scholarly journals A Single Pulse of Agrin Triggers a Pathway That Acts To Cluster Acetylcholine Receptors

2004 ◽  
Vol 24 (18) ◽  
pp. 7841-7854 ◽  
Author(s):  
Peggy Mittaud ◽  
Alain A. Camilleri ◽  
Raffaella Willmann ◽  
Susanne Erb-Vögtli ◽  
Steven J. Burden ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Acetylcholine Receptors ◽  
Cluster Formation ◽  
Single Pulse ◽  
Achr Cluster ◽  
Abl Kinases ◽  
Temporal Aspects ◽  
Kinase Cascade ◽  
Spatial Specificity ◽  
Achr Clustering

ABSTRACT Agrin triggers signaling mechanisms of high temporal and spatial specificity to achieve phosphorylation, clustering, and stabilization of postsynaptic acetylcholine receptors (AChRs). Agrin transiently activates the kinase MuSK; MuSK activation has largely vanished when AChR clusters appear. Thus, a tyrosine kinase cascade acts downstream from MuSK, as illustrated by the agrin-evoked long-lasting activation of Src family kinases (SFKs) and their requirement for AChR cluster stabilization. We have investigated this cascade and report that pharmacological inhibition of SFKs reduces early but not later agrin-induced phosphorylation of MuSK and AChRs, while inhibition of Abl kinases reduces late phosphorylation. Interestingly, SFK inhibition applied selectively during agrin-induced AChR cluster formation caused rapid cluster dispersal later upon agrin withdrawal. We also report that a single 5-min agrin pulse, followed by extensive washing, triggered long-lasting MuSK and AChR phosphorylation and efficient AChR clustering. Following the pulse, MuSK phosphorylation increased and, beyond a certain level, caused maximal clustering. These data reveal novel temporal aspects of tyrosine kinase action in agrin signaling. First, during AChR cluster formation, SFKs initiate early phosphorylation and an AChR stabilization program that acts much later. Second, a kinase mechanism rapidly activated by agrin acts thereafter autonomously in agrin's absence to further increase MuSK phosphorylation and cluster AChRs.

scholarly journals Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells.

1993 ◽  
Vol 120 (1) ◽  
pp. 185-195 ◽  
Author(s):  
L P Baker ◽  
H B Peng
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Acetylcholine Receptor ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Cluster Formation ◽  
Protein S ◽  
Muscle Cells ◽  
Nerve Muscle ◽  
Achr Clustering

Aggregation of the nicotinic acetylcholine receptor (AChR) at sites of nerve-muscle contact is one of the earliest events to occur during the development of the neuromuscular junction. The stimulus presented to the muscle by nerve and the mechanisms underlying postsynaptic differentiation are not known. The purpose of this study was to examine the distribution of phosphotyrosine (PY)-containing proteins in cultured Xenopus muscle cells in response to AChR clustering stimuli. Results demonstrated a distinct accumulation of PY at AChR clusters induced by several stimuli, including nerve, the culture substratum, and polystyrene microbeads. AChR microclusters formed by external cross-linking did not show PY colocalization, implying that the accumulation of PY in response to clustering stimuli was not due to the aggregation of basally phosphorylated AChRs. A semi-quantitative determination of the time course for development of PY labeling at bead contacts revealed early PY accumulation within 15 min of contact before significant AChR aggregation. At later stages (within 15 h), the AChR signal came to approximate the PY signal. We have reported the inhibition of bead-induced AChR clustering in response to beads by a tyrphostin tyrosine kinase inhibitor (RG50864) (Peng, H. B., L. P. Baker, and Q. Chen. 1991. Neuron. 6:237-246). RG50864 also inhibited PY accumulation at bead contacts, providing evidence for tyrosine kinase activation in response to the bead stimulus. These results suggest that tyrosine phosphorylation may play an important role in the generative stages of cluster formation, and may involve protein(s) other than or in addition to AChRs.

scholarly journals Increased expression of the 43-kD protein disrupts acetylcholine receptor clustering in myotubes

1993 ◽  
Vol 122 (1) ◽  
pp. 169-179 ◽  
Author(s):  
CM Yoshihara ◽  
ZW Hall
Keyword(s):  
Acetylcholine Receptor ◽  
Cluster Formation ◽  
Muscle Cells ◽  
Postsynaptic Membrane ◽  
Achr Cluster ◽  
Number Of Clusters ◽  
Vertebrate Muscle ◽  
Peripheral Membrane Protein ◽  
Achr Clustering

The 43-kD protein is a peripheral membrane protein that is in approximately 1:1 stoichiometry with the acetylcholine receptor (AChR) in vertebrate muscle cells and colocalizes with it in the postsynaptic membrane. To investigate the role of the 43-kD protein in AChR clustering, we have isolated C2 muscle cell lines in which some cells overexpress the 43-kD protein. We find that myotubes with increased levels of the 43-kD protein have small AChR clusters and that those with the highest levels of expression have a drastically reduced number of clusters. Our results suggest that the 1:1 stoichiometry of AChR and 43-kD protein found in muscle cells is important for AChR cluster formation.

Laminin and alpha7beta1 integrin regulate agrin-induced clustering of acetylcholine receptors

2000 ◽  
Vol 113 (16) ◽  
pp. 2877-2886 ◽  
Author(s):  
D.J. Burkin ◽  
J.E. Kim ◽  
M. Gu ◽  
S.J. Kaufman
Keyword(s):  
Motor Neurons ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Cluster Formation ◽  
Site Directed Mutagenesis ◽  
Synaptic Membrane ◽  
Before And After ◽  
Stable Association ◽  
Achr Clustering ◽  
Alpha7beta1 Integrin

The clustering of acetylcholine receptors (AChRs) in the post-synaptic membrane of skeletal muscle is an early developmental event in the formation of the neuromuscular junction. Several studies show that laminin, as well as neural agrin, can induce AChR clustering in C2C12 myofibers. We recently showed that specific isoforms of the alpha7beta1 integrin (a receptor normally found at neuromuscular junctions) colocalize and physically interact with AChR clusters in a laminin-dependent fashion. In contrast, induction with agrin alone fails to promote localization of the integrin with AChR clusters. Together both agrin and laminin enhance the interaction of the integrin with AChRs and their aggregation into clusters. To further understand this mechanism we investigated cluster formation and the association of the alpha7beta1 integrin and AChR over time following induction with laminin and/or agrin. Our results show that the alpha7beta1 integrin associates with AChRs early during the formation of the post-synaptic membrane and that laminin modulates this recruitment. Laminin induces a rapid stable association of the integrin and AChRs and this association is independent of clustering. In addition to laminin-1, merosin (laminin-2/4) is present both before and after formation of neuromuscular junctions and also promotes AChR clustering and colocalization with the integrin as well as synergism with agrin. Using site directed mutagenesis we demonstrate that a tyrosine residue in the cytoplasmic domain of both (α)7A and (α)7B chains regulates the localization of the integrin with AChR clusters. We also provide evidence that laminin, through its association with the alpha7beta1 integrin, reduces by 20-fold the concentration of agrin required to promote AChR clustering and accelerates the formation of clusters. Thus laminin, agrin and the alpha7beta1 integrin act in a concerted manner early in the development of the post-synaptic membrane, with laminin priming newly formed myofibers to rapidly and vigorously respond to low concentrations of neural agrin produced by innervating motor neurons.

scholarly journals Acetylcholine Receptor (AChR) Clustering Is Regulated Both by Glycogen Synthase Kinase 3β (GSK3β)-dependent Phosphorylation and the Level of CLIP-associated Protein 2 (CLASP2) Mediating the Capture of Microtubule Plus-ends

2014 ◽  
Vol 289 (44) ◽  
pp. 30857-30867 ◽  
Author(s):  
Sreya Basu ◽  
Stefan Sladecek ◽  
Hayley Pemble ◽  
Torsten Wittmann ◽  
Johan A. Slotman ◽  
...  
Keyword(s):  
Cluster Size ◽  
Acetylcholine Receptors ◽  
Glycogen Synthase ◽  
Phosphorylation Sites ◽  
Two Dimensional ◽  
Receptor Density ◽  
Wild Type ◽  
Achr Cluster ◽  
Capture And Release ◽  
Achr Clustering

The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2–9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2–8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.

Molecular events in synaptogenesis: nerve-muscle adhesion and postsynaptic differentiation

1988 ◽  
Vol 254 (3) ◽  
pp. C345-C364 ◽  
Author(s):  
R. J. Bloch ◽  
D. W. Pumplin
Keyword(s):  
Cell Surface ◽  
Muscle Cell ◽  
Acetylcholine Receptors ◽  
Membrane Skeleton ◽  
Postsynaptic Membrane ◽  
Molecular Assembly ◽  
Trophic Factors ◽  
Achr Cluster ◽  
Nerve Muscle ◽  
Achr Clustering

The clustering of acetylcholine receptors (AChR) in the postsynaptic membrane of newly innervated muscle fibers is one of the earliest events in the development of the vertebrate neuromuscular junction. Here, we describe two hypotheses that can account for AChR clustering in response to innervation. The "trophic factor" hypothesis proposes that the neuron releases a soluble factor that interacts with the muscle cell in a specific manner and that this interaction results in the local accumulation of AChR. The "contact and adhesion" hypothesis proposes that the binding of the nerve to the muscle cell surface is itself sufficient to induce AChR clustering, without the participation of soluble factors. We present a model for the molecular assembly of AChR clusters based on the contact and adhesion hypothesis. The model involves the sequential assembly of three distinct membrane domains. The first domain to form serves to attach microfilaments to the cytoplasmic surface of the muscle cell membrane at sites of muscle-nerve adhesion. The second domain to form is clathrin-coated membrane; it serves as a site of insertion of additional membrane elements, including AChR. Upon insertion of AChR into the cell surface, a membrane skeleton assembles by anchoring itself to the AChR. The skeleton, composed in part of actin and spectrin, binds and immobilizes significant numbers of AChR, thereby forming the third membrane domain of the AChR cluster. We make several predictions that should distinguish this model of AChR clustering from one that invokes soluble, trophic factors.

scholarly journals A Role of Tyrosine Phosphatase in Acetylcholine Receptor Cluster Dispersal and Formation

1998 ◽  
Vol 141 (7) ◽  
pp. 1613-1624 ◽  
Author(s):  
Zhengshan Dai ◽  
H. Benjamin Peng
Keyword(s):  
Acetylcholine Receptor ◽  
Hot Spots ◽  
Hot Spot ◽  
Tyrosine Phosphatase ◽  
Cluster Formation ◽  
Muscle Cells ◽  
Achr Cluster ◽  
Discrete Manner ◽  
Achr Clustering

Innervation of the skeletal muscle involves local signaling, leading to acetylcholine receptor (AChR) clustering, and global signaling, manifested by the dispersal of preexisting AChR clusters (hot spots). Receptor tyrosine kinase (RTK) activation has been shown to mediate AChR clustering. In this study, the role of tyrosine phosphatase (PTPase) in the dispersal of hot spots was examined. Hot spot dispersal in cultured Xenopus muscle cells was initiated immediately upon the presentation of growth factor–coated beads that induce both AChR cluster formation and dispersal. Whereas the density of AChRs decreased with time, the fine structure of the hot spot remained relatively constant. Although AChR, rapsyn, and phosphotyrosine disappeared, a large part of the original hot spot–associated cytoskeleton remained. This suggests that the dispersal involves the removal of a key linkage between the receptor and its cytoskeletal infrastructure. The rate of hot spot dispersal is inversely related to its distance from the site of synaptic stimulation, implicating the diffusible nature of the signal. PTPase inhibitors, such as pervanadate or phenylarsine oxide, inhibited hot spot dispersal. In addition, they also affected the formation of new clusters in such a way that AChR microclusters extended beyond the boundary set by the clustering stimuli. Furthermore, by introducing a constitutively active PTPase into cultured muscle cells, hot spots were dispersed in a stimulus- independent fashion. This effect of exogenous PTPase was also blocked by pervanadate. These results implicate a role of PTPase in AChR cluster dispersal and formation. In addition to RTK activation, synaptic stimulation may also activate PTPase which acts globally to destabilize preexisting AChR hot spots and locally to facilitate AChR clustering in a spatially discrete manner by countering the action of RTKs.

scholarly journals Laminin-1 redistributes postsynaptic proteins and requires rapsyn, tyrosine phosphorylation, and Src and Fyn to stably cluster acetylcholine receptors

2002 ◽  
Vol 157 (5) ◽  
pp. 883-895 ◽  
Author(s):  
P. Angelo Marangi ◽  
Simon T. Wieland ◽  
Christian Fuhrer
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Acetylcholine Receptors ◽  
Synapse Formation ◽  
Cluster Formation ◽  
Neuromuscular Synapse ◽  
Achr Cluster ◽  
Signaling Mechanisms ◽  
Neuromuscular Synaptogenesis ◽  
Laminin 1

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 β and δ 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.

scholarly journals SHP2 inhibitor protects AChRs from effects of myasthenia gravis MuSK antibody

2019 ◽  
Vol 7 (1) ◽  
pp. e645 ◽  
Author(s):  
Saif Huda ◽  
Michelangelo Cao ◽  
Anna De Rosa ◽  
Mark Woodhall ◽  
Pedro M. Rodriguez Cruz ◽  
...  
Keyword(s):  
Myasthenia Gravis ◽  
Cluster Formation ◽  
Inhibitory Effect ◽  
C2c12 Myotubes ◽  
Achr Cluster ◽  
Src Homology 2 ◽  
Src Homology ◽  
Src Homology 2 Domain ◽  
Achr Clustering

ObjectiveTo determine whether an SRC homology 2 domain-containing phosphotyrosine phosphatase 2 (SHP2) inhibitor would increase muscle-specific kinase (MuSK) phosphorylation and override the inhibitory effect of MuSK-antibodies (Abs).MethodsThe effect of the SHP2 inhibitor NSC-87877 on MuSK phosphorylation and AChR clustering was tested in C2C12 myotubes with 31 MuSK-myasthenia gravis (MG) sera and purified MuSK-MG IgG4 preparations.ResultsIn the absence of MuSK-MG Abs, NSC-87877 increased MuSK phosphorylation and the number of AChR clusters in C2C12 myotubes in vitro and in DOK7-overexpressing C2C12 myotubes that form spontaneous AChR clusters. In the presence of MuSK-MG sera, the AChR clusters were reduced, as expected, but NSC-87877 was able to protect or restore the clusters. Two purified MuSK-MG IgG4 preparations inhibited both MuSK phosphorylation and AChR cluster formation, and in both, clusters were restored with NSC-87877.ConclusionsStimulating the agrin-LRP4-MuSK-DOK7 AChR clustering pathway with NSC-87877, or other drugs, could represent a novel therapeutic approach for MuSK-MG and could potentially improve other NMJ disorders with reduced AChR numbers or disrupted NMJs.

scholarly journals Postsynaptic Abnormalities at the Neuromuscular Junctions of Utrophin-deficient Mice

1997 ◽  
Vol 136 (4) ◽  
pp. 883-894 ◽  
Author(s):  
Anne E. Deconinck ◽  
Allyson C. Potter ◽  
Jonathon M. Tinsley ◽  
Sarah J. Wood ◽  
Ruth Vater ◽  
...  
Keyword(s):  
Protein Complex ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Transmembrane Protein ◽  
Cluster Formation ◽  
Congenital Myasthenic Syndrome ◽  
Achr Cluster ◽  
Myasthenic Syndrome ◽  
Dystrophin Protein ◽  
Deficient Mice

Utrophin is a dystrophin-related cytoskeletal protein expressed in many tissues. It is thought to link F-actin in the internal cytoskeleton to a transmembrane protein complex similar to the dystrophin protein complex (DPC). At the adult neuromuscular junction (NMJ), utrophin is precisely colocalized with acetylcholine receptors (AChRs) and recent studies have suggested a role for utrophin in AChR cluster formation or maintenance during NMJ differentiation. We have disrupted utrophin expression by gene targeting in the mouse. Such mice have no utrophin detectable by Western blotting or immunocytochemistry. Utrophindeficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChRs (α-bungarotoxin [α-BgTx] binding reduced to ∼60% normal) and decreased postsynaptic folding, though only minimal electrophysiological changes. Utrophin is thus not essential for AChR clustering at the NMJ but may act as a component of the postsynaptic cytoskeleton, contributing to the development or maintenance of the postsynaptic folds. Defects of utrophin could underlie some forms of congenital myasthenic syndrome in which a reduction of postsynaptic folds is observed.

scholarly journals A Functional Role for Specific Spliced Variants of the α7β1 Integrin in Acetylcholine Receptor Clustering

1998 ◽  
Vol 143 (4) ◽  
pp. 1067-1075 ◽  
Author(s):  
Dean J. Burkin ◽  
Maojian Gu ◽  
Bradley L. Hodges ◽  
James T. Campanelli ◽  
Stephen J. Kaufman
Keyword(s):  
Skeletal Muscle ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Cluster Formation ◽  
Cytoplasmic Domain ◽  
Laminin Receptor ◽  
Early Event ◽  
Low Concentrations ◽  
High Concentrations ◽  
Achr Clustering

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the α7β1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, α7A and α7B, and the extracellular spliced forms, α7X1 and α7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the α7β1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-α7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-α7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the α7A and α7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the α7X2 extracellular domain were active. These results demonstrate that the α7β1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the α7 chain, and that laminin, agrin, and the α7β1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.

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