AChR phosphorylation and aggregation induced by an agrin fragment that lacks the binding domain for alpha-dystroglycan.

Agrin induces both phosphorylation and aggregation of nicotinic acetylcholine receptors (AChRs) when added to myotubes in culture, apparently by binding to a specific receptor on the myotube surface. One such agrin receptor is alpha-dystroglycan, although binding to alpha-dystroglycan appears not to mediate AChR aggregation. To determine whether agrin-induced AChR phosphorylation is mediated by alpha-dystroglycan or by a different agrin receptor, fragments of recombinant agrin that differ in affinity for alpha-dystroglycan were examined for their ability to induce AChR phosphorylation and aggregation in mouse C2 myotubes. The carboxy-terminal 95 kDa agrin fragment agrin-c95(A0B0), which binds to alpha-dystroglycan with high affinity, failed to induce AChR phosphorylation and aggregation. In contrast, agrin-c95(A4B8) which binds less strongly to alpha-dystroglycan, induced both phosphorylation and aggregation, as did a small 21 kDa fragment of agrin, agrin-c21(B8), that completely lacks the binding domain for alpha-dystroglycan. We conclude that agrin-induced AChR phosphorylation and aggregation are triggered by an agrin receptor that is distinct from alpha-dystroglycan.     

Nitric oxide and cyclic GMP regulate early events in agrin signaling in skeletal muscle cells

Agrin released from motor nerve terminals directs differentiation of the vertebrate neuromuscular junction (NMJ). Activity of nitric oxide synthase (NOS), guanylate cyclase (GC), and cyclic GMP-dependent protein kinase (PKG) contributes to agrin signaling in embryonic frog and chick muscle cells. Stimulation of the NO/cyclic GMP (cGMP) pathway in embryos potentiates agrin's ability to aggregate acetylcholine receptors (AChRs) at NMJs. Here we investigated the timing and mechanism of NO and cGMP action. Agrin increased NO levels in mouse C2C12 myotubes. NO donors potentiated agrin-induced AChR aggregation during the first 20 min of agrin treatment, but overnight treatment with NO donors inhibited agrin activity. Adenoviruses encoding siRNAs against each of three NOS isoforms reduced agrin activity, indicating that these isoforms all contribute to agrin signaling. Inhibitors of NOS, GC, or PKG reduced agrin-induced AChR aggregation in mouse muscle cells by ∼ 50%. However, increased activation of the GTPase Rac1, an early step in agrin signaling, was dependent on NOS activity and was mimicked by NO donors and a cGMP analog. Our results indicate that stimulation of the NO/cGMP pathway is important during the first few minutes of agrin signaling and is required for agrin-induced Rac1 activation, a key step leading to reorganization of the actin cytoskeleton and subsequent aggregation of AChRs on the surface of skeletal muscle cells.     

Agrin fragments differentially induce ectopic aggregation of acetylcholine receptors in myotomal muscles of Xenopus embryos

Agrin is an extracellular synaptic protein that organizes the postsynaptic apparatus, including acetylcholine receptors (AChRs), of the neuromuscular junction. The COOH-terminal portion of agrin has full AChR-aggregating activity in culture, and includes three globular domains, G1, G2, and G3. Portions of the agrin protein containing these domains bind to different cell surface proteins of muscle cells, including alpha-dystroglycan (G1-G2) and heparan sulfate proteoglycans (G2), whereas the G3 domain is sufficient to aggregate AChRs. We sought to determine whether the G1 and G2 domains of agrin potentiate agrin activity in vivo, as they do in culture. Fragments from the COOH-terminal of a neuronal agrin isoform (4,8) containing G3, both G2 and G3, or all three G domains were overexpressed in Xenopus embryos during neuromuscular synapse formation in myotomal muscles. RNA encoding these fragments of rat agrin was injected into one-cell embryos. All three fragments increased the ectopic aggregation of AChRs in noninnervated regions near the center of myotomes. Surprisingly, ectopic aggregation was more pronounced after overexpression of the smallest fragment, which lacks the heparin- and alpha-dystroglycan-binding domains. Synaptic AChR aggregation was decreased in embryos overexpressing the fragments, suggesting a competition between endogenous agrin secreted by nerve terminals and exogenous agrin fragments secreted by muscle cells. These results suggest that binding of the larger agrin fragments to alpha-dystroglycan and/or heparan sulfate proteoglycans may sequester the fragments and inhibit their activity in embryonic muscle. These intermolecular interactions may regulate agrin activity and differentiation of the neuromuscular junction in vivo.     

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.     

Staurosporine inhibits agrin-induced acetylcholine receptor phosphorylation and aggregation

Agrin, a protein that mediates nerve-induced acetylcholine receptor (AChR) aggregation at developing neuromuscular junctions, has been shown to cause an increase in phosphorylation of the beta, gamma, and delta subunits of AChRs in cultured myotubes. As a step toward understanding the mechanism of agrin-induced AChR aggregation, we examined the effects of inhibitors of protein kinases on AChR aggregation and phosphorylation in chick myotubes in culture. Staurosporine, an antagonist of both protein serine and tyrosine kinases, blocked agrin-induced AChR aggregation in a dose-dependent manner; 50% inhibition occurred at approximately 2 nM. The extent of inhibition was independent of agrin concentration, suggesting an effect downstream of the interaction of agrin with its receptor. Staurosporine blocked agrin-induced phosphorylation of the AChR beta subunit, which occurs at least in part on tyrosine residues, but did not reduce phosphorylation of the gamma and delta subunits, which occurs on serine/threonine residues. Staurosporine also prevented the agrin- induced decrease in the rate at which AChRs are extracted from intact myotubes by mild detergents. H-7, an antagonist of protein serine kinases, inhibited agrin-induced phosphorylation of the gamma and delta subunits but did not block agrin-induced phosphorylation of the AChR beta subunit, AChR aggregation, or the decrease in AChR extractability. The results provide support for the hypothesis that tyrosine phosphorylation of the beta subunit plays a role in agrin-induced AChR aggregation.     

Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions.

To test the hypothesis that agrin mediates motor neuron-induced aggregation of acetylcholine receptors (AChRs) in skeletal muscle fibers and to determine whether the agrin active in this process is released by motor neurons, we raised polyclonal antibodies to purified ray agrin that blocked its receptor aggregating activity. When the antibodies were applied to chick motor neuron--chick myotube cocultures, they inhibited the formation of AChR aggregates at and near neuromuscular contacts, demonstrating that agrin plays a role in the induction of the aggregates. Rat motor neurons, like chick motor neurons, induce AChR aggregates on chick myotubes. This effect was not inhibited by our antibodies, indicating that, although the antibodies inhibited the activity of chick agrin, they did not have a similar effect on rat agrin. We conclude that agrin released by rat motor neurons induced the chick myotubes to aggregate AChRs.     

Synaptic localization and axonal targeting of agrin secreted by ventral spinal cord neurons in culture

Agrin secreted by motor neurons is a critical signal for postsynaptic differentiation at the developing neuromuscular junction. We used cultures of chick ventral spinal cord neurons with rat myotubes and immunofluorescence with species-specific antibodies to determine the distribution of agrin secreted by neurons and compare it to the distribution of agrin secreted by myotubes. In addition, we determined the distribution of agrin secreted by isolated chick ventral spinal cord neurons and rat motor neurons grown on a substrate that binds agrin. In cocultures, neuronal agrin was concentrated along axons at sites of axon-induced acetylcholine receptor (AChR) aggregation and was found at every such synaptic site, consistent with its role in synaptogenesis. Smaller amounts of agrin were found on dendrites and cell bodies and rarely were associated with AChR aggregation. Muscle agrin, recognized by an antibody against rat agrin, was found at nonsynaptic sites of AChR aggregation but was not detected at synaptic sites, in contrast to neuronal agrin. In cultures of isolated chick neurons or rat motor neurons, agrin was deposited relatively uniformly around axons and dendrites during the first 2-3 days in culture. In older cultures, agrin immunoreactivity was markedly more intense around axons than dendrites, indicating that motor neurons possess an intrinsic, developmentally regulated program to target agrin secretion to axons.     

Guanylate cyclase and cyclic GMP-dependent protein kinase regulate agrin signaling at the developing neuromuscular junction

During formation of the neuromuscular junction (NMJ), agrin secreted by motor axons signals the embryonic muscle cells to organize a postsynaptic apparatus including a dense aggregate of acetylcholine receptors (AChRs). Agrin signaling at the embryonic NMJ requires the activity of nitric oxide synthase (NOS). Common downstream effectors of NOS are guanylate cyclase (GC), which synthesizes cyclic GMP, and cyclic GMP-dependent protein kinase (PKG). Here we show that GC and PKG are important for agrin signaling at the embryonic NMJ of the frog, Xenopus laevis. Inhibitors of both GC and PKG reduced endogenous AChR aggregation in embryonic muscles by 50-85%, and blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. A cyclic GMP analog, 8-bromo-cyclic GMP, increased endogenous AChR aggregation in embryonic muscles to 3- to 4-fold control levels. Overexpression of either GC or PKG in embryos increased AChR aggregate area by 60-170%, whereas expression of a dominant negative form of GC inhibited endogenous aggregation by 50%. These results indicate that agrin signaling in embryonic muscle cells requires the activity of GC and PKG as well as NOS.     

Calcium plays a critical role in determining the acetylcholine receptor-clustering activities of alternatively spliced isoforms of Agrin

Neural agrin, an extracellular matrix protein secreted by motor neurons, plays a key role in clustering of nicotinic acetylcholine receptors (AChR) on postsynaptic membranes of the neuromuscular junction. The action of agrin is critically dependent on an eight-amino acid insert (z8 insert) in the third of three consecutive laminin-like globular (G3) domains near the C terminus of neural agrin. Alternatively spliced agrin isoforms in non-neural tissue including muscle lack the z8 insert and are biologically inactive. Extracellular calcium has been shown to be imperative for the AChR-clustering activity of neural agrin. It is unclear, however, whether calcium preferentially interacts with the neural isoform or whether it acts solely as an intracellular messenger that mediates agrin signaling. Here, we report the G3 domain of rat neural agrin (AgG3z8) expressed in Pichia pastoris promoted AChR clustering on surface of C2C12 myotubes in a calcium-dependent manner. Direct binding of calcium to AgG3z8 was demonstrated by trypsin digestion and thermal denaturation experiments. Moreover, calcium induced a significant change in the conformation of AgG3z8, and the effect was correlated with an enhanced binding affinity of the protein to muscle receptor. Mutation of calcium-binding residues in the G3 domain diminished the conformational change of neural agrin, reduced its binding affinity to muscle membrane, and inhibited AChR-clustering activity. Conversely, the G3 domain of muscle agrin (AgG3z0) displayed little structural change in the presence of calcium, bound poorly to muscle surface, and was inactive in AChR-clustering assays. We conclude that distinct interactions of the G3 domain with calcium determine the biological activities of alternatively spliced agrin isoforms during synapse formation.     

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.     

Agrin induces phosphorylation of the nicotinic acetylcholine receptor.

Agrin causes acetylcholine receptors (AChRs) on chick myotubes in culture to aggregate, forming specializations that resemble the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction. Here we report that treating chick myotubes with agrin caused an increase in phosphorylation of the AChR beta, gamma, and delta subunits. H-7, a potent inhibitor of several protein serine kinases, blocked agrin-induced phosphorylation of the gamma and delta subunits, but did not prevent either agrin-induced AChR aggregation or phosphorylation of the beta subunit. Experiments with anti-phosphotyrosine antibodies demonstrated that agrin caused an increase in tyrosine phosphorylation of the beta subunit that began within 30 min of adding agrin to the myotube cultures, reached a plateau by 3 hr, and was blocked by treatments known to block agrin-induced AChR aggregation. Anti-phosphotyrosine antibodies labeled agrin-induced specializations as they do the postsynaptic apparatus. These results suggest that agrin-induced tyrosine phosphorylation of the beta subunit may play a role in regulating AChR distribution.     

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."     

An amino-terminal extension is required for the secretion of chick agrin and its binding to extracellular matrix

Agrin is an extracellular matrix (ECM) protein with a calculated relative molecular mass of more than 200 kD that induces the aggregation of acetylcholine receptors (AChRs) at the neuromuscular junction. This activity has been mapped to its COOH terminus. In an attempt to identify the functions of the NH2-terminal end, we have now characterized full-length chick agrin. We show that chick agrin encoded by a previously described cDNA is not secreted from transfected cells. Secretion is achieved with a construct that includes an additional 350 bp derived from the 5' end of chick agrin mRNA. Recombinant agrin is a heparan sulfate proteoglycan (HSPG) of more than 400 kD with glycosaminoglycan side chains attached only to the NH2-terminal half. Endogenous agrin in tissue homogenates also has an apparent molecular mass of > 400 kD. While the amino acid sequence encoded by the 350-bp extension has no homology to published rat agrin, it includes a stretch of 15 amino acids that is 80% identical to a previously identified bovine HSPG. The extension is required for binding of agrin to ECM. AChR aggregates induced by recombinant agrin that includes the extension are considerably smaller than those induced by agrin fragments, suggesting that binding of agrin to ECM modulates the size of receptor clusters. In addition, we found a site encoding seven amino acids at the NH2-terminal end of agrin that is alternatively spliced. While motor neurons express the splice variant with the seven amino acid long insert, muscle cells mainly synthesize isoforms that lack this insert. In conclusion, the cDNAs described here code for chick agrin that has all the characteristics previously allocated to endogenous agrin.     

Recombinant neuromuscular synapses

The developing neuromuscular junction has provided an important paradigm for studying synapse formation. An outstanding feature of neuromuscular differentiation is the aggregation of acetylcholine receptors (AChRs) at high density in the postsynaptic membrane. While AChR aggregation is generally believed to be induced by the nerve, the mechanisms underlying aggregation remain to be clarified. A 43-kD protein (43k) normally associated with the cytoplasmic aspect of AChR clusters has long been suspected of immobilizing AChRs by linking them to the cytoskeleton. In recent studies, the AChR clustering activity of 43k has, at last, been demonstrated by expressing recombinant AChR and 43k in non-muscle cells. Mutagenesis of 43k has revealed distinct domains within the primary structure which may be responsible for plasma membrane targeting and AChR binding. Other lines of study have provided clues as to how nerve-derived (extracellular) AChR-cluster inducing factors such as agrin might activate 43k-driven postsynaptic membrane specialization.     

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.     

The COOH-terminal domain of agrin signals via a synaptic receptor in central nervous system neurons

Agrin is a motor neuron-derived factor that directs formation of the postsynaptic apparatus of the neuromuscular junction. Agrin is also expressed in the brain, raising the possibility that it might serve a related function at neuron-neuron synapses. Previously, we identified an agrin signaling pathway in central nervous system (CNS) neurons, establishing the existence of a neural receptor that mediates responses to agrin. As a step toward identifying this agrin receptor, we have characterized the minimal domains in agrin that bind and activate it. Structures required for agrin signaling in CNS neurons are contained within a 20-kD COOH-terminal fragment of the protein. Agrin signaling is independent of alternative splicing at the z site, but requires sequences that flank it because their deletion results in a 15-kD fragment that acts as an agrin antagonist. Thus, distinct regions within agrin are responsible for receptor binding and activation. Using the minimal agrin fragments as affinity probes, we also studied the expression of the agrin receptor on CNS neurons. Our results show that both agrin and its receptor are concentrated at neuron-neuron synapses. These data support the hypothesis that agrin plays a role in formation and/or function of CNS synapses.     

Regulation of the interaction of nicotinic acetylcholine receptors with the cytoskeleton by agrin-activated protein tyrosine kinase

Agrin induces the accumulation of nicotinic acetylcholine receptors (AChRs) in the myofiber membrane at synaptic sites in vertebrate skeletal muscle and causes an increase in tyrosine phosphorylation of the AChR beta subunit. To examine further the mechanism of agrin- induced AChR phosphorylation and the relationship between changes in protein phosphorylation and AChR aggregation, the effect of the protein tyrosine phosphatase inhibitor sodium pervanadate was tested on chick myotubes in culture. Pervanadate caused an increase in the phosphotyrosine content of a variety of proteins, including the AChR. Pervanadate also prevented agrin-induced AChR aggregation and slowed the rate at which AChRs were extracted from intact myotubes by mild detergent treatment. The rate at which phosphorylation of the AChR beta subunit and receptor detergent extractability changed following pervanadate-induced phosphatase inhibition was increased by agrin, indicating that agrin activates a protein tyrosine kinase rather than inhibiting a protein tyrosine phosphatase. The present results, taken together with previous findings on the inhibition of agrin-induced AChR aggregation by protein kinase inhibitors, demonstrate that protein tyrosine phosphorylation regulates the formation and stability of AChR aggregates, apparently by strengthening the interaction between AChRs and the cytoskelton.     

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.     

Localized acetylcholine receptor clustering dynamics in response to microfluidic focal stimulation with agrin

Agrin is a proteoglycan secreted by the motor neuron's growing axon terminal upon contact with the muscle during embryonic development. It was long thought that agrin's role was to trigger the clustering of acetylcholine receptors (AChRs) to nascent synapse sites. However, agrin-predating, protosynaptic AChR clusters are present well before innervation in the embryo and in myotube cultures, yet no role has been conclusively ascribed to agrin. We used a microfluidic device to focally deliver agrin to protosynaptic AChR clusters in micropatterned myotube cultures. The distribution of AChRs labeled with fluorescent bungarotoxin was imaged at various time points over >24 h. We find that a 4-h focal application of agrin (100 nM) preferentially reduces AChR loss at agrin-exposed clusters by 17% relative to the agrin-deprived clusters on the same myotube. In addition, the focal application increases the addition of AChRs preferentially at the clusters by 10% relative to the agrin-exposed, noncluster areas. Taken together, these findings suggest that a focal agrin stimulus can play a key stabilizing role in the aggregation of AChRs at the early stages of synapse formation. This methodology is generally applicable to various developmental processes and cell types, including neurons and stem cells.