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At least 109 records · Page 6Linked to original sources
PubMed2000

Nitric oxide is a downstream mediator of agrin-induced acetylcholine receptor aggregation.

The synaptic basal lamina protein, agrin, is required for the formation of the neuromuscular junction. Agrin signals through a muscle-specific receptor tyrosine kinase (MuSK) initiating a cascade of events that lead to the aggregation of acetylcholine receptors (AChR) at the postsynaptic site. Another important synaptic signalling molecule is nitric oxide (NO), which is produced by the enzyme, nitric oxide synthase (NOS). We investigated the interaction between the agrin signalling cascade and the NO signalling cascade by treating cultured myotubes with agrin, NOS inhibitors, and NO donors. NOS inhibitors prevented agrin induced AChR aggregation and phosphorylation of the AChR beta subunit. Furthermore, NO donors induced AChR aggregation in the absence of agrin, as well as phosphorylation of the AChR beta subunit. These results demonstrate a role for NO as a downstream mediator of agrin induced AChR aggregation and AChR beta subunit phosphorylation at the neuromuscular junction.

Agrin↗
PubMed1995

Acetylcholine receptor aggregation at nerve-muscle contacts in mammalian cultures: induction by ventral spinal cord neurons is specific to axons.

We used a novel mammalian coculture system to study ACh receptor (AChR) redistribution and synaptic structure at nerve-muscle contacts. Ventral spinal cord (VSC) neurons were plated on cultures containing extensive myotubes but few fibroblasts. Neurite-induced redistribution of AChRs occurred within 6 hr after plating neurons and was maximal between 36-48 hr. This AChR redistribution appeared in two patterns: (1) AChR density at sites directly apposed to the neurite where neurites crossed preexisting AChR patches was sharply reduced, (2) Newly aggregated AChRs formed swaths lateral to the neurite path. VSC neurons induced more AChR aggregation than hippocampal, superior cervical ganglion and dorsal root ganglion neurons. The 43 and 58 kDa postsynaptic proteins were colocalized with AChR-enriched domains in all VSC neurite-induced aggregates whereas the colocalization of laminin was variable. Electron microscopy of regions with neurite-induced AChR aggregation showed postsynaptic membrane specializations characteristic of developing synapses and, in older cultures, features of more mature synaptic structure. Thus, the coculture system is useful for studying early stages of neuromuscular junction (NMJ) formation. Neurites in these cocultures were identified as axons or dendrites by morphological criteria and by their immunoreactivity for synaptophysin and phosphorylated heavy neurofilament subunits or for microtubule associated protein 2 (MAP2), respectively. Axons showed a 10-fold higher induction of AChR aggregation than did dendrites. Thus, at least one essential signaling molecule necessary for the induction of AChR aggregation at sites of interaction with muscle appears to be expressed in a polarized fashion in developing VSC neurons.

Animals↗
PubMed1994

Microglia with an endothelin ETB receptor aggregate in rat hippocampus CA1 subfields following transient forebrain ischemia.

We examined endothelin (ET) receptors in the hippocampus CA1 subfields of stroke-prone spontaneously hypertensive rats subjected to a 10-min bilateral carotid occlusion and reperfusion. When delayed neuronal death had occurred in the pyramidal cell layer at 7 days after transient forebrain ischemia, the quantitative receptor autoradiographic method we used revealed a dramatic increase in number of 125I-ET-1 binding sites in the hippocampus CA1 subfields. The highest number of de novo binding sites appeared in the area corresponding anatomically to the pyramidal cell layer with neuronal death. These binding sites were characteristically the ETB receptor. The de novo 125I-ET-1 binding was mainly present on microglia aggregating with a high density in the damaged pyramidal cell layer. As ET-1- and ET-3-like immunoreactivities were highly expressed within astrocytes in damaged neural tissue, the possibility that microglia with the ETB receptor are activated to participate in the pathophysiology of ischemia-related neural tissue damage by astrocytic ET-1 and ET-3 produced in response to transient forebrain ischemia would have to be considered.

Amino Acid Sequence↗
PubMed2006

Casein kinase 2-dependent serine phosphorylation of MuSK regulates acetylcholine receptor aggregation at the neuromuscular junction.

The release of Agrin by motoneurons activates the muscle-specific receptor tyrosine kinase (MuSK) as the main organizer of subsynaptic specializations at the neuromuscular junction. MuSK downstream signaling is largely undefined. Here we show that protein kinase CK2 interacts and colocalizes with MuSK at post-synaptic specializations. We observed CK2-mediated phosphorylation of serine residues within the kinase insert (KI) of MuSK. Inhibition or knockdown of CK2, or exchange of phosphorylatable serines by alanines within the KI of MuSK, impaired acetylcholine receptor (AChR) clustering, whereas their substitution by residues that imitate constitutive phosphorylation led to aggregation of AChRs even in the presence of CK2 inhibitors. Impairment of AChR cluster formation after replacement of MuSK KI with KIs of other receptor tyrosine kinases correlates with potential CK2-dependent serine phosphorylation within KIs. MuSK activity was unchanged but AChR stability decreased in the presence of CK2 inhibitors. Muscle-specific CK2beta knockout mice develop a myasthenic phenotype due to impaired muscle endplate structure and function. This is the first description of a regulatory cross-talk between MuSK and CK2 and of a role for the KI of the receptor tyrosine kinase MuSK for the development of subsynaptic specializations.

Amino Acid Sequence↗
PubMed1999

Herpes simplex virus thymidine kinase/ganciclovir-induced apoptosis involves ligand-independent death receptor aggregation and activation of caspases.

Suicide gene therapy systems such as the herpes simplex thymidine kinase/ganciclovir system (TK/GCV) may kill cancer cells by apoptosis through as yet undefined mechanisms. Here we show that TK/GCV treatment induces p53 accumulation and increases cell surface expression of CD95 and tumor necrosis factor receptor, which is likely to involve p53-mediated translocation of CD95 to the cell surface. TK/GCV-induced apoptosis involves CD95-L-independent CD95 aggregation leading to the formation of a Fas-associated death domain protein (FADD) and caspase-8-containing, death-inducing signaling complex. Dominant negative FADD, the caspase-8 inhibitor zIETD-fmk [Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone], and zVAD-fmk (Z-Val-Ala-Asp-fluoromethylketone) partially abrogate TK/GCV-induced apoptosis. In addition to apoptosis induction, TK/GCV treatment strongly sensitizes for CD95-L-, TNF-, and TNF-related, apoptosis-inducing, ligand (TRAIL)-induced cell death in constitutively resistant cells. These findings may be used to increase the efficacy of TK/GCV and other suicide gene therapy systems for the treatment of cancer.

Adaptor Proteins, Signal Transducing↗
PubMed1984

Acetylcholine receptor aggregation parallels the deposition of a basal lamina proteoglycan during development of the neuromuscular junction.

To determine the time course of synaptic differentiation, we made successive observations on identified, nerve-contacted muscle cells developing in culture. The cultures had either been stained with fluorescent alpha-bungarotoxin, or were maintained in the presence of a fluorescent monoclonal antibody. These probes are directed at acetylcholine receptors (AChR) and a basal lamina proteoglycan, substances that show nearly congruent surface organizations at the adult neuromuscular junction. In other experiments individual muscle cells developing in culture were selected at different stages of AChR accumulation and examined in the electron microscope after serial sectioning along the entire path of nerve-muscle contact. The results indicate that the nerve-induced formation of AChR aggregates and adjacent plaques of proteoglycan is closely coupled throughout early stages of synapse formation. Developing junctional accumulations of AChR and proteoglycan appeared and grew progressively, throughout a perineural zone that extended along the muscle surface for several micrometers on either side of the nerve process. Unlike junctional AChR accumulations, which disappeared within a day of denervation, both junctional and extrajunctional proteoglycan deposits were stable in size and morphology. Junctional proteoglycan deposits appeared to correspond to discrete ultrastructural plaques of basal lamina, which were initially separated by broad expanses of lamina-free muscle surface. The extent of this basal lamina, and a corresponding thickening of the postsynaptic membrane, also increased during the accumulation of AChR and proteoglycan along the path of nerve contact. Presynaptic differentiation of synaptic vesicle clusters became detectable at the developing neuromuscular junction only after the formation of postsynaptic plaques containing both AChR and proteoglycan. It is concluded that motor nerves induce a gradual formation and growth of AChR aggregates and stable basal lamina proteoglycan deposits on the muscle surface during development of the neuromuscular junction.

Animals↗
PubMed1999

Dystrophin is required for organizing large acetylcholine receptor aggregates.

Dystrophin is a cytoplasmic protein underlying the plasma membrane in normal skeletal muscle. Its absence leads to muscle degeneration as seen in Duchenne muscular dystrophy (DMD) and in mdx mice. One puzzling question in the study of dystrophinopathies is that in mdx muscles the neuromuscular junctions (NMJs) show little, if any, developmental defect, but morphological and functional abnormalities of NMJs are obvious after muscle damage and regeneration begin. This phenomenon leads us to hypothesize that dystrophin may be required for endplate maintenance and/or endplate remodeling in regenerating fibers. Here we show that the absence of dystrophin causes NMJ fragmentation in adult muscle fibers, and greatly reduces both spontaneous and agrin-induced acetylcholine receptor (AChR) clustering activities on cultured myotubes derived from satellite cells. The lower AChR clustering in mdx myotubes originates in the smaller size of each cluster and from a 72% reduction in the occurrence of large (> 10 micron 2) AChR clusters. Our results suggest dystrophin is involved in organizing small AChR clusters into large AChR aggregates during muscle regeneration, although it is not required for initiating the original AChR clustering activity.

Age Factors↗
PubMed1992

Comparative studies of isolated CD3: CD8, CD3: CD3, and monovalent CD3 binding on CD8+ T-cell activation: model of progressive T-cell receptor aggregation synergism.

The TCR and CD8 complexes of CD8+ T cells bind to different regions of MHC class I molecules and both play important roles in the response of the CD8+ T cells to Ag/MHC on APCs. In this report, we mimicked common MHC binding with an anti-CD3:anti-CD8 (CD3,8) BSMAB to isolate the effect of CD3: CD8 pairing, compared this with the effect of CD3: CD3 pairing by the parental bivalent anti-CD3 MAB, and with monovalent anti-CD3 binding by an anti-CD3: anti-CD4 (CD3,4) BSMAB. CD3: CD8 pairing induced an increase in cytosolic free [Ca2+] 1.5 to 3.0-fold greater than the increase induced by CD3: CD3 pairing whereas monovalent CD3 binding induced only 20%-30% of the increase. Postbinding receptor migration studies suggested that microaggregation increased from monovalent CD3 binding to CD3: CD3 pairing to CD3: CD8 pairing. Further studies revealed that progressively higher concentrations of antibodies were needed from CD3,8 to CD3,3 to CD3,4 to initiate the same degree of DNA synthesis. These results demonstrated that Ti/CD3 and CD8 can indeed be bridged by a single molecule. A model of direct CD8: CD3 synergism was raised as a possible explanation for the enhanced activation induced by CD3: CD8 pairing. The observed parallel between all three parameters and the number of TCRs that can be directly linked by the Abs raised a nonmutually exclusive model whereby CD3 binding induces activated TCR intermediaries (aTCRi) that progressively synergize with other adjacent aTCRis. In this model, this dominant inter-aTCRi synergism may be enhanced by the di- and multimeric CD8 alpha chains serving as aTCRi-aggregation foci.

Antigen-Antibody Reactions↗
PubMed1984

Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates.

Monoclonal antibodies recognizing laminin, heparan sulfate proteoglycan, fibronectin, and two apparently novel connective tissue components have been used to examine the organization of extracellular matrix of skeletal muscle in vivo and in vitro. Four of the five monoclonal antibodies are described for the first time here. Immunocytochemical experiments with frozen-sectioned muscle demonstrated that both the heparan sulfate proteoglycan and laminin exhibited staining patterns identical to that expected for components of the basal lamina. In contrast, the remaining matrix constituents were detected in all regions of muscle connective tissue: the endomysium, perimysium, and epimysium. Embryonic muscle cells developing in culture elaborated an extracellular matrix, each antigen exhibiting a unique distribution. Of particular interest was the organization of extracellular matrix on myotubes: the build-up of matrix components was most apparent in plaques overlying clusters of an integral membrane protein, the acetylcholine receptor (AChR). The heparan sulfate proteoglycan was concentrated at virtually all AChR clusters and showed a remarkable level of congruence with receptor organization; laminin was detected at 70-95% of AChR clusters but often was not completely co-distributed with AChR within the cluster; fibronectin and the two other extracellular matrix antigens occurred at approximately 20, 8, and 2% of the AChR clusters, respectively, and showed little or no congruence with AChR. From observations on the distribution of extracellular matrix components in tissue cultured fibroblasts and myogenic cells, several ideas about the organization of extracellular matrix are suggested. (a) Congruence between AChR clusters and heparan sulfate proteoglycan suggests the existence of some linkage between the two molecules, possibly important for regulation of AChR distribution within the muscle membrane. (b) The qualitatively different patterns of extracellular matrix organization over myotubes and fibroblasts suggest that each of these cell types uses somewhat different means to regulate the assembly of extracellular matrix components within its domain. (c) The limited co-distribution of different components within the extracellular matrix in vitro and the selective immune precipitation of each antigen from conditioned medium suggest that each extracellular matrix component is secreted in a form that is not complexed with other matrix constituents.

Animals↗
PubMed1992

Phospholipase C-gamma 1 is translocated to the membrane of rat basophilic leukemia cells in response to aggregation of IgE receptors.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells results in the rapid hydrolysis of membrane inositol phospholipids by phospholipase C (PLC). Although at least seven isoenzymes of PLC have been characterized in different mammalian cells, the isoenzyme involved in Fc epsilon RI-mediated signal transduction and the mechanism of its activation have not been demonstrated. We now report that PLC-gamma 1 is translocated to the membrane of mast cells after aggregation of Fc epsilon RI. Activation of rat basophilic leukemia cells, a rat mast cell line, with oligomeric IgE resulted in an increase in PLC activity in washed membrane preparations in a cell free assay containing exogenous [3H]phosphatidylinositol (PI). The increase in PLC activity has the same dose-response to oligomeric IgE as receptor mediated hydrolysis of inositol lipids (PI hydrolysis) in intact cells. Analysis by Western blot probed with anti-PLC-gamma 1 antibody revealed that there is a three- to fourfold increase in PLC-gamma 1 in membranes from activated cells. The increase in PLC activity is augmented a further 20% by the addition of orthovanadate to the incubation medium suggesting that a tyrosine phosphatase is involved in the down-regulation of this phenomenon. These findings demonstrate translocation of PLC-gamma 1 to the membrane following activation of a receptor which does not contain intrinsic tyrosine kinase activity. Activation of PLC-gamma 1 by this pathway may account for Fc epsilon RI-mediated PI hydrolysis.

Animals↗
PubMed1994

Localization and alternative splicing of agrin mRNA in adult rat brain: transcripts encoding isoforms that aggregate acetylcholine receptors are not restricted to cholinergic regions.

Agrin is a protein implicated in the formation and maintenance of the neuromuscular junction. In addition to motor neurons, agrin mRNA has been detected in the brains of embryonic rat and chick and adult marine ray, suggesting that this molecule may also be involved in the formation of synapses between neurons. As a step toward understanding agrin's role in the CNS, we utilized Northern blot and in situ hybridization techniques to analyze the regional distribution and cellular localization of agrin mRNA in the spinal cord and brain of adult rats. The results of these studies indicate that the agrin mRNA is expressed predominantly by neurons broadly distributed throughout the adult CNS. Moreover, expression of agrin mRNA is not restricted to cholinergic structures or regions of the brain receiving cholinergic input. Recently, RNA isolated from rat embryonic spinal cord was shown to contain four alternatively spliced agrin mRNAs, referred to as agrin0, agrin8, agrin11, and agrin19, each of which encodes agrin proteins that are active in acetylcholine receptor aggregating assays (Ferns et al., 1992). Using the polymerase chain reaction we demonstrate that all four of these agrin transcripts are expressed within the adult CNS. Agrin0, agrin8, and agrin19 were present in all regions analyzed. In contrast, agrin11 was detected only in forebrain. Results of these studies indicate that both the level of expression and pattern of alternative splicing of agrin mRNA are differentially regulated in the brain. The broad and predominantly neuronal distribution of agrin mRNA in the adult brain suggests that, in addition to its role at the neuromuscular junction, agrin may play a role in formation and maintenance of synapses between neurons in the CNS.

Agrin↗
PubMed1993

Collagen induced human platelet aggregation: serotonin receptor antagonism retards aggregate growth in vitro.

OBJECTIVE: The role of platelet derived serotonin (5-HT) in collagen induced human platelet aggregation was examined both in hirudinised plasma and whole blood. Hirudin was used to maintain normocalcaemia, thereby avoiding erroneous findings often obtained with citrated blood. METHODS: Platelet aggregation, aggregate growth, and primary platelet aggregation were quantified by optical aggregometry and platelet counting respectively. RESULTS: In plasma, platelet aggregation induced by 5-HT (3 microM) in the presence of adrenaline (1 microM) was inhibited by more than 85% by ICI 170809 (3 microM, IC50 0.1 microM), a specific platelet 5-HT2 antagonist. With collagen (0.5 micrograms.ml-1), ICI 170809 retarded the rate of aggregation by 30%, whereas aspirin abolished the response. In contrast, ICI 170809 inhibited collagen induced primary aggregation by less than 10% in both plasma and whole blood. CONCLUSIONS: (1) 5-HT contributes to collagen induced aggregate growth and 5-HT2 receptor antagonism with ICI 170809 retards the rate of growth. (2) This could explain the efficacy of 5-HT2 antagonists in limiting coronary thrombosis despite the limited role of 5-HT in primary aggregation.

Apyrase↗
PubMed1982

Aggregation of acetylcholine receptors in nerve-muscle cocultures is decreased by inhibitors of collagen production.

Coculturing of rat embryonic muscle cells with spinal cord explants resulted in the formation of large numbers of acetylcholine receptor aggregates on the myotube surface, compared to those found on muscle cells grown in the absence of nervous tissue. Remarkably fewer receptor aggregates were formed when, upon addition of nerve explants, these cocultures were treated with either cis-hydroxyproline (a specific inhibitor of collagen production) or collagenase. The possibility is raised that collagen participates in the aggregation of acetylcholine receptors and in synapse formation.

Animals↗
PubMed1997

Aggregation of PDGF-beta receptors in human skin fibroblasts: characterization by image correlation spectroscopy (ICS).

Receptor aggregation is believed to be an important, early step when growth factors such as PDGF stimulate proliferation and differentiation of cell populations. To investigate receptor aggregation, we utilized a novel biophysical technique, image correlation spectroscopy, to study the distribution and aggregation state of PDGF-beta receptors on the surface of human dermal fibroblasts under various experimental conditions. It was found that the cell surface receptors were pre-clustered at 4 degrees C and receptor aggregation increased for samples measured at 37 degrees C. Treatment with PDGF-BB had no measurable effect on the receptor aggregation state. The results also indicate that additions of 10% serum or an inhibitor of tyrosine kinase activity, may disperse the receptors. The results of this study are consistent with organization of PDGF-beta receptors in pre-existing membrane domains.

Becaplermin↗