Agrin induces alpha-actinin, filamin, and vinculin to co-localize with AChR clusters on cultured chick myotubes.

Agrin induces discrete high-density patches of acetylcholine receptors (AChRs) and other synaptic components on cultured myotubes in a manner that resembles synaptic differentiation. Furthermore, agrin-like molecules are present at developing neuromuscular junctions in vivo. This provides us with a unique opportunity to manipulate AChR patching in order to examine the role of cytoskeletal components. Cultured chick myotubes were fixed and labeled to visualize the distributions of actin, alpha-actinin, filamin, tropomyosin, and vinculin. Overnight exposure to agrin caused a small amount of alpha-actinin, filamin, and vinculin to reorganize into discrete clusters. Double-labeling studies revealed that 78% of the AChR clusters were associated with detectable concentrations of filamin, 70% with alpha-actinin, and 58% with vinculin. Filamin even showed congruence to AChRs within clustered regions. By contrast, actin (visualized with fluorescein-phalloidin) and tropomyosin did not show specific associations with agrin-induced AChR clusters. The accumulation of cytoskeletal components at AChRs clusters raised the possibility that cytoskeletal rearrangements direct AChR clustering. However, a time course of agrin-induced clustering that focused on filamin revealed that most of the early AChR clusters (3-6 h) were not associated with detectable amounts of cytoskeletal material. The accumulation of cytoskeletal material at later times (12-18 h) may imply a role in maintenance and stabilization, but it appears unlikely that these cytoskeletal elements initiate AChR clustering on myotubes.     

Agrin regulates rapsyn interaction with surface acetylcholine receptors,and this underlies cytoskeletal anchoring and clustering

The acetylcholine receptor (AChR)-associated protein rapsyn is essential for neuromuscular synapse formation and clustering of AChRs, but its mode of action remains unclear. We have investigated whether agrin, a key nerve-derived synaptogenic factor, influences rapsyn-AChR interactions and how this affects clustering and cytoskeletal linkage of AChRs. By precipitating AChRs and probing for associated rapsyn, we found that in denervated diaphragm rapsyn associates with synaptic as well as with extrasynaptic AChRs showing that rapsyn interacts with unclustered AChRs in vivo. Interestingly, synaptic AChRs are associated with more rapsyn suggesting that clustering of AChRs may require increased interaction with rapsyn. In similar experiments in cultured myotubes, rapsyn interacted with intracellular AChRs and with unclustered AChRs at the cell surface, although surface interactions are much more prominent. Remarkably, agrin induces recruitment of additional rapsyn to surface AChRs and clustering of AChRs independently of the secretory pathway. This agrin-induced increase in rapsyn-AChR interaction strongly correlates with clustering, because staurosporine and herbimycin blocked both the increase and clustering. Conversely, laminin and calcium induced both increased rapsyn-AChR interaction and AChR clustering. Finally, time course experiments revealed that the agrin-induced increase occurs with AChRs that become cytoskeletally linked, and that this precedes receptor clustering. Thus, we propose that neural agrin controls postsynaptic aggregation of the AChR by enhancing rapsyn interaction with surface AChRs and inducing cytoskeletal anchoring and that this is an important precursor step for AChR clustering.     

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.     

Association of cytoskeletal proteins with newly formed acetylcholine receptor aggregates induced by embryonic brain extract

Aggregates of acetylcholine receptors (AChR) in muscle cell membranes are associated with accumulations of certain cytoskeletal and peripheral membrane proteins. We treated cultured rat myotubes briefly with embryonic brain extract (EBX) to promote AChR aggregation and determined the distribution of several of these proteins at early stages of aggregation. EBX-treated and control cultures were stained with tetramethylrhodamine-alpha-bungarotoxin to identify AChR aggregates and were then frozen and sectioned on a cryostat. These sections were stained with primary antibodies and fluoresceinated secondary antibodies to localize cytoskeletal proteins. The distributions of AChRs and cytoskeletal proteins was examined qualitatively and analyzed by a semiquantitative assay. Qualitatively, the 43K protein had a distribution that was virtually identical to that of AChR in both control and EBX-treated cultures, and it always colocalized with early AChR aggregates. The 58K protein similarly colocalized with early AChR aggregates, but it was also in aggregate-free areas of muscle membrane. The association of vinculin with the aggregates was quantitatively similar to that of the 43K and 58K proteins, but, qualitatively, its distribution did not follow that of the AChR as closely. Like the 58K protein and vinculin, alpha-actinin, filamin, and actin were concentrated in AChR aggregates and were also enriched elsewhere. However, they were less closely associated with the aggregates, both quantitatively and qualitatively. These results show that AChR aggregates induced by EBX tend to be enriched in the same cytoskeletal proteins that are present at the neuromuscular junction in vivo and at AChR clusters formed at sites of cell-substrate adhesion in vitro. Semiquantitative analysis also revealed that the fractional area of the cell surface associated with vinculin, alpha-actinin, and the 58K protein was the same in controls and EBX-treated myotubes, although the area enriched in AChR and the 43K protein increased about three-fold upon EBX treatment. These results suggest that AChR aggregates may form preferentially in membrane regions that are already enriched in these proteins.     

Lithium inhibits a late step in agrin-induced AChR aggregation

Agrin activates an intracellular signaling pathway to induce the formation of postsynaptic specializations on muscle fibers. In myotubes in culture, this pathway has been shown to include autophosphorylation of the muscle-specific kinase MuSK, activation of Src-family kinases, tyrosine phosphorylation of the acetylcholine receptor (AChR) beta subunit, a decrease in receptor detergent extractability, and the accumulation of AChRs into high-density aggregates. Here we report that treating chick myotubes with lithium prevented any detectable agrin-induced change in AChR distribution without affecting the number of AChRs or the agrin-induced change in AChR tyrosine phosphorylation and detergent extractability. Lithium treatment also increased the rate at which AChR aggregates disappeared when agrin was removed. The effects of lithium developed slowly over the course of approximately 12 h. Thus, sensitivity to lithium identifies a late step in the agrin signaling pathway, after agrin-induced MuSK and AChR phosphorylation, that is necessary for the recruitment of AChRs into visible aggregates.     

Mechanism of agrin-induced acetylcholine receptor aggregation.

Agrin induces the formation of specializations on chick myotubes in culture at which several components of the postsynaptic apparatus accumulate, including acetylcholine receptors (AChRs). Agrin also induces AChR phosphorylation. Several lines of evidence suggest that agrin-induced phosphorylation of tyrosine residues in the beta subunit of the AChR is an early step in receptor aggregation: agrin-induced phosphorylation and aggregation have the same dose dependence; treatments that prevent aggregation block phosphorylation; phosphorylation begins before any detectable change in receptor distribution, reaches a maximum hours before aggregation is complete, and declines slowly together with the disappearance of aggregates after agrin is withdrawn; agrin slows the rate at which receptors are solubilized from intact myotubes by detergent extraction; and the change in receptor extractability parallels the change in phosphorylation. A model for agrin-induced AChR aggregation is presented in which phosphorylation of AChRs by an agrin-activated protein tyrosine kinase causes receptors to become attached to the cytoskeleton, which reduces their mobility and detergent extractability, and leads to the accumulation of receptors in the vicinity of the activated kinase, forming an aggregate.     

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.     

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.     

Acetylcholine receptors are required for agrin-induced clustering of postsynaptic proteins.

We have investigated the role of acetylcholine receptors (AChRs) in an early step of postsynaptic assembly at the neuromuscular synapse, the clustering of postsynaptic proteins induced by nerve-released agrin. To achieve this, we used two variants of C2 myotubes virtually lacking AChRs and C2 cells in which surface AChRs were down-regulated by AChR antibodies. In all cases, agrin caused normal clustering of the agrin receptor component MuSK, alpha-dystrobrevin and utrophin, but failed to aggregate AChRs, alpha- and beta-dystroglycan, syntrophin isoforms and rapsyn, an AChR-anchoring protein necessary for postsynaptic assembly and AChR clustering. In C2 variants, the stability of rapsyn was decreased, whereas in antibody-treated cells, rapsyn efficiently co-localized with remaining AChRs in microaggregates. Upon ectopic injection into myofibers in vivo, rapsyn did not form clusters in the absence of AChRs. These results show that AChRs and rapsyn are interdependent components of a pre-assembled protein complex that is required for agrin-induced clustering of a full set of postsynaptic proteins, thus providing evidence for an active role of AChRs in postsynaptic assembly.     

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.     

CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles

A hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to the adult NMJ are not yet understood in detail. We previously showed that microtubule (MT) capture by the plus end–tracking protein CLASP2 regulates AChR density at agrin-induced AChR clusters in cultured myotubes via PI3 kinase acting through GSK3β. Here we show that knockdown of the CLASP2-interaction partner LL5β by RNAi and forced expression of a CLASP2 fragment blocking the CLASP2/LL5β interaction inhibit microtubule capture. The same treatments impair focal vesicle delivery to the clusters. Consistent with these findings, knockdown of LL5β at the NMJ in vivo reduces the density and insertion of AChRs into the postsynaptic membrane. MT capture and focal vesicle delivery to agrin-induced AChR clusters are also inhibited by microtubule- and actin-depolymerizing drugs, invoking both cytoskeletal systems in MT capture and in the fusion of AChR vesicles with the cluster membrane. Combined our data identify a transport system, organized by agrin through PI3 kinase, GSK3β, CLASP2, and LL5β, for precise delivery of AChR vesicles from the subsynaptic nuclei to the overlying synaptic membrane.     

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.     

Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin

Neuregulin (NRG)/ErbB signaling is involved in numerous developmental processes in the nervous system, including synapse formation and function in the central nervous system. Although intensively investigated, its role at the neuromuscular synapse has remained elusive. Here, we demonstrate that loss of neuromuscular NRG/ErbB signaling destabilized anchoring of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane and that this effect was caused by dephosphorylation of α-dystrobrevin1, a component of the postsynaptic scaffold. Specifically, in mice in which NRG signaling to muscle was genetically or pharmacologically abolished, postsynaptic AChRs moved rapidly from the synaptic to the perisynaptic membrane, and the subsynaptic scaffold that anchors the AChRs was impaired. These defects combined compromised synaptic transmission. We further show that blockade of NRG/ErbB signaling abolished tyrosine phosphorylation of α-dystrobrevin1, which reduced the stability of receptors in agrin-induced AChR clusters in cultured myotubes. Our data indicate that NRG/ErbB signaling maintains high efficacy of synaptic transmission by stabilizing the postsynaptic apparatus via phosphorylation of α-dystrobrevin1.     

Acetylcholine receptor clustering is required for the accumulation and maintenance of scaffolding proteins

The maintenance of a high density of postsynaptic receptors is essential for proper synaptic function. At the neuromuscular junction, acetylcholine receptor (AChR) aggregation is induced by nerve-clustering factors and mediated by scaffolding proteins. Although the mechanisms underlying AChR clustering have been extensively studied, the role that the receptors themselves play in the clustering process and how they are organized with scaffolding proteins is not well understood. Here, we report that the exposure of AChRs labeled with Alexa 594 conjugates to relatively low-powered laser light caused an effect similar to chromaphore-assisted light inactivation (CALI) , which resulted in the unexpected dissipation of the illuminated AChRs from clusters on cultured myotubes. This technique enabled us to demonstrate that AChR removal from illuminated regions induced the removal of scaffolding proteins and prevented the accumulation of new AChRs and associated scaffolding proteins. Further, the dissipation of clustered AChRs and scaffold was spatially restricted to the illuminated region and had no effect on neighboring nonilluminated AChRs. These results provide direct evidence that AChRs are essential for the local maintenance and accumulation of intracellular scaffolding proteins and suggest that the scaffold is organized into distinct modular units at AChR clusters.     

Mechanism of agrin-induced acetylcholine receptor aggregation

Agrin induces the formation of specializations on chick myotubes in culture at which several components of the postsynaptic apparatus accumulate, including acetylcholine receptors (AChRs). Agrin also induces AChR phosphorylation. Several lines of evidence suggest that agrininduced phosphorylation of tyrosine residues in the β subunit of the AChR is an early step in receptor aggregation: agrin-induced phosphorylation and aggregation have the same dose dependence; treatments that prevent aggregation block phosphorylation; phosphorylation begins before any detectable change in receptor distribution, reaches a maximum hours before aggregation is complete, and declines slowly together with the disappearance of aggregates after agrin is withdrawn; agrin slows the rate at which receptors are solubilized from intact myotubes by detergent extraction; and the change in receptor extractability parallels the change in phosphorylation. A model for agrin-induced AChR aggregation is presented in which phosphorylation of AChRs by an agrin-activated protein tyrosine kinase causes receptors to become attached to the cytoskeleton, which reduces their mobility and detergent extractability, and leads to the accumulation of receptors in the vicinity of the activated kinase, forming an aggregate. © 1992 John Wiley & Sons, Inc.     

Localization of Myosin,Actin, α-Actinin,Tropomyosin and Vinculin in Surface-Activated,Spreading Human Platelets

We observed the localization of the contractile proteins myosin, filamentous actin, α-actinin, tropomyosin, and vinculin in surface-activated, spreading human platelets using a single fluorescence staining procedure and conventional fluorescence microscopy. Myosin was distributed in a speckled pattern that extended radially from the granulomere. F-actin demonstrated cable-networks. Tropomyosin and α-actinin occurred in a punctuate distribution, and vinculin was localized at adhesion sites. Although myosin, F-actin, α-actinin, tropomyosin, and vinculin were not studied in resting platelets, our data support the idea that these contractile proteins are reorganized and reassembled in activated platelets during platelet function.     

Phosphoinositide 3-kinase acts through RAC and Cdc42 during agrin-induced acetylcholine receptor clustering

The formation of the neuromuscular junction (NMJ) is regulated by the nerve-derived heparan sulfate proteoglycan agrin and the muscle-specific kinase MuSK. Agrin induces a signal transduction pathway via MuSK, which promotes the reorganization of the postsynaptic muscle membrane. Activation of MuSK leads to the phosphorylation and redistribution of acetylcholine receptors (AChRs) and other postsynaptic proteins to synaptic sites. The accumulation of high densities of AChRs at postsynaptic regions represents a hallmark of NMJ formation and is required for proper NMJ function. Here we show that phosphoinositide 3-kinase (PI3-K) represents a component of the agrin/MuSK signaling pathway. Muscle cells treated with specific PI3-K inhibitors are unable to form full-size AChR clusters in response to agrin and AChR phosphorylation is reduced. Moreover, agrin-induced activation of Rac and Cdc42 is impaired in the presence of PI3-K inhibitors. PI3-K is localized to the postsynaptic muscle membrane consistent with a role during agrin/MuSK signaling. These results put PI3-K downstream of MuSK as regulator of AChR phosphorylation and clustering. Its role during agrin-stimulated Rac and Cdc42 activation suggests a critical function during cytoskeletal reorganizations, which lead to the redistribution of actin-anchored AChRs.     

Cooperative regulation by Rac and Rho of agrin-induced acetylcholine receptor clustering in muscle cells

A key aspect of neuromuscular synapse formation is the clustering of muscle acetylcholine receptors (AChR) at synaptic sites in response to neurally secreted agrin. Agrin-induced AChR clustering in cultured myotubes proceeds via the initial formation of small microclusters, which then aggregate to form AChR clusters. Here we show that the coupling of agrin signaling to AChR clustering is dependent on the coordinated activities of Rac and Rho GTPases. The addition of agrin induces the sequential activation of Rac and Rho in C2 muscle cells. The activation of Rac is rapid and transient and constitutes a prerequisite for the subsequent activation of Rho. This temporal pattern of agrin-induced Rac and Rho activation reflects their respective roles in AChR cluster formation. Whereas agrin-induced activation of Rac is necessary for the initial phase of AChR cluster formation, which involves the aggregation of diffuse AChR into microclusters, Rho activation is crucial for the subsequent condensation of these microclusters into full-size AChR clusters. Co-expression of constitutively active forms of Rac and Rho is sufficient to induce the formation of mature AChR clusters in the absence of agrin. These results establish that Rac and Rho play distinct but complementary roles in the mechanism of agrin-induced AChR clustering.     

Agrin-induced activation of acetylcholine receptor-bound Src family kinases requires Rapsyn and correlates with acetylcholine receptor clustering

During neuromuscular synaptogenesis, neurally released agrin induces aggregation and tyrosine phosphorylation of acetylcholine receptors (AChRs) by acting through both the receptor tyrosine kinase MuSK (muscle-specific kinase) and the AChR-associated protein, rapsyn. To elucidate this signaling mechanism, we examined tyrosine phosphorylation of AChR-associated proteins, particularly addressing whether agrin activates Src family kinases bound to the AChR. In C2 myotubes, agrin induced tyrosine phosphorylation of these kinases, of AChR-bound MuSK, and of the AChR beta and delta subunits, as observed in phosphotyrosine immunoblotting experiments. Kinase assays revealed that the activity of AChR-associated Src kinases was increased by agrin, whereas phosphorylation of the total cellular kinase pool was unaffected. In both rapsyn-deficient myotubes and staurosporine-treated C2 myotubes, where AChRs are not clustered, agrin activated MuSK but did not cause either Src family or AChR phosphorylation. In S27 mutant myotubes, which fail to aggregate AChRs, no agrin-induced phosphorylation of AChR-bound Src kinases, MuSK, or AChRs was observed. These results demonstrate first that agrin leads to phosphorylation and activation of AChR-associated Src-related kinases, which requires rapsyn, occurs downstream of MuSK, and causes AChR phosphorylation. Second, this activation intimately correlates with AChR clustering, suggesting that these kinases may play a role in agrin-induced AChR aggregation by forming an AChR-bound signaling cascade.