Acetylcholine receptor-aggregating proteins are associated with the extracellular matrix of many tissues in Torpedo

The synaptic basal lamina, a component of extracellular matrix (ECM) in the synaptic cleft at the neuromuscular junction, directs the formation of new postsynaptic specializations, including the aggregation of acetylcholine receptors (AChRs), during muscle regeneration in adult animals. Although the molecular basis of this phenomenon is unknown, it is mimicked by AChR-aggregating proteins in ECM-enriched fractions from muscle and the synapse-rich electric organ of the ray Torpedo californica. Molecules immunologically similar to these proteins are concentrated in the synaptic basal lamina at neuromuscular junctions of the ray and frog. Here we demonstrate that immunologically, chemically, and functionally similar AChR-aggregating proteins are also associated with the ECM of several other tissues in Torpedo. Monoclonal antibodies against the AChR-aggregating proteins from electric organ intensely stained neuromuscular junctions and the ventral surfaces of electrocytes, structures with a high density of AChRs. However, they also labeled many other structures which have basal laminae, including the extrajunctional perimeters of skeletal muscle fibers, smooth and cardiac muscle cells, Schwann cell sheaths in peripheral nerves, walls of some blood vessels, and epithelial basement membranes in the gut, skin, and heart. Some structures with basal laminae did not stain with the antibodies; e.g., the dorsal surfaces of electrocytes. Bands of similar molecular weight were detected by the antibodies on Western blots of extracts of ECM-enriched fractions from electric organ and several other tissues. Proteins from all tissues examined, enriched from these extracts by affinity chromatography with the monoclonal antibodies, aggregated AChRs on cultured myotubes. Thus, similar AChR- aggregating proteins are associated with the extracellular matrix of many Torpedo tissues. The broad distribution of these proteins suggests they have functions in addition to AChR aggregation.     

Identification of agrin, a synaptic organizing protein from Torpedo electric organ

Extracts of the electric organ of Torpedo californica contain a proteinaceous factor that causes the formation of patches on cultured myotubes at which acetylcholine receptors (AChR), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) are concentrated. Results of previous experiments indicate that this factor is similar to the molecules in the synaptic basal lamina that direct the aggregation of AChR and AChE at regenerating neuromuscular junctions in vivo. We have purified the active components in the extracts 9,000-fold. mAbs against four different epitopes on the AChR/AChE/BuChE-aggregating molecules each immunoprecipitated four polypeptides from electric organ extracts, with molecular masses of 150, 135, 95, and 70 kD. Gel filtration chromatography of electric organ extracts revealed two peaks of AChR/AChE/BuChE-aggregation activity; one comigrated with the 150-kD polypeptide, the other with the 95-kD polypeptide. The 135- and 70-kD polypeptides did not cause AChR/AChE/BuChE aggregation. Based on these molecular characteristics and on the pattern of staining seen in sections of muscle labeled with the mAbs, we conclude that the electric organ-aggregating factor is distinct from previously identified molecules, and we have named it "agrin."     

The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscle

If skeletal muscles are damaged in ways that spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fiber plasma membrane is characterized by infoldings and a high concentration of acetylcholine receptors (AChRs). The aim of this study was to determine whether or not the synaptic portion of the myofiber basal lamina sheath plays a direct role in the formation of the subsynaptic apparatus on regenerating myofibers, a question raised by the results of earlier experiments. The junctional region of the frog cutaneous pectoris muscle was crushed or frozen, which resulted in disintegration and phagocytosis of all cells at the synapse but left intact much of the myofiber basal lamina. Reinnervation was prevented. When new myofibers developed within the basal lamina sheaths, patches of AChRs and infoldings formed preferentially at sites where the myofiber membrane was apposed to the synaptic region of the sheaths. Processes from unidentified cells gradually came to lie on the presynaptic side of the basal lamina at a small fraction of the synaptic sites, but there was no discernible correlation between their presence and the effectiveness of synaptic sites in accumulating AChRs. We therefore conclude that molecules stably attached to the myofiber basal lamina at synaptic sites direct the formation of subsynaptic apparatus in regenerating myofibers. An analysis of the distribution of AChR clusters at synaptic sites indicated that they formed as a result of myofiber-basal lamina interactions that occurred at numerous places along the synaptic basal lamina, that their presence was not dependent on the formation of plasma membrane infoldings, and that the concentration of receptors within clusters could be as great as the AChR concentration at normal neuromuscular junctions.     

Motor neurons contain agrin-like molecules

Molecules antigenically similar to agrin, a protein extracted from the electric organ of Torpedo californica, are highly concentrated in the synaptic basal lamina of neuromuscular junctions in vertebrate skeletal muscle. On the basis of several lines of evidence it has been proposed that agrin-like molecules mediate the nerve-induced formation of acetylcholine receptor (AChR) and acetylcholinesterase (AChE) aggregates on the surface of muscle fibers at developing and regenerating neuromuscular junctions and that they help maintain these postsynaptic specializations in the adult. Here we show that anti-agrin monoclonal antibodies selectively stain the cell bodies of motor neurons in embryos and adults, and that the stain is concentrated in the Golgi apparatus. We also present evidence that motor neurons in both embryos and adults contain molecules that cause the formation of AChR and AChE aggregates on cultured myotubes and that these AChR/AChE-aggregating molecules are antigenically similar to agrin. These findings are consistent with the hypothesis that agrin-like molecules are synthesized by motor neurons, and are released from their axon terminals to become incorporated into the synaptic basal lamina where they direct the formation of synapses during development and regeneration.     

Aggregating factor from Torpedo electric organ induces patches containing acetylcholine receptors, acetylcholinesterase, and butyrylcholinesterase on cultured myotubes

A factor in extracts of the electric organ of Torpedo californica causes the formation of clusters of acetylcholine receptors (AChRs) and aggregates of acetylcholinesterase (AChE) on myotubes in culture. In vivo, AChRs and AChE accumulate at the same locations on myofibers, as components of the postsynaptic apparatus at neuromuscular junctions. The aim of this study was to compare the distribution of AChRs, AChE, and butyrylcholinesterase (BuChE), a third component of the postsynaptic apparatus, on control and extract-treated myotubes. Electric organ extracts induced the formation of patches that contained high concentrations of all three molecules. The extract-induced aggregation of AChRs, AChE, and BuChE occurred in defined medium, and these components accumulated in patches simultaneously. Three lines of evidence indicate that a single factor in the extracts induced the aggregation of all three components: the dose dependence for the formation of patches of AChRs was the same as that for patches of AChE and BuChE; the AChE- and BuChE-aggregating activities co-purified with the AChR-aggregating activity; and all three aggregating activities were immunoprecipitated at the same titer by a monoclonal antibody against the AChR-aggregating factor. We have shown previously that this monoclonal antibody binds to molecules concentrated in the synaptic cleft at neuromuscular junctions. Taken together, these results suggest that during development and regeneration of myofibers in vivo, the accumulation at synaptic sites of at least three components of the postsynaptic apparatus, AChRs, AChE, and BuChE, are all triggered by the same molecule, a molecule similar if not identical to the electric organ aggregating factor.     

Comparison of agrin-like proteins from the extracellular matrix of chicken kidney and muscle with neural agrin, a synapse organizing protein.

Agrin is a synapse-organizing protein that is concentrated in embryonic motor neurons and the synaptic basal lamina of the neuromuscular junction. Agrin or closely related proteins are also associated with most other basal laminae. Here I report that the major agrin-like proteins from the nervous system and other tissues of the chicken are immunochemically and biochemically similar. Four major agrin-like proteins of approximately 60, 72, 80, and 90 kDa were identified on immunoblots of agrin preparations from both neural and non-neural tissues. Agrin-like proteins from embryonic chicken brain and adult kidney were similar in amino acid composition. Rabbit antisera against each of the kidney proteins labeled basement membranes of several tissues, as well as spinal cord motor neurons. These antibodies specifically precipitated and inhibited acetylcholine receptor (AChR)-aggregating activity from the chicken nervous system and Torpedo electric organ. Thus, the agrin-like proteins of non-neural tissues in the chicken are closely related to agrin from the nervous system. However, the AChR-aggregating activity of chicken agrin preparations differed depending on the tissue of origin. Agrin enriched by immunoaffinity chromatography from the central nervous system induced large numbers of AChR aggregates on cultured myotubes. In contrast, agrin preparations from other chicken tissues induced dramatically fewer and smaller AChR aggregates. The difference in biological activity between these agrin preparations may reflect differential inactivation or the existence of tissue- or cell-specific isoforms of agrin.     

The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering.

Agrin derived from Torpedo electric organ induces the clustering of acetylcholine receptors (AChRs) on cultured myotubes. As a first step toward characterizing the plasma membrane receptor for agrin, we have examined agrin binding to cultured myotubes. Agrin binding is saturable as measured by radioimmunoassay and, like agrin-induced AChR clustering, requires extracellular calcium. Immunofluorescence shows that on myotubes incubated with agrin at 4 degrees C, agrin binds in a uniform, finely punctate pattern that correlates poorly with the distribution of AChRs. Myotubes stimulated with agrin at 37 degrees C for greater than or equal to 2 hr show a coclustering of agrin binding sites and AChRs. By contrast, if anti-AChR antibodies are used either to cluster or to internalize AChRs, the distribution and number of agrin binding sites remain unchanged. The aggregation and calcium dependence of the putative agrin receptor may represent important control points in postsynaptic differentiation.     

The antigenic structure of the acetylcholine receptor from Torpedo californica

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization. Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.     

Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester

Agrin, a protein extracted from the electric organ of Torpedo californica, induces the formation of specializations on cultured chick myotubes that resemble the postsynaptic apparatus at the neuromuscular junction. The aim of the studies reported here was to characterize the effects of agrin on the distribution of acetylcholine receptors (AChRs) and cholinesterase as a step toward determining agrin's mechanism of action. When agrin was added to the medium bathing chick myotubes small (less than 4 micron 2) aggregates of AChRs began to appear within 2 h and increased rapidly in number until 4 h. Over the next 12-20 h the number of aggregates per myotube decreased as the mean size of each aggregate increased to approximately 15 micron 2. The accumulation of AChRs into agrin-induced aggregates occurred primarily by lateral migration of AChRs already in the myotube plasma membrane at the time agrin was added to the cultures. Aggregates of AChRs and cholinesterase remained as long as agrin was present in the medium; if agrin was removed the number of aggregates declined slowly. The formation and maintenance of agrin-induced AChR aggregates required Ca++, Co++ and Mn++ inhibited agrin-induced AChR aggregation and increased the rate of aggregate dispersal. Mg++ and Sr++ could not substitute for Ca++. Agrin-induced receptor aggregation also was inhibited by phorbol 12-myristate 13-acetate, an activator of protein kinase C, and by inhibitors of energy metabolism. The similarities between agrin's effects on cultured myotubes and events that occur during formation of neuromuscular junctions support the hypothesis that axon terminals release molecules similar to agrin that induce the differentiation of the postsynaptic apparatus.     

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

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.     

Evidence for Structural Dissimilarity in the Neurotransmitter Binding Region of Purified Acetylcholine Receptors from Human Muscle and Torpedo Electric Organ

Acetylcholine receptor (AChR) purified from human skeletal muscle affinity-alkylated with bromoacetyl[methyl-3H]choline bromide ([3H]BAC) in mildly reducing conditions to yield a specifically radiolabeled polypeptide, Mr 44,000, the alpha-subunit. The binding of [125I]alpha-bungarotoxin to AChR was completely inhibited by affinity-alkylation, indicating that the human AChR's binding site for alpha-bungarotoxin is closely associated with the alpha-subunit's acetylcholine binding site. Structures in the vicinity of the alpha-bungarotoxin binding sites of AChRs from human muscle and Torpedo electric organ were compared by varying the conditions of alkylation. Under optimal conditions of reduction and alkylation, both human and Torpedo AChR incorporated BAC in equivalence to the number of alpha-bungarotoxin binding sites. However, with limited conditions of reduction but sufficient BAC to alkylate 100% of the alpha-bungarotoxin binding sites of human AChR, only 71% of the Torpedo AChR's binding sites were alkylated. In optimal conditions of reduction but with the minimal concentration of BAC that permitted 100% alkylation of the human AChR's alpha-bungarotoxin sites, only 74% of the Torpedo AChR's binding sites were alkylated. These data suggest that the neurotransmitter binding region of human muscle AChR is structurally dissimilar from that of Torpedo electric organ, having a higher binding affinity for BAC and an adjacent disulfide bond that is more readily accessible to reducing agents.     

Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies

A collection of 126 monoclonal antibodies (mAbs) made against acetylcholine receptors (AChRs) from the electric organs of Torpedo californica or Electrophorus electricus was tested for cross-reactivity with AChRs in cryostat sections of skeletal muscle from Rana pipiens and Xenopus laevis by indirect immunofluorescence. 49 mAbs (39%) cross-reacted with AChRs from Rana, and 25 mAbs (20%) cross-reacted with AChRs from Xenopus. mAbs specific for each of the four subunits of electric organ AChR (alpha, beta, gamma, delta) cross-reacted with AChRs from each amphibian species. mAbs cross-reacting with Xenopus AChRs were, with one exception, a subset of the mAbs cross-reacting with Rana AChRs. The major difference detected between the two species was in binding by mAbs specific for the main immunogenic region (MIR) of the alpha-subunit. Whereas 22 of 33 anti-MIR mAbs tested cross-reacted with Rana AChRs, only one of these mAbs cross-reacted with Xenopus AChRs. Some (32) of the cross-reacting mAbs were tested for binding to AChRs in intact muscle. 21 of these mAbs bound to AChRs only when membranes were made permeable with saponin. Electron microscopy using immunoperoxidase or colloidal gold techniques revealed that these mAbs recognize cytoplasmic determinants and that mAbs that do not require saponin in order to bind AChRs in intact muscle recognize extracellular determinants. These results suggest that AChRs in skeletal muscle of Rana and Xenopus are composed of subunits corresponding to the alpha-, beta-, gamma-, and delta-subunits of AChRs from fish electric organs. The subunit specificity of mAbs whose binding was examined by electron microscopy suggests that parts of each subunit (alpha, beta, gamma, delta) are exposed on the cytoplasmic surface and that, as in AChRs from fish electric organs and mammalian muscle, the MIR on alpha-subunits of Rana AChRs is exposed on the extracellular surface.     

Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle

Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.     

Control of the Amount of a 34K Ca2+-Dependent Membrane Binding Protein (Calelectrin)

A 34,000-Da Ca2+-dependent membrane binding protein (34K) was purified from the electric organ of Torpedo marmorata. Specific antibodies to this protein were raised in rabbits, and radioimmunoassay was used to test the presence of 34K in different tissues of Torpedo as well as in other species. In Torpedo, not only the electric organ, but also the muscle, the spleen, and the liver contained 34K antigenicity. Blood was the only tissue in which 34K antigenicity could not be detected. A 34,000-Da protein (Mr 32,000-36,000) that bound to Torpedo acetylcholine receptor (AChR)-rich membrane in a Ca2+-dependent manner and cross-reacted with anti-(Torpedo 34K) antibody was found in chicken muscle, rat muscle, marine mollusk (Aplysia) central ganglia, and rat and human brain. The concentration of 34K seems to be controlled during development. Chicken 34K antigenicity reached a peak on embryonic day 18, declined, and finally gained its maximal value after synaptic maturation. The AChR concentration in chicken legs also changed in the course of muscle development, although it showed a peak on embryonic day 12 and then declined rapidly. In rat diaphragm, both AChRs and 34K were concentrated in the subsynaptic region. Transection of the phrenic nerve induced the synthesis of AChRs in postsynaptic muscle fibers. This operation did not increase the amount of 34K in the diaphragm. On the contrary, it reduced 34K content to the extrasynaptic level. Taken together, these results support the idea that 34K is an important structural constituent of mature synapses, an observation suggesting the involvement of this protein in the function of the mature synapse.     

A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle

To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43- and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87- and 270-kD proteins were cleaved by Ca++- activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.     

Species specificity of anti-acetylcholine receptor antibodies elicited by synthetic peptides

Two peptides corresponding to amino acid residues 351-368 of the alpha-subunits of Torpedo and human acetylcholine receptor (AChR) were synthesized. These peptides contain a segment (residues 355-364) which displays the greatest variability in amino acid sequence between the two species. Antibodies elicited against the two peptides cross-reacted with the respective native AChRs and were shown to be species specific by radioimmunoassay, immunoblotting, and immunofluorescence microscopy. Thus, antibodies against the Torpedo peptide cross-reacted with Torpedo AChR but did not bind to mammalian or chicken AChR. Antibodies against the human peptide proved to be specific probes for mammalian muscle AChR. They cross-reacted with mammalian AChR (human, calf, mouse, and rat) but not with Torpedo or chicken AChR. These antibodies were also shown to react preferentially with the extrajunctional form of muscle AChR, as compared to their reactivity with junctional muscle AChR. In immunofluorescence experiments, the anti-human peptide antibody stained AChR aggregates in sectioned or ethanol-permeabilized rat and mouse myotubes grown in culture but did not stain living myotubes. This indicates that the sequence 351-368 of the alpha-subunit of mammalian AChR is on the cytoplasmic face of muscle cell membranes, as predicted theoretically.     

Nicotine decreases agrin signaling and acetylcholine receptor clustering in C2C12 myotube culture

The clustering of acetylcholine receptors (AChRs) in skeletal muscle fibers is a critical event in neuromuscular synaptogenesis. AChRs in concert with other molecules form postsynaptic scaffolds in response to agrin released from motor neurons as motor neurons near skeletal muscle fibers in development. Agrin drives an intracellular signaling pathway that precedes AChR clustering and includes the tyrosine phosphorylation of AChRs. In C2C12 myotube culture, agrin application stimulates the agrin signaling pathway and AChR clustering. Previous studies have determined that the frequency of spontaneous AChR clustering is decreased and AChRs are partially inactivated when bound by the acetylcholine agonist nicotine. We hypothesized that nicotine interferes with AChR clustering and consequent postsynaptic scaffold formation. In the present study, C2C12 myoblasts were cultured with growth medium to stimulate proliferation and then differentiation medium to stimulate fusion into myotubes. They were bathed in a physiologically relevant concentration of nicotine and then subject to agrin treatment after myotube formation. Our results demonstrate that nicotine decreases agrin-induced tyrosine phosphorylation of AChRs and decreases the frequency of spontaneous as well as agrin-induced AChR clustering. We conclude that nicotine interferes with postsynaptic scaffold formation by preventing the tyrosine phosphorylation of AChRs, an agrin signaling event that precedes AChR clustering.     

Acetylcholine receptor clustering in C2 muscle cells requires chondroitin sulfate

Proteoglycans have been implicated in the clustering of acetylcholine receptors (AChRs) on cultured myotubes and at the neuromuscular junction. We report that the presence of chondroitin sulfate is associated with the ability of cultured myotubes to form spontaneous clusters of AChRs. Three experimental manipulations of wild type C2 cells in culture were found to affect both glycosaminoglycans (GAGs) and AChR clustering in concert. Chlorate was found to have dose-dependent negative effects both on GAG sulfation and on the frequency of AChR clusters. When extracellular calcium was raised from 1.8 to 6.8 mM in cultures of wild-type C2 myotubes, increases were observed both in the level of cell layer-associated chondroitin sulfate and in the frequency of AChR clusters. Culture of wild-type C2 myotubes in the presence of chondroitinase ABC eliminated cell layer-associated chondroitin sulfate while leaving heparan sulfate intact and simultaneously prevented the formation of AChR clusters. Treatment with either chlorate or chondroitinase inhibited AChR clustering only if begun prior to the spontaneous formation of clusters. We propose that chondroitin sulfate plays an essential role in the initiation of AChR clustering and in the early events of synapse formation on muscle. © 1995 John Wiley & Sons, Inc.     

Effect of agrin on the distribution of acetylcholine receptors and sodium channels on adult skeletal muscle fibers in culture

We used the loose patch voltage clamp technique and rhodamine-conjugated alpha-bungarotoxin to study the regulation of Na channel (NaCh) and acetylcholine receptor (AChR) distribution on dissociated adult skeletal muscle fibers in culture. The aggregate of AChRs and NaChs normally found in the postsynaptic membrane of these cells gradually fragmented and dispersed from the synaptic region after several days in culture. This dispersal was the result of the collagenase treatment used to dissociate the cells, suggesting that a factor associated with the extracellular matrix was responsible for maintaining the high concentration of AchRs and NaChs at the neuromuscular junction. We tested whether the basal lamina protein agrin, which has been shown to induce the aggregation of AChRs on embryonic myotubes, could similarly influence the distribution of NaChs. By following identified fibers, we found that agrin accelerated both the fragmentation of the endplate AChR cluster into smaller patches as well as the appearance of new AChR clusters away from the endplate. AChR patches which were fragments of the original endplate retained a high density of NaChs, but no new NaCh hotspots were found elsewhere on the fiber, including sites of newly formed AChR clusters. The results are consistent with the hypothesis that extracellular signals regulate the distribution of AChRs and NaChs on skeletal muscle fibers. While agrin probably serves this function for the AChR, it does not appear to play a role in the regulation of the NaCh distribution.