scholarly journals Acetylcholine receptor-aggregating activity of agrin isoforms and mapping of the active site.

1995 ◽  
Vol 128 (4) ◽  
pp. 625-636 ◽  
Author(s):  
M Gesemann ◽  
A J Denzer ◽  
M A Ruegg
Keyword(s):  
Amino Acids ◽  
Alternative Splicing ◽  
Neuromuscular Junction ◽  
Motor Neurons ◽  
Active Site ◽  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Effective Concentration ◽  
Achr Clustering

Agrin is a basal lamina protein that induces aggregation of acetylcholine receptors (AChRs) and other molecules at the developing neuromuscular junction. Alternative splicing of chick agrin mRNA at two sites, A and B, gives rise to eight possible isoforms of which five are expressed in vivo. Motor neurons express high levels of isoforms with inserts at sites A and B, muscle cells synthesize isoforms that lack amino acids at the B-site. To obtain further insights into the mechanism of agrin-induced AChR aggregation, we have determined the EC50 (effective concentration to induce half-maximal AChR clustering) of each agrin isoform and of truncation mutants. On chick myotubes, EC50 of the COOH-terminal, 95-kD fragment of agrinA4B8 was approximately 35 pM, of agrinA4B19 approximately 110 pM and of agrinA4B11 approximately 5 nM. While some AChR clusters were observed with 64 nM of agrinA4B0, no activity was detected for agrinA0B0. Recombinant full-length chick agrin and a 100-kD fragment of ray agrin showed similar EC50 values. A 45-kD, COOH-terminal fragment of agrinA4B8 retained high activity (EC50 approximately equal to 130 pM) and a 21-kD fragment was still active, but required higher concentrations (EC50 approximately equal to 13 nM). Unlike the 45-kD fragment, the 21-kD fragment neither bound to heparin nor did heparin inhibit its capability to induce AChR aggregation. These data show quantitatively that agrinA4B8 and agrinA4B19, expressed in motor neurons, are most active, while no activity is detected in agrinA0B0, the dominant isoform synthesized by muscle cells. Furthermore, our results show that a fragment comprising site B8 and the most COOH-terminal G-like domain is sufficient for this activity, and that agrin domains required for binding to heparin and those for AChR aggregation are distinct from each other.

scholarly journals Processing of ARIA and release from isolated nerve terminals

1999 ◽  
Vol 354 (1381) ◽  
pp. 411-416 ◽  
Author(s):  
Bomie Han ◽  
Gerald D. Fischbach
Keyword(s):  
Neuromuscular Junction ◽  
Action Potential ◽  
Acetylcholine Receptors ◽  
Molecular Layer ◽  
Postsynaptic Membrane ◽  
High Density ◽  
Small Contribution ◽  
Presynaptic Nerve Terminal ◽  
Voltage Gated

The neuromuscular junction is a specialized synapse in that every action potential in the presynaptic nerve terminal results in an action potential in the postsynaptic membrane, unlike most interneuronal synapses where a single presynaptic input makes only a small contribution to the population postsynaptic response. The postsynaptic membrane at the neuromuscular junction contains a high density of neurotransmitter (acetylcholine) receptors and a high density of voltage–gated Na + channels. Thus, the large acetylcholine activated current occurs at the same site where the threshold for action potential generation is low. Acetylcholine receptor inducing activity (ARIA), a 42 kD protein, that stimulates synthesis of acetylcholine receptors and voltage–gated Na + channels in cultured myotubes, probably plays the same roles at developing and mature motor endplates in vivo . ARIA is synthesized as part of a larger, transmembrane, precursor protein called proARIA. Delivery of ARIA from motor neuron cell bodies in the spinal cord to the target endplates involves several steps, including proteolytic cleavage of proARIA. ARIA is also expressed in the central nervous system and it is abundant in the molecular layer of the cerebellum. In this paper we describe our first experiments on the processing and release of ARIA from subcellular fractions containing synaptosomes from the chick cerebellum as a model system.

scholarly journals In vivo recovery of muscle contraction after alpha-bungarotoxin binding.

1975 ◽  
Vol 66 (1) ◽  
pp. 209-213 ◽  
Author(s):  
H C Fertuck ◽  
W Woodward ◽  
M M Salpeter
Keyword(s):  
Fine Structure ◽  
Neuromuscular Junction ◽  
Muscle Contraction ◽  
Acetylcholine Receptors ◽  
Actinomycin D ◽  
Recovery Period ◽  
Em Autoradiography ◽  
Alpha Bungarotoxin ◽  
In Vivo Recovery

Acetylcholine receptors were inactivated in vivo at the mouse neuromuscular junction using alpha-bungarotoxin (alpha-BTX). It was found that neurally produced muscle contraction recovered within 4-8 days (halftime similar to 3 days). Actinomycin D interfered with this recovery, but did not affect normal nerve-stimulated muscle contraction. If the response was initially eliminated by [125-I]alpha-BTX and the end plates examined by EM autoradiography, no evidence of mass internalization of bound radioactivity during recovery was seen. The fine structure of the end plates and muscle was unaltered during the post-alpha-BTX recovery period.

Incorporation of C14-amino acids into protein of isolated diaphragms: effect of epinephrine and norepinephrine

1960 ◽  
Vol 198 (1) ◽  
pp. 54-56 ◽  
Author(s):  
Ira G. Wool
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glucose Concentration ◽  
Muscle Protein ◽  
Effective Concentration ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Minimal Effective Concentration

When diaphragms isolated from normal rats were incubated with a C14-amino acid the addition of epinephrine or norepinephrine decreased incorporation of C14 into muscle protein. The inhibition occurred whether epinephrine was added in vitro or administered in vivo. The minimal effective concentration of epinephrine in vitro was 0.1 µg/ml. When the glucose concentration in the medium was raised to 300 mg % or more the epinephrine induced inhibition of amino acid incorporation into muscle protein was no longer observed.

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 Calpain 3 Is Activated through Autolysis within the Active Site and Lyses Sarcomeric and Sarcolemmal Components

2003 ◽  
Vol 23 (24) ◽  
pp. 9127-9135 ◽  
Author(s):  
Mathieu Taveau ◽  
Nathalie Bourg ◽  
Guillaume Sillon ◽  
Carinne Roudaut ◽  
Marc Bartoli ◽  
...  
Keyword(s):  
Active Site ◽  
Adaptive Response ◽  
Focal Adhesions ◽  
Muscle Cells ◽  
Cysteine Proteases ◽  
Activation Mechanism ◽  
Calpain 3 ◽  
Endogenous Proteins ◽  
Lines Of Evidence

ABSTRACT Calpain 3 (Capn3) is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. This enigmatic protease has many unique features among the calpain family and, importantly, mutations in Capn3 have been shown to be responsible for limb girdle muscular dystrophy type 2A. Here we demonstrate that the Capn3 activation mechanism is similar to the universal activation of caspases and corresponds to an autolysis within the active site of the protease. We undertook a search for substrates in immature muscle cells, as several lines of evidence suggest that Capn3 is mostly in an inactive state in muscle and needs a signal to be activated. In this model, Capn3 proteolytic activity leads to disruption of the actin cytoskeleton and disorganization of focal adhesions through cleavage of several endogenous proteins. In addition, we show that titin, a previously identified Capn3 partner, and filamin C are further substrates of Capn3. Finally, we report that Capn3 colocalizes in vivo with its substrates at various sites along cytoskeletal structures. We propose that Capn3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli, establishing Capn3 as a muscle cytoskeleton regulator.

scholarly journals Regulation of the Rapsyn Promoter by Kaiso and δ-Catenin

2004 ◽  
Vol 24 (16) ◽  
pp. 7188-7196 ◽  
Author(s):  
Marianna Rodova ◽  
Kevin F. Kelly ◽  
Michael VanSaun ◽  
Juliet M. Daniel ◽  
Michael J. Werle
Keyword(s):  
Transcription Factor ◽  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Consensus Sequence ◽  
Specific Protein ◽  
C2c12 Cells ◽  
Congenital Myasthenic Syndrome ◽  
P120 Catenin ◽  
Myasthenic Syndrome

ABSTRACT Rapsyn is a synapse-specific protein that is required for clustering acetylcholine receptors at the neuromuscular junction. Analysis of the rapsyn promoter revealed a consensus site for the transcription factor Kaiso within a region that is mutated in a subset of patients with congenital myasthenic syndrome. Kaiso is a POZ-zinc finger family transcription factor which recognizes the specific core consensus sequence CTGCNA (where N is any nucleotide). Previously, the only known binding partner for Kaiso was the cell adhesion cofactor, p120 catenin. Here we show that δ-catenin, a brain-specific member of the p120 catenin subfamily, forms a complex with Kaiso. Antibodies against Kaiso and δ-catenin recognize proteins in the nuclei of C2C12 myocytes and at the postsynaptic domain of the mouse neuromuscular junction. Endogenous Kaiso in C2C12 cells coprecipitates with the rapsyn promoter in vivo as shown by chromatin immunoprecipitation assay. Minimal promoter assays demonstrated that the rapsyn promoter can be activated by Kaiso and δ-catenin; this activation is apparently muscle specific. These results provide the first experimental evidence that rapsyn is a direct sequence-specific target of Kaiso and δ-catenin. We propose a new model of synapse-specific transcription that involves the interaction of Kaiso, δ-catenin, and myogenic transcription factors at the neuromuscular junction.

scholarly journals Set2-Dependent K36 Methylation Is Regulated by Novel Intratail Interactions within H3

2009 ◽  
Vol 29 (24) ◽  
pp. 6413-6426 ◽  
Author(s):  
James N. Psathas ◽  
Suting Zheng ◽  
Song Tan ◽  
Joseph C. Reese
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Chromatin Remodeling ◽  
Posttranslational Modifications ◽  
Active Site ◽  
Transcription Initiation ◽  
Gene Activation ◽  
N Terminus

ABSTRACT Posttranslational modifications to histones have been studied extensively, but the requirement for the residues within the tails for different stages of transcription is less clear. Using RNR3 as a model, we found that the residues within the N terminus of H3 are predominantly required for steps after transcription initiation and chromatin remodeling. Specifically, deleting as few as 20 amino acids, or substituting glutamines for lysines in the tail, greatly impaired K36 methylation by Set2. The mutations to the tail described here preserve the residues predicted to fill the active site of Set2, and the deletion mimics the recently described cleavage of the H3 tail that occurs during gene activation. Importantly, maintaining the charge of the unmodified tail by arginine substitutions preserves Set2 function in vivo. The H3 tail is dispensable for Set2 recruitment to genes but is required for the catalytic activity of Set2 in vitro. We propose that Set2 activity is controlled by novel intratail interactions which can be influenced by modifications and changes to the structure of the H3 tail to control the dynamics and localization of methylation during elongation.

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 Stabilization of Acetylcholine Receptors by Exogenous ATP and Its Reversal by cAMP and Calcium

1997 ◽  
Vol 138 (1) ◽  
pp. 159-165 ◽  
Author(s):  
James P. O'Malley ◽  
Charlotte T. Moore ◽  
Miriam M. Salpeter
Keyword(s):  
Neuromuscular Junction ◽  
Half Life ◽  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Muscle Membrane ◽  
Intracellular Camp ◽  
Signaling Systems ◽  
Camp Analogue ◽  
Degradation Rates ◽  
The Stability

Innervation of the neuromuscular junction (nmj) affects the stability of acetylcholine receptors (AChRs). A neural factor that could affect AChR stabilization was studied using cultured muscle cells since they express two distinct populations of AChRs similar to those seen at the nmjs of denervated muscle. These two AChR populations are (in a ratio of 9 to 1) a rapidly degrading population (Rr) with a degradation half-life of ∼1 d and a slowly degrading population (Rs) that can alternate between an accelerated form (half-life ∼3–5 d) and a stabilized form (half-life ∼10 d), depending upon the state of innervation of the muscle. Previous studies have shown that elevation of intracellular cAMP can stabilize the Rs, but not the Rr. We report here that in cultured rat muscle cells, exogenous ATP stabilized the degradation half-life of Rr and possibly also the Rs. Furthermore, pretreatment with ATP caused more stable AChRs to be inserted into the muscle membrane. Thus, in the presence of ATP, the degradation rates of the Rr and Rs overlap. This suggests that ATP released from the nerve may play an important role in the regulation of AChR degradation. Treatment with either the cAMP analogue dibutyryl-cAMP (dB-cAMP) or the calcium mobilizer ryanodine caused the ATP-stabilized Rr to accelerate back to a half-life of 1 d. Thus, at least three signaling systems (intracellular cAMP, Ca2+, and extracellular ATP) have the potential to interact with each other in the building of an adult neuromuscular junction.

scholarly journals Activity-Dependent Regulation of the Binomial Parameters p and n at the Mouse Neuromuscular Junction In Vivo

2010 ◽  
Vol 104 (5) ◽  
pp. 2352-2358 ◽  
Author(s):  
Xueyong Wang ◽  
Qingbo Wang ◽  
Kathrin L. Engisch ◽  
Mark M. Rich
Keyword(s):  
Neuromuscular Junction ◽  
Muscle Activity ◽  
Acetylcholine Receptors ◽  
Quantal Content ◽  
Spontaneous Release ◽  
Postsynaptic Activity ◽  
Activity Dependent

Block of neurotransmission at the mammalian neuromuscular junction triggers an increase in the number of vesicles released (quantal content). The increase occurs whether nerve and muscle activity are both blocked by placement of a tetrodotoxin (TTX) containing cuff on the nerve or whether muscle activity is selectively blocked by injection of α-bungarotoxin (BTX). We used ANOVA to examine whether the mechanism underlying the increase in quantal content differed between the two types of activity blockade. We found that TTX-induced blockade increased the probability of release ( p), whereas BTX-induced blockade increased the number of releasable vesicles ( n). The lack of increase in p when postsynaptic activity was blocked with BTX suggests that block of presynaptic activity triggers the increase. To determine whether n is regulated by mismatch of pre- and postsynaptic activity introduced by BTX injection we combined BTX and TTX and still found an increase in n. We conclude that block of acetylcholine binding to acetylcholine receptors during spontaneous release triggers the increase in n.

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