these phenotypes is unknown (for review, see ref. 1). The giant DMD gene spans approximately 2,000 kilobases (kb) (0.05% of the human genome) and encodes a 14-kb mRNA. The tissue-specificity of its expression has not been precisely determined. Monaco et al., using Northern blots, reported expression of the gene in human fetal skeletal muscle and small intestine but not in human fetal brain, or in human cultured myoblasts and transformed B and T cells. More recently, expression was detected in mouse skeletal and cardiac muscle, but not in mouse brain. Here we show, using a ribonuclease protection assay, that the DMD gene is developmentally regulated in rat and mouse myogenic cell cultures, and that it is expressed in rat and mouse striated muscle, in mouse smooth muscle and in rat, mouse and rabbit brain. We could not detect transcripts in other non-muscle tissues.
Article
Complementary DNA clones were isolated that represent the 5' terminal 2.5 kilobases of the murine Duchenne muscular dystrophy (Dmd) messenger RNA (mRNA). Mouse Dmd mRNA was detectable in skeletal and cardiac muscle and at a level approximately 90 percent lower in brain. Dmd mRNA is also present, but at much lower than normal levels, in both the muscle and brain of three different strains of dystrophic mdx mice. The identification of Dmd mRNA in brain raises the possibility of a relation between human Duchenne muscular dystrophy (DMD) gene expression and the mental retardation found in some DMD males. These results also provide evidence that the mdx mutations are allelic variants of mouse Dmd gene mutations.