Conjugation of LG domains of agrins and perlecan to polymerizing laminin-2 promotes acetylcholine receptor clustering

Neuromuscular junction (NMJ) assembly is characterized by the clustering and neuronal alignment of acetylcholine receptors (AChRs). In this study we have addressed post-synaptic contributions to assembly that may arise from the NMJ basement membrane with cultured myotubes. We show that the cell surface-binding LG domains of non-neural (muscle) agrin and perlecan promote AChR clustering in the presence of laminin-2. This type of AChR clustering occurs with a several hour lag, requires muscle-specific kinase (MuSK), and is accompanied by tyrosine phosphorylation of MuSK and betaAChR. It also requires conjugation of the agrin or perlecan to laminin together with laminin polymerization. Furthermore, AChR clustering can be mimicked with antibody binding to non-neural agrin, supporting a mechanism of ligand aggregation. Neural agrin, in addition to its unique ability to cluster AChRs through its B/z sequence insert, also exhibits laminin-dependent AChR clustering, the latter enhancing and stabilizing its activity. Finally, we show that type IV collagen, which lacks clustering activity on its own, stabilizes laminin-dependent AChR clusters. These findings provide evidence for cooperative and partially redundant MuSK-dependent functions of basement membrane in AChR assembly that can enhance neural agrin activity yet operate in its absence. Such interactions may contribute to the assembly of aneural AChR clusters that precede neural agrin release as well as affect later NMJ development.     

Agrin Binds to the Nerve–Muscle Basal Lamina via Laminin

Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel™. The first 130 amino acids from the NH₂ terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH₂-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel™ and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH₂-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.     

Dystroglycan binding to laminin α1LG4 module influences epithelial morphogenesis of salivary gland and lung in vitro

Dystroglycan is a receptor for the basement membrane components laminin-1, -2, perlecan, and agrin. Genetic studies have revealed a role for dystroglycan in basement membrane formation of the early embryo. Dystroglycan binding to the E3 fragment of laminin-1 is involved in kidney epithelial cell development, as revealed by antibody perturbation experiments. E3 is the most distal part of the carboxyterminus of laminin alpha1 chain, and is composed of two laminin globular (LG) domains (LG4 and LG5). Dystroglycan-E3 interactions are mediated solely by discrete domains within LG4. Here we examined the role of this interaction for the development of mouse embryonic salivary gland and lung. Dystroglycan mRNA was expressed in epithelium of developing salivary gland and lung. Immunofluorescence demonstrated dystroglycan on the basal side of epithelial cells in these tissues. Antibodies against dystroglycan that block binding of alpha-dystroglycan to laminin-1 perturbed epithelial branching morphogenesis in salivary gland and lung organ cultures. Inhibition of branching morphogenesis was also seen in cultures treated with polyclonal anti-E3 antibodies. One monoclonal antibody (mAb 200) against LG4 blocked interactions between a-dystroglycan and recombinant laminin alpha1LG4-5, and also inhibited salivary gland and lung branching morphogenesis. Three other mAbs, also specific for the alpha1 carboxyterminus and known not to block branching morphogenesis, failed to block binding of alpha-dystroglycan to recombinant laminin alpha1LG4-5. These findings clarify why mAbs against the carboxyterminus of laminin alpha1 differ in their capacity to block epithelial morphogenesis and suggest that dystroglycan binding to alpha1LG4 is important for epithelial morphogenesis of several organs.     

Non-muscle alpha-dystroglycan is involved in epithelial development

The dystroglycan complex is a transmembrane linkage between the cytoskeleton and the basement membrane in muscle. One of the components of the complex, alpha-dystroglycan binds both laminin of muscle (laminin-2) and agrin of muscle basement membranes. Dystroglycan has been detected in nonmuscle tissues as well, but the physiological role in nonmuscle tissues has remained unknown. Here we show that dystroglycan during mouse development in nonmuscle tissues is expressed in epithelium. In situ hybridization revealed strong expression of dystroglycan mRNA in all studied epithelial sheets, but not in endothelium or mesenchyme. Conversion of mesenchyme to epithelium occurs during kidney development, and the embryonic kidney was used to study the role of alpha-dystroglycan for epithelial differentiation. During in vitro culture of the metanephric mesenchyme, the first morphological signs of epithelial differentiation can be seen on day two. Northern blots revealed a clear increase in dystroglycan mRNA on day two of in vitro development. A similar increase of expression on day two was previously shown for laminin alpha 1 chain. Immunofluorescence showed that dystroglycan is strictly located on the basal side of developing kidney epithelial cells. Monoclonal antibodies known to block binding of alpha-dystroglycan to laminin-1 perturbed development of epithelium in kidney organ culture, whereas control antibodies did not do so. We suggest that the dystroglycan complex acts as a receptor for basement membrane components during epithelial morphogenesis. It is likely that this involves binding of alpha- dystroglycan to E3 fragment of laminin-1.     

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

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 beta and delta 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.     

A stoichiometric complex of neurexins and dystroglycan in brain

In nonneuronal cells, the cell surface protein dystroglycan links the intracellular cytoskeleton (via dystrophin or utrophin) to the extracellular matrix (via laminin, agrin, or perlecan). Impairment of this linkage is instrumental in the pathogenesis of muscular dystrophies. In brain, dystroglycan and dystrophin are expressed on neurons and astrocytes, and some muscular dystrophies cause cognitive dysfunction; however, no extracellular binding partner for neuronal dystroglycan is known. Regular components of the extracellular matrix, such as laminin, agrin, and perlecan, are not abundant in brain except in the perivascular space that is contacted by astrocytes but not by neurons, suggesting that other ligands for neuronal dystroglycan must exist. We have now identified alpha- and beta-neurexins, polymorphic neuron-specific cell surface proteins, as neuronal dystroglycan receptors. The extracellular sequences of alpha- and beta-neurexins are largely composed of laminin-neurexin-sex hormone-binding globulin (LNS)/laminin G domains, which are also found in laminin, agrin, and perlecan, that are dystroglycan ligands. Dystroglycan binds specifically to a subset of the LNS domains of neurexins in a tight interaction that requires glycosylation of dystroglycan and is regulated by alternative splicing of neurexins. Neurexins are receptors for the excitatory neurotoxin alpha-latrotoxin; this toxin competes with dystroglycan for binding, suggesting overlapping binding sites on neurexins for dystroglycan and alpha-latrotoxin. Our data indicate that dystroglycan is a physiological ligand for neurexins and that neurexins' tightly regulated interaction could mediate cell adhesion between brain cells.     

Disruption of perlecan binding and matrix assembly by post-translational or genetic disruption of dystroglycan function

Dystroglycan is a cell-surface matrix receptor that requires LARGE-dependent glycosylation for laminin binding. Although the interaction of dystroglycan with laminin has been well characterized, less is known about the role of dystroglycan glycosylation in the binding and assembly of perlecan. We report reduced perlecan-binding activity and mislocalization of perlecan in the LARGE-deficient Large(myd) mouse. Cell-surface ligand clustering assays show that laminin polymerization promotes perlecan assembly. Solid-phase binding assays provide evidence for the first time of a trimolecular complex formation of dystroglycan, laminin and perlecan. These data suggest functional disruption of the trimolecular complex in glycosylation-deficient muscular dystrophy.     

Combination of agrin and laminin increase acetylcholine receptor clustering and enhance functional neuromuscular junction formation In vitro

Clustering of acetylcholine receptors (AChR) at the postsynaptic membrane is a crucial step in the development of neuromuscular junctions (NMJ). During development and after denervation, aneural AChR clusters form on the sarcolemma. Recent studies suggest that these receptors are critical for guiding and initiating synaptogenesis. The aim of this study is to investigate the effect of agrin and laminin‐1; agents with known AChR clustering activity; on NMJ formation and muscle maturation. Primary myoblasts were differentiated in vitro on collagen, laminin or collagen and laminin‐coated surfaces in the presence or absence of agrin and laminin. The pretreated cells were then subject to innervation by PC12 cells. The number of neuromuscular junctions was assessed by immunocytochemical co‐localization of AChR clusters and the presynaptic marker synaptophysin. Functional neuromuscular junctions were quantitated by analysis of the level of spontaneous as well as neuromuscular blocker responsive contractile activity and muscle maturation was assessed by the degree of myotube striation. Agrin alone did not prime muscle for innervation while a combination of agrin and laminin pretreatment increased the number of neuromuscular junctions formed and enhanced acetylcholine based neurotransmission and myotube striation. This study has direct clinical relevance for treatment of denervation injuries and creating functional neuromuscular constructs for muscle tissue repair. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 551–565, 2016     

Agrin-Deficient Myotube Retains Its Acetylcholine Receptor Aggregation Ability when Challenged with Agrin

Abstract: Agrin is a synapse-organizing molecule that mediates the nerve-induced aggregation of acetylcholine receptors (AChRs) and other postsynaptic components at the developing and regenerating vertebrate neuromuscular junctions. At the neuromuscular junction, three different cell types can express agrin, i.e., neuron, muscle, and Schwann cell. Several lines of evidence suggested that neuron-derived agrin is the AChR-aggregating factor, but the possible roles of muscle-derived agrin in the formation of AChR aggregate are not known. By using the recombinant DNA method, a clonal stable C2C12 cell line transfected with antisense agrin cDNA was created. RNA dot blot and western blot analysis indicated that the expression of agrin in the transfected cell was abolished by DNA transfection. When the agrin-deficient C2C12 cells were induced to form myotubes and subsequently cocultured with agrin cDNA transfected fibroblasts, AChR aggregates were formed in the cocultures. In addition, acetylcholinesterase (AChE) aggregates in agrin-deficient myotubes were also induced by exogenous agrin and the AChE aggregates were colocalized with the AChR aggregates. The agrin-deficient myotubes could also respond to neuron-induced AChR aggregation after coculturing with neuroblastoma cells. Thus, the agrin-deficient myotubes retain their ability to exhibit the agrin- or neuron-induced AChR aggregation. This result suggests that the formation of postsynaptic specializations during development and regeneration is mediated by neuron-derived agrin but not the agrin from muscle.     

Assembly of laminin polymers is dependent on beta1-integrins

Recent reports suggest that laminin deposition is controlled by the cell via specific receptors, one of which is dystroglycan. In this study, the involvement of beta1-integrins in this process was investigated by comparing beta1-integrin-deficient cells of different phenotypes with their normal counterparts. Normal embryonic stem (ES) cells and embryoid bodies (EBs) derived from them were found to deposit cell-associated laminin into fibrillar networks, and in the EBs a basement membrane was assembled under the primitive endoderm. beta1-deficient ES cells and their EBs formed only small amounts of dot-like laminin deposits. Skeletal myotubes formed after prolonged differentiation in EBs were found to be surrounded by laminin, nidogen, and perlecan by immunofluorescent staining irrespective of the presence of beta1-integrins on the myotubes. However, at the electron microscope level only very thin sheet-like structures were detected close to the beta1-deficient myotubes, while the wt myotubes formed thick basement membranes. An epithelial cell line, GE11, derived from the beta1-integrin-deficient ES cells was also unable to assemble laminin on the cell surface, while transfection of the cells with the integrin beta1 subunit resulted in formation of a dense laminin network. Taken together, these results suggest that dystroglycan and beta1-integrins can both contribute to the recruitment of laminin to cell surfaces and that integrins are required at a subsequent step in the formation of basement membranes.     

Dystroglycan overexpression in vivo alters acetylcholine receptor aggregation at the neuromuscular junction

Dystroglycan is a member of the transmembrane dystrophin glycoprotein complex in muscle that binds to the synapse-organizing molecule agrin. Dystroglycan binding and AChR aggregation are mediated by two separate domains of agrin. To test whether dystroglycan plays a role in receptor aggregation at the neuromuscular junction, we overexpressed it by injecting rabbit dystroglycan RNA into one- or two-celled Xenopus embryos. We measured AChR aggregation in myotomes by labeling them with rhodamine-alpha-bungarotoxin followed by confocal microscopy and image analysis. Dystroglycan overexpression decreased AChR aggregation at the neuromuscular junction. This result is consistent with dystroglycan competition for agrin without signaling AChR aggregation. It also supports the hypothesis that dystroglycan is not the myotube-associated specificity component, (MASC) a putative coreceptor needed for agrin to activate muscle-specific kinase (MuSK) and signal AChR aggregation. Dystroglycan was distributed along the surface of muscle membranes, but was concentrated at the ends of myotomes, where AChRs normally aggregate at synapses. Overexpressed dystroglycan altered AChR aggregation in a rostral-caudal gradient, consistent with the sequential development of neuromuscular synapses along the embryo. Increasing concentrations of dystroglycan RNA did not further decrease AChR aggregation, but decreased embryo survival. Development often stopped during gastrulation, suggesting an essential, nonsynaptic role of dystroglycan during this early period of development.     

Basal lamina components are concentrated in premuscle masses and at early acetylcholine receptor clusters in chick embryo hindlimb muscles

As an initial step in characterizing the function of basal lamina components during muscle cell differentiation and innervation in vivo, we have determined immunohistochemically the pattern of expression of three components--laminin, proteins related to agrin (an acetylcholine receptor (AChR)-aggregating protein), and a heparan sulfate proteoglycan--during the development of chick embryo hindlimb muscles. Monoclonal antibodies against agrin were used to purify the protein from the Torpedo ray and to characterize agrin-like proteins from embryonic and adult chicken. In early hindlimb buds (stage 19), antibodies against laminin and agrin stained the ectodermal basement membrane and bound to limb mesenchyme with a generalized, punctate distribution. However, as dorsal and ventral premuscle masses condensed (stage 22-23), mesenchymal immunoreactivity for laminin and agrin-like proteins, but not the proteoglycan, became concentrated in these myogenic regions. Significantly, the preferential accumulation of these molecules in myogenic regions of the limb preceded by 1-2 days the appearance of muscle-specific proteins, myoblast fusion, and muscle innervation. All three basal lamina components were preferentially associated with all AChR clusters from the time we first observed them on newly formed myotubes at stage 26. Localization of these antigens in three-dimensional collagen gel cultures of limb mesenchyme, explanted prior to innervation of the limb, paralleled the staining patterns seen during limb development in the embryo. These results indicate that basal lamina molecules intrinsic to limb mesenchyme are early markers for myogenic and synaptic differentiation, and suggest that these components play important roles during the initial phases of myogenesis and synaptogenesis.     

Transmembrane mechanisms in the assembly of the postsynaptic apparatus at the neuromuscular junction

The vertebrate neuromuscular junction (NMJ) is marked by molecular specializations that include postsynaptic clusters of acetylcholine receptor (AChR) and acetylcholinesterase (AChE). Whereas AChRs are aggregated in the postsynaptic muscle membrane to a density of 10,000/mum(2), AChE is concentrated, also to a high density, in the synaptic basement membrane (BM). In recent years considerable progress has been made in understanding the cellular and molecular mechanisms of AChR clustering. It is known that during the early stages of motoneuron-muscle interaction, the nerve-secreted proteoglycan agrin activates the muscle-specific kinase MuSK, which leads to the formation of a postsynaptic cytoskeletal scaffold that immobilizes and concentrates AChRs through a process generally accepted to involve diffusion-mediated trapping of the receptors. We have recently tested this diffusion-trap model at the single molecule level for the first time by using quantum-dot labeling to track individual AChRs during NMJ development. Our results showed that single AChRs exhibit Brownian-type movement, with diffusion coefficients of 10(-11) to 10(-9)cm(2)/s, until they become immobilized at "traps" assembled in response to synaptogenic stimuli. Thus, free diffusion of AChRs is an integral part of their clustering mechanism. What is the mechanism for AChE clustering? We previously showed that the A(12) asymmetric form of AChE binds to perlecan, a heparan-sulfate proteoglycan which in turn interacts with the transmembrane dystroglycan complex. Through this linkage AChE becomes bound to the muscle membrane and, like AChRs, may exhibit lateral mobility along the membrane. Consistent with this idea, pre-existent AChE at the cell surface becomes clustered together with AChRs following synaptogenic stimulation. Future studies testing diffusion-mediated trapping of AChE should provide insights into the synaptic localization of BM-bound molecules at the NMJ.     

Alpha-galactosidase stimulates acetylcholine receptor aggregation in skeletal muscle cells via PNA-binding carbohydrates

Aggregation of nicotinic acetylcholine receptors (AChRs) in skeletal muscle is an essential step in the formation of the mammalian neuromuscular junction. While proteins that bind to myotube receptors such as agrin and laminin can stimulate AChR aggregation in cultured myotubes, removal of cell surface sialic acids stimulates aggregation in a ligand-independent manner. Here, we show that removal of cell surface alpha-galactosides also stimulates AChR aggregation in the absence of added laminin or agrin. AChR aggregation stimulated by alpha-galactosidase was blocked by peanut agglutinin (PNA), which binds to lactosamine-containing disaccharides, but not by the GalNAc-binding lectin Vicia villosa agglutinin (VVA-B4). AChR aggregation stimulated by alpha-galactosidase potentiated AChR clustering induced by either neural agrin or laminin-1 and could be inhibited by muscle agrin. These data suggest that capping of cell surface lactosamines or N-acetyllactosamines with alpha-galactose affects AChR aggregation much as capping with sialic acids does.     

Recycling of acetylcholine receptors at ectopic postsynaptic clusters induced by exogenous agrin in living rats

During the development of the neuromuscular junction, motor axons induce the clustering of acetylcholine receptors (AChRs) and increase their metabolic stability in the muscle membrane. Here, we asked whether the synaptic organizer agrin might regulate the metabolic stability and density of AChRs by promoting the recycling of internalized AChRs, which would otherwise be destined for degradation, into synaptic sites. We show that at nerve-free AChR clusters induced by agrin in extrasynaptic membrane, internalized AChRs are driven back into the ectopic synaptic clusters where they intermingle with pre-existing and new receptors. The extent of AChR recycling depended on the strength of the agrin stimulus, but not on the development of junctional folds, another hallmark of mature postsynaptic membranes. In chronically denervated muscles, in which both AChR stability and recycling are significantly decreased by muscle inactivity, agrin maintained the amount of recycled AChRs at agrin-induced clusters at a level similar to that at denervated original endplates. In contrast, AChRs did not recycle at agrin-induced clusters in C2C12 or primary myotubes. Thus, in muscles in vivo, but not in cultured myotubes, neural agrin promotes the recycling of AChRs and thereby increases their metabolic stability.     

Agrin-induced reorganization of extracellular matrix components on cultured myotubes: relationship to AChR aggregation

Agrin, an extracellular matrix-associated protein extracted from synapse-rich tissues, induces the accumulation of acetylcholine receptors (AChRs) and other synaptic components into discrete patches on cultured myotubes. The appearance of agrin-like molecules at neuromuscular junctions suggests that it may direct synaptic organization in vivo. In the present study we examined the role of extracellular matrix components in agrin-induced differentiation. We used immunohistochemical techniques to visualize the spatial and temporal distribution of laminin, a heparan sulfate proteoglycan (HSPG), fibronectin, and type IV collagen on cultured chick myotubes during agrin-induced aggregation of AChRs. Myotubes displayed significant amounts of laminin and HSPG, lesser amounts of type IV collagen, and little, if any, fibronectin. Agrin treatment caused cell surface laminin and HSPG to patch, while collagen and fibronectin distributions were generally unaffected. Many of the agrin-induced laminin and HSPG patches colocalized with AChR patches, raising the possibility of a causal relationship between matrix patching and AChR accumulations. However, patching of AChRs (complete within a few hours) preceded that of laminin or HSPG (not complete until 15-20 h), making it unlikely that matrix accumulations initiate AChR patching at agrin-induced sites. Conversely, when AChR patching was blocked by treatment with anti-AChR antibody mAb 35, agrin was still able to effect patching of laminin and HSPG. Taken together, these findings suggest that agrin-induced accumulations of AChR and laminin/HSPG are not mechanistically linked.     

Role of lipid rafts in agrin-elicited acetylcholine receptor clustering

Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.     

A Functional Role for Specific Spliced Variants of the α7β1 Integrin in Acetylcholine Receptor 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.     

Overexpression of rapsyn inhibits agrin-induced acetylcholine receptor clustering in muscle cells

Rapsyn is a protein on the cytoplasmic face of the postsynaptic membrane of skeletal muscle that is essential for clustering acetylcholine receptors (AChR). Here we show that transfection of rapsyn cDNA can restore AChR clustering function to muscle cells cultured from rapsyn deficient (KORAP) mice. KORAP myotubes displayed no AChR aggregates before or after treatment with neural agrin. After transfection with rapsyn expression plasmid, some KORAP myotubes expressed rapsyn at physiological levels. These formed large AChR-rapsyn clusters in response to agrin, just like wild-type myotubes. KORAP myotubes that overexpressed rapsyn formed only scattered AChR-rapsyn microaggregates, irrespective of agrin treatment. KORAP cells were then transfected with mutant forms of rapsyn. A deletion mutant lacking residues 16–254 formed rapsyn microaggregates, but failed to aggregate AChRs. Substitution mutation to the C-terminal serine phosphorylation site of rapsyn (M43^D405,D406) did not impair the response to agrin, showing that differential phosphorylation of this site is unlikely to mediate agrin-induced clustering. The results indicate that rapsyn expression is essential for agrin-induced AChR clustering but that its overexpression inhibits this pathway. The approach of using rapsyn-deficient muscle cells opens the way for defining the role of rapsyn in agrin-induced AChR clustering.     

Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo.

Aggregation of acetylcholine receptors (AChRs) in muscle fibers by nerve-derived agrin plays a key role in the formation of neuromuscular junctions. So far, the effects of agrin on muscle fibers have been studied in culture systems, transgenic animals, and in animals injected with agrin--cDNA constructs. We have applied purified recombinant chick neural and muscle agrin to rat soleus muscle in vivo and obtained the following results. Both neural and muscle agrin bind uniformly to the surface of innervated and denervated muscle fibers along their entire length. Neural agrin causes a dose-dependent appearance of AChR aggregates, which persist > or = 7 wk after a single application. Muscle agrin does not cluster AChRs and at 10 times the concentration of neural agrin does not reduce binding or AChR-aggregating activity of neural agrin. Electrical muscle activity affects the stability of agrin binding and the number, size, and spatial distribution of the neural agrin--induced AChR aggregates. Injected agrin is recovered from the muscles together with laminin and both proteins coimmunoprecipitate, indicating that agrin binds to laminin in vivo. Thus, the present approach provides a novel, simple, and efficient method for studying the effects of agrin on muscle under controlled conditions in vivo.