scholarly journals Faculty Opinions recommendation of Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs.

2021 ◽  
Author(s):  
Alessandra Cambi
Keyword(s):  
Achr Clustering

scholarly journals Extracellular synaptic factors induce clustering of acetylcholine receptors stably expressed in fibroblasts.

1991 ◽  
Vol 115 (1) ◽  
pp. 165-177 ◽  
Author(s):  
D S Hartman ◽  
N S Millar ◽  
T Claudio
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Hybrid Cell ◽  
Muscle Cells ◽  
Specific Gene ◽  
Temperature Sensitive ◽  
Antigenic Modulation ◽  
Achr Clustering

The clustering of nicotinic acetylcholine receptors (AChRs) is one of the first events observed during formation of the neuromuscular junction. To determine the mechanism involved in AChR clustering, we established a nonmuscle cell line (mouse fibroblast L cells) that stably expresses just one muscle-specific gene product, the AChR. We have shown that when Torpedo californica AChRs are expressed in fibroblasts, their immunological, biochemical, and electrophysiological properties all indicate that fully functional cell surface AChRs are produced. In the present study, the cell surface distribution and stability of Torpedo AChRs expressed in fibroblasts (AChR-fibroblasts) were analyzed and shown to be similar to nonclustered AChRs expressed in muscle cells. AChR-fibroblasts incubated with antibodies directed against the AChR induced the formation of small AChR microclusters (less than 0.5 micron 2) and caused an increase in the internalization rate and degradation of surface AChRs (antigenic modulation) in a manner similar to that observed in muscle cells. Two disparate sources of AChR clustering factors, extracellular matrix isolated from Torpedo electric organ and conditioned media from a rodent neuroblastoma-glioma hybrid cell line, each induced large (1-3 microns 2), stable AChR clusters with no change in the level of surface AChR expression. By exploiting the temperature-sensitive nature of Torpedo AChR assembly, we were able to demonstrate that factor-induced clusters were produced by mobilization of preexisting surface AChRs, not by directed insertion of newly synthesized AChRs. AChR clusters were never observed in the absence of extracellular synaptic factors. Our results suggest that these factors can interact directly with the AChR.

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.

scholarly journals Participation of calcium and calmodulin in the formation of acetylcholine receptor clusters.

1984 ◽  
Vol 98 (2) ◽  
pp. 550-557 ◽  
Author(s):  
H B Peng
Keyword(s):  
Acetylcholine Receptor ◽  
Divalent Cations ◽  
Simple Procedure ◽  
Latex Beads ◽  
Immunofluorescence Studies ◽  
Cellular Machinery ◽  
The Stability ◽  
Receptor Clusters ◽  
Myotomal Muscle ◽  
Achr Clustering

The formation of acetylcholine receptor (AChR) clusters can be experimentally induced in cultured Xenopus myotomal muscle cells by positive polypeptide-coated latex beads (Peng, H.B., P.-C. Cheng, and P.W. Luther, 1981, Nature [Lond.], 292:831-834). This provides a simple procedure for studying the cellular process of AChR clustering. In this study, the involvement of calcium and calmodulin in this process was examined. A deprivation in extracellular calcium by calcium-free medium or by the addition of calcium antagonists such as divalent cations Co2+ and Ni2+ (1-5 mM) or organic compounds verapamil and D-600 (0.1-0.5 mM) suppressed the formation of AChR clusters induced by the latex beads in a largely reversible manner. Antagonists against calmodulin, including trifluoperazine (1-5 microM) and the naphthalene sulfonamide W-7 (20 microM), also suppressed AChR clustering. However, the effect of W-7 was much weaker than that of trifluoperazine (TFP). Although the formation of AChR clusters is inhibited by these drugs, the stability of the existent clusters is relatively insensitive to them. These data suggest that the clustering of AChR involves a Ca2+ and calmodulin-activated process. Immunofluorescence studies using an antibody against calmodulin indicate that calmodulin is diffusely distributed in the cytoplasm in addition to its localization at the I-bands. Thus I propose that a local rise in intracellular calcium caused by a locally applied stimulus, exemplified here by the polypeptide-coated latex beads, may trigger the formation of AChR clusters. Furthermore, the cellular machinery for this process may involve calmodulin and is diffusely distributed in the muscle cell.

scholarly journals MyoD and Myogenin are Required for Agrin‐induced AChR Clustering

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Erica E. Anderson ◽  
Wade A. Grow
Keyword(s):  
Achr Clustering

Globular domains of agrin are functional units that collaborate to induce acetylcholine receptor clustering

1999 ◽  
Vol 112 (8) ◽  
pp. 1213-1223 ◽  
Author(s):  
T. Cornish ◽  
J. Chi ◽  
S. Johnson ◽  
Y. Lu ◽  
J.T. Campanelli
Keyword(s):  
Amino Acid ◽  
Acetylcholine Receptors ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Physical Separation ◽  
Surface Receptors ◽  
Physical Interactions ◽  
Functional Units ◽  
Covalently Linked ◽  
Achr Clustering

Agrin, an extracellular matrix protein involved in neuromuscular junction formation, directs clustering of postsynaptic molecules, including acetylcholine receptors (AChRs). This activity resides entirely in the C-terminal portion of the protein, which consists of three laminin-like globular domains (G-domains: G1, G2 and G3) and four EGF-like repeats. Additionally, alternate mRNA splicing yields G-domain variants G2(0,4) with 0- or 4-amino-acid inserts, and G3(0, 8,11,19) with 0-, 8-, 11- or 19-amino-acid inserts. In order to better understand the contributions of individual domains and alternate splicing to agrin activity, single G-domains and covalently linked pairs of G-domains were expressed as soluble proteins and their AChR clustering activity measured on cultured C2 myotubes. These analyses reveal the following: (1) While only G3(8) exhibits detectable activity by itself, all G-domains studied (G1, G2(0), G2(4), G3(0) and G3(8)) enhance G3(8) activity when physically linked to G3(8). This effect is most pronounced when G2(4) is linked to G3(8) and is independent of the order of the G-domains. (2) The deletion of EGF-like repeats enhances activity. (3) Increasing the physical separation between linked G1 and G3(8) domains produces a significant increase in activity; similar alterations to linked G2 and G3(8) domains are without effect. (4) Clusters induced by two concatenated G3(8) domains are significantly smaller than all other agrin forms studied. These data suggest that agrin G-domains are the functional units which interact independently of their specific organization to yield AChR clustering. G-domain synergism resulting in biological output could be due to physical interactions between G-domains or, alternatively, independent interactions of G-domains with cell surface receptors which require spatially localized coactivation for optimal signal transduction.

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.

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