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Christian Fuhrer - One of the best experts on this subject based on the ideXlab platform.

  • diverse molecular mechanisms involved in achr deficiency due to Rapsyn mutations
    Brain, 2006
    Co-Authors: John Cossins, Hayley Spearman, Christian Fuhrer, Angela Vincent, G Burke, Susan Maxwell, Somai Man, J B M Kuks, Jackie Palace, David Beeson
    Abstract:

    Congenital myasthenic syndromes are inherited disorders of neuromuscular transmission characterized by fatigable muscle weakness. Autosomal recessive acetylcholine receptor (AChR) deficiency syndromes, in which levels of this receptor at the neuromuscular junction are severely reduced, may be caused by mutations within genes encoding the AChR or the AChR-clustering protein, Rapsyn. Most patients have mutations within the Rapsyn coding region and are either homozygous for N88K or heteroallelic for N88K and a second mutation. In some cases the second allele carries a null mutation but in many the mutations are missense, and are located in different functional domains. Little is known about the functional effects of these mutations, but we hypothesize that they would have an effect on AChR clustering by a variety of mechanisms that might correlate with disease severity. Here we expressed RAPSN mutations A25V, N88K, R91L, L361R and K373del in TE671 cells and in Rapsyn-/- myotubes to determine their pathogenic mechanisms. The A25Vmutation impaired colocalization of Rapsyn with AChR and prevented agrin-induced AChR clusters in Rapsyn-/- myotubes. In TE671 cells, R91L reduced the ability of Rapsyn to self-associate, and K373del-Rapsyn was significantly less stable than wild-type. The effects of mutations L361R and N88K were more subtle: in TE671 cells, in comparison with wild-type Rapsyn, L361R-Rapsyn showed reduced expression/stability, and both N88K-Rapsyn and L361R-Rapsyn showed significantly reduced co-localization with AChR. N88K-Rapsyn and L361R-Rapsyn could effectively mediate agrin-induced AChR clusters, but these were reduced in number and were less stable than with wild-type Rapsyn. The disease severity of patients harbouring the compound allelic mutations was greater than that of patients with homozygous Rapsyn mutation N88K, suggesting that the second mutant allele may largely determine severity.

  • the postsynaptic submembrane machinery at the neuromuscular junction requirement for Rapsyn and the utrophin dystrophin associated complex
    Journal of Neurocytology, 2003
    Co-Authors: Glen B Banks, Christian Fuhrer, Marvin E Adams, Stanley C Froehner
    Abstract:

    Neuromuscular synapse formation is brought about by a complex bi-directional exchange of information between the innervating motor neuron and its target skeletal muscle fiber. Agrin, a heparin sulfate proteoglycan, is released from the motor nerve terminal to activate its muscle-specific kinase (MuSK) receptor that leads to a second messenger cascade requiring Rapsyn to ultimately bring about AChR clustering in the muscle membrane. Rapsyn performs many functions in skeletal muscle. First, Rapsyn and AChRs co-target to the postsynatic apparatus. Second, Rapsyn may self associate to stabilize and promote AChR clustering. Third, Rapsyn is essential for AChR cluster formation. Fourth, Rapsyn is required to transduce the agrin-evoked MuSK phosphorylation signal to AChRs. Finally, Rapsyn links AChRs to the utrophin-associated complex, which appears to be required for AChR stabilization as well as maturation of the neuromuscular junction. Proteins within the utrophin-associated complex such as alpha-dystrobrevin and alpha-syntrophin are also important for signaling events that affect neuromuscular synapse stability and function. Here we review our current understanding of the role of the postsynaptic-submembrane machinery involving Rapsyn and the utrophin-associated complex at the neuromuscular synapse. In addition we briefly review how these studies of the neuromuscular junction relate to GABAergic and glycinergic synapses in the CNS.

  • agrin regulates Rapsyn interaction with surface acetylcholine receptors and this underlies cytoskeletal anchoring and clustering
    Journal of Biological Chemistry, 2003
    Co-Authors: Martijn Moransard, Lucia S Borges, Raffaella Willmann, Angelo P Marangi, Hans Rudolf Brenner, Michael J Ferns, Christian Fuhrer
    Abstract:

    The acetylcholine receptor (AChR)-associated protein Rapsyn is essential for neuromuscular synapse formation and clustering of AChRs, but its mode of action remains unclear. We have investigated whether agrin, a key nerve-derived synaptogenic factor, influences Rapsyn-AChR interactions and how this affects clustering and cytoskeletal linkage of AChRs. By precipitating AChRs and probing for associated Rapsyn, we found that in denervated diaphragm Rapsyn associates with synaptic as well as with extrasynaptic AChRs showing that Rapsyn interacts with unclustered AChRs in vivo. Interestingly, synaptic AChRs are associated with more Rapsyn suggesting that clustering of AChRs may require increased interaction with Rapsyn. In similar experiments in cultured myotubes, Rapsyn interacted with intracellular AChRs and with unclustered AChRs at the cell surface, although surface interactions are much more prominent. Remarkably, agrin induces recruitment of additional Rapsyn to surface AChRs and clustering of AChRs independently of the secretory pathway. This agrin-induced increase in Rapsyn-AChR interaction strongly correlates with clustering, because staurosporine and herbimycin blocked both the increase and clustering. Conversely, laminin and calcium induced both increased Rapsyn-AChR interaction and AChR clustering. Finally, time course experiments revealed that the agrin-induced increase occurs with AChRs that become cytoskeletally linked, and that this precedes receptor clustering. Thus, we propose that neural agrin controls postsynaptic aggregation of the AChR by enhancing Rapsyn interaction with surface AChRs and inducing cytoskeletal anchoring and that this is an important precursor step for AChR clustering.

  • laminin 1 redistributes postsynaptic proteins and requires Rapsyn tyrosine phosphorylation and src and fyn to stably cluster acetylcholine receptors
    Journal of Cell Biology, 2002
    Co-Authors: Angelo P Marangi, Simon T Wieland, Christian Fuhrer
    Abstract:

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

  • agrin induced activation of acetylcholine receptor bound src family kinases requires Rapsyn and correlates with acetylcholine receptor clustering
    Journal of Biological Chemistry, 2001
    Co-Authors: Peggy Mittaud, Angelo P Marangi, Susanne Erbvogtli, Christian Fuhrer
    Abstract:

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

Richard L Huganir - One of the best experts on this subject based on the ideXlab platform.

  • identification of a torpedo homolog of sam68 that interacts with the synapse organizing protein Rapsyn
    FEBS Letters, 1998
    Co-Authors: Eric T Fung, Anthony Lanahan, Paul F Worley, Richard L Huganir
    Abstract:

    Nicotinic acetylcholine receptors (nAChRs) are initially expressed diffusely on the surface of myotubes and, in response to neuronally derived factors, cluster at the endplate to a final concentration of approximately 10000/microm2. The synaptic peripheral membrane protein Rapsyn has been shown to mediate clustering of nAChRs in several systems. Here we describe the use of the yeast two-hybrid system to identify proteins that can interact with Rapsyn. One of the clones we have identified is a Torpedo californica homolog of the Src-associated in mitosis protein (Sam68). We further show that Sam68, like Rapsyn, is localized at the neuromuscular junction.

  • the synapse associated protein Rapsyn regulates tyrosine phosphorylation of proteins colocalized at nicotinic acetylcholine receptor clusters
    Molecular and Cellular Neuroscience, 1996
    Co-Authors: Elizabeth D Apel, John P. Merlie, Carol Doherty, Peter W Hoffman, Richard L Huganir
    Abstract:

    Abstract Protein tyrosine phosphorylation has been suggested to play an important role in the clustering of the nicotinic acetylcholine receptor (AChR) at the developing neuromuscular junction. Recent studies have shown that the 43-kDa synapse-associated protein Rapsyn induces clustering of the AChR in heterologous expression systems. In this study we examined whether tyrosine phosphorylation is involved in this Rapsyn-induced AChR clustering. Rapsyn-induced AChR clusters in fibroblasts contain phosphotyrosine, as detected using immunofluorescent labeling with anti-phosphotyrosine antibodies. No anti-phosphotyrosine staining of Rapsyn clusters is seen in the absence of AChR expression, indicating that the AChR is required for the appearance of phosphotyrosine at clusters. In addition, coexpression of Rapsyn with the AChR induces the tyrosine phosphorylation of the β amd δ subunits of the AChR. Surprisingly, mutation of the tyrosine phosphorylation sites in the AChR did not inhibit Rapsyn-induced clustering of the AChR and clusters of the mutant AChRs still contained high levels of phosphotyrosine. Experiments with single AChR subunits demonstrate that the α subunit of the AChR appears to be necessary and sufficient for codistribution of phosphotyrosine with Rapsyn-induced clusters of AChR subunits. Finally, transfection of cells with Rapsyn activates cellular protein tyrosine kinase activity, resulting in the tyrosine phosphorylation of several membrane-associated proteins. These results suggest that Rapsyn may therefore regulate clustering at least in part by regulating the tyrosine phosphorylation of cellular proteins.

  • Rapsyn clusters and activates the synapse specific receptor tyrosine kinase musk
    Neuron, 1996
    Co-Authors: Susan K H Gillespie, Sudha Balasubramanian, Eric T Fung, Richard L Huganir
    Abstract:

    Abstract Nerve-induced clustering of the nicotinic acetylcholine receptor (AChR) requires Rapsyn, a synaptic peripheral membrane protein, as well as protein-tyrosine kinase activity. Here, we show that Rapsyn induces the clustering of the synapse-specific receptor-tyrosine kinase MuSK in transfected QT-6 fibroblasts. Furthermore, Rapsyn stimulates the autophosphorylation of MuSK, leading to a subsequent MuSK-dependent increase in cellular tyrosine phosphorylation. Moreover, Rapsyn-activated MuSK specifically phosphorylated the AChR β subunit, the same subunit that is tyrosine phosphorylated during innervation or agrin treatment of muscle. These results suggest Rapsyn may mediate the synaptic localization of MuSK in muscle and that MuSK may play an important role in the agrin-induced clustering of the AChR.

Andrew G Engel - One of the best experts on this subject based on the ideXlab platform.

  • myasthenic syndrome due to defects in Rapsyn clinical and molecular findings in 39 patients
    Neurology, 2009
    Co-Authors: Margherita Milone, Kinji Ohno, Xinming Shen, Duygu Selcen, Joan M Brengman, Susan T Iannaccone, C M Harper, Andrew G Engel
    Abstract:

    The congenital myasthenic syndromes (CMS) are heterogenous disorders of neuromuscular transmission caused by defects in presynaptic, synaptic, and postsynaptic proteins of the neuromuscular junction.1 Among the 295 patients with CMS investigated at the Mayo Clinic, 15% carry mutations in Rapsyn, a postsynaptic protein.1 Rapsyn binds to the long cytoplasmic loop of the AChR subunits2 and is essential for clustering and anchoring AChR in the postsynaptic membrane. Rapsyn is composed of several functionally distinct regions (figure 1A): a myristoylated N-terminal is required for membrane interaction3; 7 tetratricopeptide repeats (TPR) are important for Rapsyn self-aggregation4 and binding to the cytoplasmic portion of the muscle-specific kinase MuSK5; the coiled-coil domain interacts with the cytoplasmic loops of AChR subunits6; and the C-terminal domain binds to β dystroglycan and thereby links the Rapsyn-AChR complex to the cytoskeleton.7 Mutations in Rapsyn compromise the safety margin of neuromuscular transmission by causing endplate (EP) AChR deficiency.8 Figure 1 Genetic analysis of Rapsyn The clinical spectrum of Rapsyn-CMS varies from severe hypotonia and arthrogryposis at birth to mild limb muscle weakness. Early and late-onset phenotypes have been described.9 Episodic respiratory crises and lack of ophthalmoparesis have been reported as hallmarks of Rapsyn-CMS that differentiate it from the CMS caused by low expressor mutations in the AChR ɛ subunit.10 Here we review our findings in 39 Rapsyn-CMS patients investigated at the Mayo Clinic.

  • novel truncating rapsn mutations causing congenital myasthenic syndrome responsive to 3 4 diaminopyridine
    Neuromuscular Disorders, 2004
    Co-Authors: Brenda Banwell, Kinji Ohno, Joern P Sieb, Andrew G Engel
    Abstract:

    Abstract Rapsyn is essential for clustering the acetylcholine receptor at the postsynaptic membrane of the neuromuscular junction. Direct sequencing of RAPSN in two children with congenital myasthenic syndromes with no mutation in any of the AChR subunits identified two heterozygous recessive mutations in each: a previously characterized N88K mutation in both, and a second frameshifting mutation in Patient (Pt) 1 and a nonsense mutation in Pt 2. An intercostal muscle biopsy in Pt 1 revealed decreased AChRs per endplate and decreased amplitude of the miniature endplate potential, predicted consequences of Rapsyn deficiency. Clinically, both children manifested with hypomotility in utero, fatigable ocular and limb weakness since birth, decreased strength during viral illness, decremental response on electromyography, and absence of AChR antibodies. Pt 1, however, had a more severe clinical course with recurrent episodes of respiratory failure, contractures, and craniofacial malformations. In both patients, treatment with pyridostigmine was of some benefit, but the addition of 3,4-diaminopyridine led to significant clinical improvement. Thus, Rapsyn deficiency predicting similar consequences at the cellular level can result in phenotypes with marked differences in severity of symptoms, risk of respiratory failure, and presence of contractures and craniofacial malformations.

  • lack of founder haplotype for the Rapsyn n88k mutation n88k is an ancient founder mutation or arises from multiple founders
    Journal of Medical Genetics, 2004
    Co-Authors: Kinji Ohno, Andrew G Engel
    Abstract:

    Mutations in RAPSN , a gene encoding Rapsyn, a molecule that clusters acetylcholine receptors at the motor endplate,1 cause endplate acetylcholine receptor deficiency.2–7 Muller and colleagues recently reported that N88K is a frequent mutation in RAPSN .5 By genotyping 17 mutant K88 chromosomes for two RAPSN polymorphisms (IVS3-11delC and 456T/C) and a microsatellite marker D11S4117 (fig 1A) in 12 patients from 10 independent central or western European families, they found that 14 of 17 mutant chromosomes shared a common haplotype and concluded that N88K arises from a common founder. Figure 1  Haplotype analysis. (A) Physical map of microsatellite markers (open circles) flanking RAPSN (closed circle) according to the NCBI uniSTS database and Map Viewer Build 33. Positions of markers are slightly different from those reported by us3 and by Muller et al ,5 because the Human Genome Project has recently been completed for this region (GenBank accession number NT_009237). Numbers in parentheses indicate genetic distances from RAPSN in centiMorgans according to the deCODE recombination map.9 The genetic distances are approximate, because RAPSN has not been positioned in any recombination map. na, Not available. p values represent Fisher’s two tailed exact test to see if a dominant genotype at each marker is linked to N88K. (B) Identified haplotypes. F1–F10 denote families 1 to 10. K and N in the haplotype names represent mutant K88 and wild type N88, respectively. Genotypes dominant in the mutant K88 chromosomes are shaded. Numbers in haplotypes represent the size of PCR product. na, Not available, because all family members were heterozygous for two morphs. In F8, all members carried morphs 260 and 264 at D11S4117. In F10, all members carried morphs 277 and 283 at D11S4174. Richard and colleagues also recently reported that N88K arises from a common founder; they …

  • sleuthing molecular targets for neurological diseases at the neuromuscular junction
    Nature Reviews Neuroscience, 2003
    Co-Authors: Andrew G Engel, Kinji Ohno, Steven M Sine
    Abstract:

    The analysis of congenital myasthenic syndromes (CMSs) has disclosed a diverse array of molecular targets at the motor endplate and has delineated their contribution to synaptic function. Clinical, electrophysiological and morphological studies have paved the way for detecting CMS-related mutations in proteins such as choline acetyltransferase, acetylcholinesterase, the acetylcholine receptor and Rapsyn, and studies of the mutant proteins have allowed us to correlate the effects of the mutations with predicted alterations in protein structure. Here, we review the symptomatology of CMSs, consider the factors that impair neuromuscular transmission, survey the mutations that have been uncovered in the different synaptic proteins, and consider the functional implications of the identified mutations.

  • Rapsyn mutations in humans cause endplate acetylcholine receptor deficiency and myasthenic syndrome
    American Journal of Human Genetics, 2002
    Co-Authors: Kinji Ohno, Andrew G Engel, Xinming Shen, Duygu Selcen, Joan M Brengman, Michel C Harper, Akira Tsujino, Margherita Milone
    Abstract:

    Congenital myasthenic syndromes (CMSs) stem from genetic defects in endplate (EP)-specific presynaptic, synaptic, and postsynaptic proteins. The postsynaptic CMSs identified to date stem from a deficiency or kinetic abnormality of the acetylcholine receptor (AChR). All CMSs with a kinetic abnormality of AChR, as well as many CMSs with a deficiency of AChR, have been traced to mutations in AChR-subunit genes. However, in a subset of patients with EP AChR deficiency, the genetic defect has remained elusive. Rapsyn, a 43-kDa postsynaptic protein, plays an essential role in the clustering of AChR at the EP. Seven tetratricopeptide repeats (TPRs) of Rapsyn subserve self-association, a coiled-coil domain binds to AChR, and a RING-H2 domain associates with β-dystroglycan and links Rapsyn to the subsynaptic cytoskeleton. Rapsyn self-association precedes recruitment of AChR to Rapsyn clusters. In four patients with EP AChR deficiency but with no mutations in AChR subunits, we identify three recessive Rapsyn mutations: one patient carries L14P in TPR1 and N88K in TPR3; two are homozygous for N88K; and one carries N88K and 553ins5, which frameshifts in TPR5. EP studies in each case show decreased staining for Rapsyn and AChR, as well as impaired postsynaptic morphological development. Expression studies in HEK cells indicate that none of the mutations hinders Rapsyn self-association but that all three diminish coclustering of AChR with Rapsyn.

William D Phillips - One of the best experts on this subject based on the ideXlab platform.

  • neural agrin increases postsynaptic ach receptor packing by elevating Rapsyn protein at the mouse neuromuscular synapse
    Developmental Neurobiology, 2008
    Co-Authors: Jennifer Brockhausen, Othon L Gervasio, Peter G Noakes, R N Cole, Shyuan T Ngo, William D Phillips
    Abstract:

    Fluorescence resonance energy transfer (FRET) experiments at neuromuscular junctions in the mouse tibialis anterior muscle show that postsynaptic acetylcholine receptors (AChRs) become more tightly packed during the first month of postnatal development. Here, we report that the packing of AChRs into postsynaptic aggregates was reduced in 4-week postnatal mice that had reduced amounts of the AChR-associated protein, Rapsyn, in the postsynaptic membrane (Rapsyn(+/-) mice). We hypothesize that nerve-derived agrin increases postsynaptic expression and targeting of Rapsyn, which then drives the developmental increase in AChR packing. Neural agrin treatment elevated the expression of Rapsyn in C2 myotubes by a mechanism that involved slowing of Rapsyn protein degradation. Similarly, exposure of synapses in postnatal muscle to exogenous agrin increased Rapsyn protein levels and elevated the intensity of anti-Rapsyn immunofluorescence, relative to AChR, in the postsynaptic membrane. This increase in the Rapsyn-to-AChR immunofluorescence ratio was associated with tighter postsynaptic AChR packing and slowed AChR turnover. Acute blockade of synaptic AChRs with a-bungarotoxin lowered the Rapsyn-to-AChR immunofluorescence ratio, suggesting that AChR signaling also helps regulate the assembly of extra Rapsyn in the postsynaptic membrane. The results suggest that at the postnatal neuromuscular synapse agrin signaling elevates the expression and targeting of Rapsyn to the postsynaptic membrane, thereby packing more AChRs into stable, functionally-important AChR aggregates. (C) 2008 Wiley Periodicals, Inc.

  • developmental increase in the amount of Rapsyn per acetylcholine receptor promotes postsynaptic receptor packing and stability
    Developmental Biology, 2007
    Co-Authors: Othon L Gervasio, Paul F Armson, William D Phillips
    Abstract:

    Neuromuscular synaptic transmission depends upon tight packing of acetylcholine receptors (AChRs) into postsynaptic AChR aggregates, but not all postsynaptic AChRs are aggregated. Here we describe a new confocal Fluorescence Resonance Energy Transfer (FRET) assay for semi-quantitative comparison of the degree to which AChRs are aggregated at synapses. During the first month of postnatal life the mouse tibialis anterior muscle showed increases both in the number of postsynaptic AChRs and the efficiency with which AChR was aggregated (by FRET). There was a concurrent two-fold increase in immunofluorescent labeling for the AChR-associated cytoplasmic protein, Rapsyn. When 1-month old muscle was denervated, postsynaptic Rapsyn immunostaining was reduced, as was the efficiency of AChR aggregation. In vivo electroporation of Rapsyn-EGFP into muscle fibers increased postsynaptic Rapsyn levels. Those synapses with higher ratios of Rapsyn-EGFP to AChR displayed a slower metabolic turnover of AChR. Conversely, the reduction of postsynaptic Rapsyn after denervation was accompanied by an acceleration of AChR turnover. Thus, a developmental increase in the amount of Rapsyn targeted to the postsynaptic membrane may drive enhanced postsynaptic AChRs aggregation and AChR stability within the postsynaptic membrane.

  • increased ratio of Rapsyn to ach receptor stabilizes postsynaptic receptors at the mouse neuromuscular synapse
    The Journal of Physiology, 2005
    Co-Authors: Othon L Gervasio, William D Phillips
    Abstract:

    The metabolic turnover of nicotinic ACh receptors (AChR) at the neuromuscular synapse is regulated over a tenfold range by innervation status, muscle electrical activity and neural agrin, but the downstream effector of such changes has not been defined. The AChR-associated protein Rapsyn is essential for forming AChR clusters during development. Here, Rapsyn was tagged with enhanced green fluorescent protein (EGFP) to begin to probe its influence at the adult synapse. In C2 myotubes, Rapsyn–EGFP participated with AChR in agrin-induced AChR cluster formation. When electroporated into the tibialis anterior muscle of young adult mice, Rapsyn–EGFP accumulated in discrete subcellular structures, many of which colocalized with Golgi markers, consistent with the idea that Rapsyn assembles with AChR in the exocytic pathway. Rapsyn–EGFP also targeted directly to the postsynaptic membrane where it occupied previously vacant Rapsyn binding sites, thereby increasing the Rapsyn to AChR ratio. At endplates displaying Rapsyn–EGFP, the metabolic turnover of AChR (labelled with rhodamine-α-bungarotoxin) was slowed. Thus, the metabolic half-life of receptors at the synapse may be modulated by local changes in the subsynaptic ratio of Rapsyn to AChR.

  • overexpression of Rapsyn modifies the intracellular trafficking of acetylcholine receptors
    Journal of Neuroscience Research, 2000
    Co-Authors: Hong Han, Shihong Yang, William D Phillips
    Abstract:

    Rapsyn is a protein that interacts with the cytoplasmic face of the nicotinic acetylcholine receptors (AChR) to cluster them within postsynaptic membrane of muscle. Here we show that intracellular AChRs are also affected by Rapsyn. When Rapsyn was co-transfected with AChR into QT-6 fibroblasts, (125)I-alpha-bungarotoxin binding indicated a reduction in the fraction of AChRs expressed on the cell surface, compared to cells expressing AChRs alone. Double fluorescent labeling showed that intracellular AChRs accumulated in patches at the cell periphery, beneath Rapsyn-associated cell surface AChR clusters. These patches were observed even when cells were grown in medium containing excess unlabelled alpha-bungarotoxin to mask internalized AChRs, suggesting that they arose from hindered trafficking of newly formed AChRs to the cell surface. Similarly, in the muscle cell line, C2, overexpression of Rapsyn resulted in the co-localization of aggregates of intracellular alpha-bungarotoxin binding sites with Rapsyn beneath cell surface AChR microaggregates. The results indicate that Rapsyn can modify the trafficking of AChRs within the cell and suggest a role in selectively targeting newly synthesized intracellular AChRs to the postsynaptic membrane.

  • overexpression of Rapsyn inhibits agrin induced acetylcholine receptor clustering in muscle cells
    Journal of Neurocytology, 1999
    Co-Authors: Hong Han, Peter G Noakes, William D Phillips
    Abstract:

    Rapsyn is a protein on the cytoplasmic face of the postsynaptic membrane of skeletal muscle that is essential for clustering acetylcholine receptors (AChR). Here we show that transfection of Rapsyn cDNA can restore AChR clustering function to muscle cells cultured from Rapsyn deficient (KORAP) mice. KORAP myotubes displayed no AChR aggregates before or after treatment with neural agrin. After transfection with Rapsyn expression plasmid, some KORAP myotubes expressed Rapsyn at physiological levels. These formed large AChR-Rapsyn clusters in response to agrin, just like wild-type myotubes. KORAP myotubes that overexpressed Rapsyn formed only scattered AChR-Rapsyn microaggregates, irrespective of agrin treatment. KORAP cells were then transfected with mutant forms of Rapsyn. A deletion mutant lacking residues 16–254 formed Rapsyn microaggregates, but failed to aggregate AChRs. Substitution mutation to the C-terminal serine phosphorylation site of Rapsyn (M43D405,D406) did not impair the response to agrin, showing that differential phosphorylation of this site is unlikely to mediate agrin-induced clustering. The results indicate that Rapsyn expression is essential for agrin-induced AChR clustering but that its overexpression inhibits this pathway. The approach of using Rapsyn-deficient muscle cells opens the way for defining the role of Rapsyn in agrin-induced AChR clustering.

Fei Chen - One of the best experts on this subject based on the ideXlab platform.

  • wnt beta catenin signaling suppresses Rapsyn expression and inhibits acetylcholine receptor clustering at the neuromuscular junction
    Journal of Biological Chemistry, 2008
    Co-Authors: Jia Wang, Nanjie Ruan, Lei Qian, Fei Chen
    Abstract:

    The dynamic interaction between positive and negative signals is necessary for remodeling of postsynaptic structures at the neuromuscular junction. Here we report that Wnt3a negatively regulates acetylcholine receptor ( AChR) clustering by repressing the expression of Rapsyn, an AChR-associated protein essential for AChR clustering. In cultured myotubes, treatment with Wnt3a or overexpression of beta-catenin, the condition mimicking the activation of the Wnt canonical pathway, inhibited Agrin-induced formation of AChR clusters. Moreover, Wnt3a treatment promoted dispersion of AChR clusters, and this effect was prevented by DKK1, an antagonist of the Wnt canonical pathway. Next, we investigated possible mechanisms underlying Wnt3a regulation of AChR clustering in cultured muscle cells. Interestingly, we found that Wnt3a treatment caused a decrease in the protein level of Rapsyn. In addition, Rapsyn promoter activity in cultured muscle cells was inhibited by the treatment with Wnt3a or beta-catenin overexpression. Forced expression of Rapsyn driven by a promoter that is not responsive to Wnt3a prevented the dispersing effect of Wnt3a on AChR clusters, suggesting that Wnt3a indeed acts to disperse AChR clusters by down-regulating the expression of Rapsyn. The role of Wnt/beta-catenin signaling in dispersing AChR clusters was also investigated in vivo by electroporation of Wnt3a or beta-catenin into mouse limb muscles, where ectopic Wnt3a or beta-catenin caused disassembly of postsynaptic apparatus. Together, these results suggest that Wnt/beta-catenin signaling plays a negative role for postsynaptic differentiation at the neuromuscular junction, probably by regulating the expression of synaptic proteins, such as Rapsyn.

  • wnt beta catenin signaling suppresses Rapsyn expression and inhibits acetylcholine receptor clustering at the neuromuscular junction
    Journal of Biological Chemistry, 2008
    Co-Authors: Jia Wang, Nanjie Ruan, Lei Qian, Fei Chen, Wenliang Lei, Zhenge Luo
    Abstract:

    The dynamic interaction between positive and negative signals is necessary for remodeling of postsynaptic structures at the neuromuscular junction. Here we report that Wnt3a negatively regulates acetylcholine receptor (AChR) clustering by repressing the expression of Rapsyn, an AChR-associated protein essential for AChR clustering. In cultured myotubes, treatment with Wnt3a or overexpression of β-catenin, the condition mimicking the activation of the Wnt canonical pathway, inhibited Agrin-induced formation of AChR clusters. Moreover, Wnt3a treatment promoted dispersion of AChR clusters, and this effect was prevented by DKK1, an antagonist of the Wnt canonical pathway. Next, we investigated possible mechanisms underlying Wnt3a regulation of AChR clustering in cultured muscle cells. Interestingly, we found that Wnt3a treatment caused a decrease in the protein level of Rapsyn. In addition, Rapsyn promoter activity in cultured muscle cells was inhibited by the treatment with Wnt3a or β-catenin overexpression. Forced expression of Rapsyn driven by a promoter that is not responsive to Wnt3a prevented the dispersing effect of Wnt3a on AChR clusters, suggesting that Wnt3a indeed acts to disperse AChR clusters by down-regulating the expression of Rapsyn. The role of Wnt/β-catenin signaling in dispersing AChR clusters was also investigated in vivo by electroporation of Wnt3a or β-catenin into mouse limb muscles, where ectopic Wnt3a or β-catenin caused disassembly of postsynaptic apparatus. Together, these results suggest that Wnt/β-catenin signaling plays a negative role for postsynaptic differentiation at the neuromuscular junction, probably by regulating the expression of synaptic proteins, such as Rapsyn.

  • Rapsyn interaction with calpain stabilizes achr clusters at the neuromuscular junction
    Neuron, 2007
    Co-Authors: Fei Chen, Zhihua Yang, Peter G Noakes, Shyuan T Ngo, Jia Wang, Nanjie Ruan, Lei Qian, Ying Huang, Claudio Schneider, Yuqiang Ding
    Abstract:

    Agrin induces, whereas acetylcholine (ACh) disperses, ACh receptor (AChR) clusters during neuromuscular synaptogenesis. Such counteractive interaction leads to eventual dispersal of nonsynaptic AChR-rich sites and formation of receptor clusters at the postjunctional membrane. However, the underlying mechanisms are not well understood. Here we show that calpain, a calcium-dependent protease, is activated by the cholinergic stimulation and is required for induced dispersion of AChR clusters. Interestingly, the AChR-associated protein Rapsyn interacted with calpain in an agrin-dependent manner, and this interaction inhibited the protease activity of calpain. Disrupting the endogenous Rapsyn/calpain interaction enhanced CCh-induced dispersion of AChR clusters. Moreover, the loss of AChR clusters in agrin mutant mice was partially rescued by the inhibition of calpain via overexpressing calpastatin, an endogenous calpain inhibitor, or injecting calpeptin, a cell-permeable calpain inhibitor. These results demonstrate that calpain participates in ACh-induced dispersion of AChR clusters, and Rapsyn stabilizes AChR clusters by suppressing calpain activity.