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.     

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.     

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.     

Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation

Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship between tyrosine phosphorylation and receptor aggregation, we have used the technique of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes treated with agrin or with pervanadate, a protein tyrosine phosphatase inhibitor. Agrin induced the formation of patches in C2 myotubes that stained intensely with anti-phosphotyrosine antibodies and within which AChRs were relatively immobile. Pervanadate, on the other hand, increased protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without affecting the mobility of other membrane proteins. Pervanadate, like agrin, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscular junctions in embryonic rat muscles also labeled with anti- phosphotyrosine antibodies, suggesting that tyrosine phosphorylation could mediate AChR aggregation in vivo as well. Thus, agrin appears to induce AChR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.     

A single pulse of agrin triggers a pathway that acts to cluster acetylcholine receptors

Agrin triggers signaling mechanisms of high temporal and spatial specificity to achieve phosphorylation, clustering, and stabilization of postsynaptic acetylcholine receptors (AChRs). Agrin transiently activates the kinase MuSK; MuSK activation has largely vanished when AChR clusters appear. Thus, a tyrosine kinase cascade acts downstream from MuSK, as illustrated by the agrin-evoked long-lasting activation of Src family kinases (SFKs) and their requirement for AChR cluster stabilization. We have investigated this cascade and report that pharmacological inhibition of SFKs reduces early but not later agrin-induced phosphorylation of MuSK and AChRs, while inhibition of Abl kinases reduces late phosphorylation. Interestingly, SFK inhibition applied selectively during agrin-induced AChR cluster formation caused rapid cluster dispersal later upon agrin withdrawal. We also report that a single 5-min agrin pulse, followed by extensive washing, triggered long-lasting MuSK and AChR phosphorylation and efficient AChR clustering. Following the pulse, MuSK phosphorylation increased and, beyond a certain level, caused maximal clustering. These data reveal novel temporal aspects of tyrosine kinase action in agrin signaling. First, during AChR cluster formation, SFKs initiate early phosphorylation and an AChR stabilization program that acts much later. Second, a kinase mechanism rapidly activated by agrin acts thereafter autonomously in agrin's absence to further increase MuSK phosphorylation and cluster AChRs.     

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.     

Acetylcholine Receptor (AChR) Clustering Is Regulated Both by Glycogen Synthase Kinase 3β (GSK3β)-dependent Phosphorylation and the Level of CLIP-associated Protein 2 (CLASP2) Mediating the Capture of Microtubule Plus-ends

The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2–9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2–8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.     

Regulation of ACh receptor clustering by the tyrosine phosphatase Shp2

At the vertebrate neuromuscular junction (NMJ), postsynaptic aggregation of muscle acetylcholine receptors (AChRs) depends on the activation of MuSK, a muscle-specific tyrosine kinase that is stimulated by neural agrin and regulated by muscle-intrinsic tyrosine kinases and phosphatases. We recently reported that Shp2, a tyrosine phosphatase containing src homology two domains, suppressed MuSK-dependent AChR clustering in cultured myotubes, but how this effect of Shp2 is controlled has remained unclear. In this study, biochemical assays showed that agrin-treatment of C2 mouse myotubes enhanced the tyrosine phosphorylation of signal regulatory protein alpha1 (SIRPalpha1), a known activator of Shp2, and promoted SIRPalpha1's interaction with Shp2. Moreover, in situ experiments revealed that treatment of myotubes with the Shp2-selective inhibitor NSC-87877 increased spontaneous and agrin-induced AChR clustering, and that AChR clustering was also enhanced in myotubes ectopically expressing inactive (dominant-negative) Shp2; in contrast, AChR clustering was reduced in myotubes expressing constitutively active Shp2. Significantly, expression of truncated (nonShp2-binding) and full-length (Shp2-binding) forms of SIRPalpha1 in myotubes also increased and decreased AChR clustering, respectively, and coexpression of truncated SIRPalpha1 with active Shp2 and full-length SIRPalpha1 with inactive Shp2 reversed the actions of the exogenous Shp2 proteins on AChR clustering. These results suggest that SIRPalpha1 is a novel downstream target of MuSK that activates Shp2, which, in turn, suppresses AChR clustering. We propose that an inhibitory loop involving both tyrosine kinases and phosphatases sets the level of agrin/MuSK signaling and constrains it spatially to help generate high-density AChR clusters selectively at NMJs.     

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.     

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.     

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.     

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.     

A Highlights from MBoC Selection: Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn–plectin 1f complexes

Mutations in the cytolinker protein plectin lead to grossly distorted morphology of neuromuscular junctions (NMJs) in patients suffering from epidermolysis bullosa simplex (EBS)-muscular dystrophy (MS) with myasthenic syndrome (MyS). Here we investigated whether plectin contributes to the structural integrity of NMJs by linking them to the postsynaptic intermediate filament (IF) network. Live imaging of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, in contrast to wild-type cells. We found plectin isoform 1f (P1f) to bridge AChRs and IFs via direct interaction with the AChR-scaffolding protein rapsyn in an isoform-specific manner; forced expression of P1f in plectin-deficient cells rescued both compromised AChR clustering and IF network anchoring. In conditional plectin knockout mice with gene disruption in muscle precursor/satellite cells (Pax7-Cre/cKO), uncoupling of AChRs from IFs was shown to lead to loss of postsynaptic membrane infoldings and disorganization of the NMJ microenvironment, including its invasion by microtubules. In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span. Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body locomotion.     

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals-possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCdelta with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior.     

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.     

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.     

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.     

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 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.