Nicotinic Acetylcholine Receptor Clusters Research Articles

The role of Rapsyn in neuromuscular junction and congenital myasthenic syndrome.

Rapsyn, an intracellular scaffolding protein associated with the postsynaptic membranes in the neuromuscular junction (NMJ), is critical for nicotinic acetylcholine receptor clustering and maintenance. Therefore, Rapsyn is essential to the NMJ formation and maintenance, and Rapsyn mutant is one of the reasons causing the pathogenies of congenital myasthenic syndrome (CMS). In addition, there is little research on Rapsyn in the central nervous system (CNS). In this review, the role of Rapsyn in the NMJ formation and the mutation of Rapsyn leading to CMS will be reviewed separately and sequentially. Finally, the potential function of Rapsyn is prospected.

Liposomal dexamethasone attenuates tourniquet‐induced ischemia‐reperfusion injury in mouse hindlimb

Tourniquet‐induced ischemia and subsequent release cause serious ischemia‐reperfusion (IR) injury in the skeletal muscle. Here, we observed the effect of liposomal dexamethasone (lipo‐Dex) in tourniquet‐Induced ischemic reperfusion injury (IR) in mouse hindlimb.Unilateral hindlimb of C57/BL6 mice was subjected to 3 hours of ischemia by placing a rubber band and followed different periods of reperfusion (1 week, 2 weeks, 4 weeks, and 6 weeks). Before tourniquet was released, lipo‐Dex (7 mg/kg, equivalent to amg/kg/day of Dex for 1 week) was administered via tail vein injection. After 3 hours of ischemia and different periods of reperfusion, gastrocnemius muscle showed significantly morphological and functional damages. TNFα and IL‐1β were overexpressed in the gastrocnemius muscle after 1, 2, 4, and 6 weeks of tourniquet‐induced IR. Treatment with Lipo‐Dex inhibited the elevation of TNFα and IL‐1β, improved structure of the muscle sarcolemma, reduced fragmentation of nicotinic acetylcholine receptor clusters, and promoted recovery of gastrocnemius muscle contraction.The data suggest that tourniquet‐related IR induces serious skeletal muscle inflammation and degradation. Treatment with lipo‐Dex suppresses IR‐induced inflammation and promotes recovery of skeletal muscle morphology and function from tourniquet‐induced IR injury.Support or Funding InformationUSAMRAA grant from Department of Defense, W81XWH‐17‐1‐0037(PI, Yulong Li).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Two proteolytic fragments of menin coordinate the nuclear transcription and postsynaptic clustering of neurotransmitter receptors during synaptogenesis between Lymnaea neurons.

Synapse formation and plasticity depend on nuclear transcription and site-specific protein targeting, but the molecular mechanisms that coordinate these steps have not been well defined. The MEN1 tumor suppressor gene, which encodes the protein menin, is known to induce synapse formation and plasticity in the CNS. This synaptogenic function has been conserved across evolution, however the underlying molecular mechanisms remain unidentified. Here, using central neurons from the invertebrate Lymnaea stagnalis, we demonstrate that menin coordinates subunit-specific transcriptional regulation and synaptic clustering of nicotinic acetylcholine receptors (nAChR) during neurotrophic factor (NTF)-dependent excitatory synaptogenesis, via two proteolytic fragments generated by calpain cleavage. Whereas menin is largely regarded as a nuclear protein, our data demonstrate a novel cytoplasmic function at central synapses. Furthermore, this study identifies a novel synaptogenic mechanism in which a single gene product coordinates the nuclear transcription and postsynaptic targeting of neurotransmitter receptors through distinct molecular functions of differentially localized proteolytic fragments.

Structure and superorganization of acetylcholine receptor–rapsyn complexes

The scaffolding protein at the neuromuscular junction, rapsyn, enables clustering of nicotinic acetylcholine receptors in high concentration and is critical for muscle function. Patients with insufficient receptor clustering suffer from muscle weakness. However, the detailed organization of the receptor-rapsyn network is poorly understood: it is unclear whether rapsyn first forms a wide meshwork to which receptors can subsequently dock or whether it only forms short bridges linking receptors together to make a large cluster. Furthermore, the number of rapsyn-binding sites per receptor (a heteropentamer) has been controversial. Here, we show by cryoelectron tomography and subtomogram averaging of Torpedo postsynaptic membrane that receptors are connected by up to three rapsyn bridges, the minimum number required to form a 2D network. Half of the receptors belong to rapsyn-connected groups comprising between two and fourteen receptors. Our results provide a structural basis for explaining the stability and low diffusion of receptors within clusters.

Abelson Family Tyrosine Kinases Regulate the Function of Nicotinic Acetylcholine Receptors and Nicotinic Synapses on Autonomic Neurons

Abelson family kinases (AFKs; Abl1, Abl2) are non-receptor tyrosine kinases (NRTKs) implicated in cancer, but they also have important physiological roles that include regulating synaptic structure and function. Recent studies using Abl-deficient mice and the antileukemia drug STI571 [imatinib mesylate (Gleevec); Novartis], which potently and selectively blocks Abl kinase activity, implicate AFKs in regulating presynaptic neurotransmitter release in hippocampus and postsynaptic clustering of nicotinic acetylcholine receptors (nAChRs) in muscle. Here, we tested whether AFKs are relevant for regulating nAChRs and nAChR-mediated synapses on autonomic neurons. AFK immunoreactivity was detected in ciliary ganglion (CG) lysates and neurons, and STI571 application blocked endogenous Abl tyrosine kinase activity. With similar potency, STI571 specifically reduced whole-cell current responses generated by both nicotinic receptor subtypes present on CG neurons (α3*- and α7-nAChRs) and lowered the frequency and amplitude of α3*-nAChR-mediated excitatory postsynaptic currents. Quantal analysis indicated that the synaptic perturbations were postsynaptic in origin, and confocal imaging experiments revealed they were unaccompanied by changes in nAChR clustering or alignment with presynaptic terminals. The results indicate that in autonomic neurons, Abl kinase activity normally supports postsynaptic nAChR function to sustain nAChR-mediated neurotransmission. Such consequences contrast with the influence of Abl kinase activity on presynaptic function and synaptic structure in hippocampus and muscle, respectively, demonstrating a cell-specific mechanism of action. Finally, because STI571 potently inhibits Abl kinase activity, the autonomic dysfunction side effects associated with its use as a chemotherapeutic agent may result from perturbed α3*- and/or α7-nAChR function.

Caveolin-3 Promotes Nicotinic Acetylcholine Receptor Clustering and Regulates Neuromuscular Junction Activity

The molecular mechanisms that regulate the organization and activity of the neuromuscular junction remain to be fully identified. Caveolae are invaginations of the plasma membrane. Caveolin-3 is the structural protein component of caveolae in muscle cells. We show that caveolin-3 is expressed at the neuromuscular junction, that it associates with the nicotinic acetylcholine receptor (nAChR), and that a lack of caveolin-3 inhibits clustering of the nAChR in myotubes. At the molecular level, we demonstrate that caveolin-3 is a novel muscle-specific kinase (MuSK) binding protein and that altered nAChR clustering in caveolin-3-lacking myotubes results from inhibition of agrin-induced phosphorylation/activation of MuSK and activation of Rac-1. Functional studies in caveolin-3 null mice show abnormal neuromuscular junction activity that is consistent with altered nAChR localization at the sarcolemma. Together, these data identify caveolin-3 as a critical component of the signaling machinery that drives nicotinic acetylcholine receptor clustering and controls neuromuscular junction function.

Control of Rapsyn Stability by the CUL-3-containing E3 Ligase Complex

Rapsyn is a postsynaptic protein required for clustering of nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction. Here we report the mechanism for posttranslational control of rapsyn protein stability. We confirmed that C18H9.7-encoded RPY-1 is a rapsyn homolog in Caenorhabditis elegans by showing that human rapsyn rescued rpy-1 mutant phenotypes in nematodes, as determined by levamisole assays and micropost array behavioral assays. We found that RPY-1 was degraded in the absence of functional UNC-29, a non-alpha subunit of the receptor, in an allele-specific manner, but not in the absence of other receptor subunits. The cytoplasmic loop of UNC-29 was found to be critical for RPY-1 stability. Through RNA interference screening, we found that UBC-1, UBC-12, NEDD-8, and RBX-1 were required for degradation of RPY-1. We identified cullin (CUL)-3 as a component of E3 ligase and KEL-8 as the substrate adaptor of RPY-1. Mammalian rapsyn was ubiquitinated by the CUL3/KLHL8-containing E3 ligase in vitro, and the knockdown of KLHL-8, a mammalian KEL-8 homolog, inhibited rapsyn ubiquitination in vivo, implying evolutionary conservation of the rapsyn stability control machinery. kel-8 suppression and rpy-1 overexpression in C. elegans produced a phenotype similar to that of a loss-of-function mutation of rpy-1, suggesting that control of rapsyn abundance is important for proper function of the receptor. Our results suggest a link between the control of rapsyn abundance and congenital myasthenic syndromes.

Localization of the membrane‐anchored MMP‐regulator RECK at the neuromuscular junctions

Nerve apposition on nicotinic acetylcholine receptor clusters and invagination of the post-synaptic membrane (i.e. secondary fold formation) occur by embryonic day 18.5 at the neuromuscular junctions (NMJs) in mouse skeletal muscles. Finding the molecules expressed at the NMJ at this stage of development may help elucidating how the strong linkage between a nerve terminal and a muscle fiber is established. Immunohistochemical analyses indicated that the membrane-anchored matrix metalloproteinase regulator RECK was enriched at the NMJ in adult skeletal muscles. Confocal and electron microscopy revealed the localization of RECK immunoreactivity in secondary folds and subsynaptic intracellular compartments in muscles. Time course studies indicated that RECK immunoreactivity becomes associated with the NMJ in the diaphragm at around embryonic day 18.5 and thereafter. These findings, together with known properties of RECK, support the hypothesis that RECK participates in NMJ formation and/or maintenance, possibly by protecting extracellular components, such as synaptic basal laminae, from proteolytic degradation.

Regulation of synaptic growth and maturation by a synapse-associated E3 ubiquitin ligase at the neuromuscular junction

The ubiquitin–proteasome pathway has been implicated in synaptic development and plasticity. However, mechanisms by which ubiquitination contributes to precise and dynamic control of synaptic development and plasticity are poorly understood. We have identified a PDZ domain containing RING finger 3 (PDZRN3) as a synapse-associated E3 ubiquitin ligase and have demonstrated that it regulates the surface expression of muscle-specific receptor tyrosine kinase (MuSK), the key organizer of postsynaptic development at the mammalian neuromuscular junction. PDZRN3 binds to MuSK and promotes its ubiquitination. Regulation of cell surface levels of MuSK by PDZRN3 requires the ubiquitin ligase domain and is mediated by accelerated endocytosis. Gain- and loss-of-function studies in cultured myotubes show that regulation of MuSK by PDZRN3 plays an important role in MuSK-mediated nicotinic acetylcholine receptor clustering. Furthermore, overexpression of PDZRN3 in skeletal muscle of transgenic mice perturbs the growth and maturation of the neuromuscular junction. These results identify a synapse-associated E3 ubiquitin ligase as an important regulator of MuSK signaling.

Lipid Rafts Serve as a Signaling Platform for Nicotinic Acetylcholine Receptor Clustering

Agrin, a motoneuron-derived factor, and the muscle-specific receptor tyrosine kinase (MuSK) are essential for the acetylcholine receptor (AChR) clustering at the postjunctional membrane. However, the underlying signaling mechanisms remain poorly defined. We show that agrin stimulates a dynamic translocation of the AChR into lipid rafts-cholesterol and sphingolipid-rich microdomains in the plasma membrane. This follows MuSK partition into lipid rafts and requires its activation. Disruption of lipid rafts inhibits MuSK activation and downstream signaling and AChR clustering in response to agrin. Rapsyn, an intracellular protein necessary for AChR clustering, is located constitutively in lipid rafts, but its interaction with the AChR is inhibited when lipid rafts are perturbed. These results reveal that lipid rafts may regulate AChR clustering by facilitating the agrin/MuSK signaling and the interaction between the receptor and rapsyn, both necessary for AChR clustering and maintenance. These results provide insight into mechanisms of AChR cluster formation.

Induction of myasthenia by immunization against muscle-specific kinase

Muscle-specific kinase (MuSK) is critical for the synaptic clustering of nicotinic acetylcholine receptors (AChRs) and plays multiple roles in the organization and maintenance of neuromuscular junctions (NMJs). MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering at the postsynaptic membrane. Although autoantibodies against the ectodomain of MuSK have been found in a proportion of patients with generalized myasthenia gravis (MG), it is unclear whether MuSK autoantibodies are the causative agent of generalized MG. In the present study, rabbits immunized with MuSK ectodomain protein manifested MG-like muscle weakness with a reduction of AChR clustering at the NMJs. The autoantibodies activated MuSK and blocked AChR clustering induced by agrin or by mediators that do not activate MuSK. Thus MuSK autoantibodies rigorously inhibit AChR clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of MG.

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.

Functional innervation of cultured human skeletal muscle proceeds by two modes with regard to agrin effects

Nerve-derived agrin is a specific isoform of agrin that promotes clustering of nicotinic acetylcholine receptors (AChR) and other components of the neuromuscular junction (NMJ). We investigated the effects of agrin on functional maturation of NMJs at the early stages of synaptogenesis in human muscle. Specifically, we assessed the importance of agrin for the differentiation of developing NMJs to the stage where they are able to transmit signals that result in contractions of myotubes. We utilized an in vitro model in which human myotubes are innervated by neurons extending from spinal cord explants of fetal rat. This model is suitable for functional studies because all muscle contractions are the result of neuromuscular transmission and can be quantitated. An anti-agrin antibody, Agr 33, was applied to co-cultures during de novo NMJ formation. Quantitative analyses demonstrated that Agr 33 reduced the number of AChR clusters to 20% and their long axes to 50% of control, yet still permitted early, NMJ-mediated muscle contractions that are normally observed in 7–10-day-old co-cultures. However, at later times of development, the same treatment completely prevented the increase in the number of contracting units as compared with untreated co-cultures. It is concluded that there are two modes of functional maturation of NMJs with regard to agrin effects: one that is insensitive and the other that is sensitive to agrin blockade. Agrin-insensitive mode is limited to the small population of NMJs that become functional at the earlier stages of functional innervation. However, most of the NMJs become contraction-competent at the later stages of the innervation process. These NMJs become functional only if agrin action is uncompromised. This is the first characterization of the contribution of agrin to NMJ development on human muscle.

Genetically dystrophic mdx/mdx mice exhibit decreased response to nicotine in passive avoidance.

mdx mice are considered as a genetic homologous of human Duchenne muscular dystrophy. Recent evidence demonstrates that in mouse sympathetic ganglion dystrophin is involved in the stabilization of nicotinic acetylcholine receptor clusters. The purpose of this study was to verify possible effects of dystrophin alterations at the central level. This was assessed by evaluating the response to nicotine administration in mdx and wild-type mice. Thus the effects of post-training nicotine administrations (0.1, 0.25 and 0.5 mg/kg) were tested in mice subjected to a passive avoidance memory task, that measures the ability of mice to remember on test day a shock received 24 h before. Nicotine enhanced memory in wild-type as well as in mdx mice. However, the doses needed to increase memory in mdx were higher than in wild-type. These results are discussed in terms of possible functional changes in central nicotinic acetylcholine receptor in mdx mice.

Clustering of nicotinic acetylcholine receptors: from the neuromuscular junction to interneuronal synapses.

Fast and accurate synaptic transmission requires high-density accumulation of neurotransmitter receptors in the postsynaptic membrane. During development of the neuromuscular junction, clustering of acetylcholine receptors (AChR) is one of the first signs of postsynaptic specialization and is induced by nerve-released agrin. Recent studies have revealed that different mechanisms regulate assembly vs stabilization of AChR clusters and of the postsynaptic apparatus. MuSK, a receptor tyrosine kinase and component of the agrin receptor, and rapsyn, an AChR-associated anchoring protein, play crucial roles in the postsynaptic assembly. Once formed, AChR clusters and the postsynaptic membrane are stabilized by components of the dystrophin/utrophin glycoprotein complex, some of which also direct aspects of synaptic maturation such as formation of postjunctional folds. Nicotinic receptors are also expressed across the peripheral and central nervous system (PNS/CNS). These receptors are localized not only at the pre- but also at the postsynaptic sites where they carry out major synaptic transmission. In neurons, they are found as clusters at synaptic or extrasynaptic sites, suggesting that different mechanisms might underlie this specific localization of nicotinic receptors. This review summarizes the current knowledge about formation and stabilization of the postsynaptic apparatus at the neuromuscular junction and extends this to explore the synaptic structures of interneuronal cholinergic synapses.

Role of Rapsyn Tetratricopeptide Repeat and Coiled-coil Domains in Self-association and Nicotinic Acetylcholine Receptor Clustering

Rapsyn, a 43-kDa peripheral membrane protein of skeletal muscle, is essential for clustering nicotinic acetylcholine receptors (nAChR) in the postsynaptic membrane. Previous studies with rapsyn NH(2)-terminal fragments fused to green fluorescent protein, expressed in 293T cells along with nAChRs, establish the following: Rapsyn-(1-90), containing the myristoylated amino terminus and two tetratricopeptide repeats (TPRs), was sufficient for self-association at the plasma membrane; rapsyn-(1-287), containing seven TPRs, did not cluster nAChRs; whereas rapsyn-(1-360)(,) containing a coiled-coil domain (rapsyn-(298-331)), clustered nAChRs. To further analyze the role of rapsyn structural domains in self-association and nAChR clustering, we have characterized the clustering properties of additional rapsyn mutants containing deletions and substitutions within the TPR and coiled-coil domains. A mutant lacking the coiled-coil domain alone (rapsyn-(black triangle288-348)), failed to cluster nAChRs. Within the coiled-coil domain neutralization of the charged side chains was tolerated, while alanine substitutions of large hydrophobic residues resulted in the loss of nAChR clustering. Rapsyn self-association requires at least two TPRs, as a single TPR (TPR1 or TPR2 alone) was not sufficient. While TPRs 1 and 2 are sufficient for self-association, they are not necessary, as TPRs 3-7 also formed clusters similar to wild-type rapsyn. Fragments containing TPRs co-localized with full-length rapsyn, while the expressed coiled-coil or RING-H2 domain did not. These results are discussed in terms of a homology model of rapsyn, based on the three-dimensional structure of the TPR domain of protein phosphatase 5.

Differential targeting of components of the dystrophin complex to the postsynaptic membrane

Accumulating evidence points to the participation of dystroglycan in the clustering of nicotinic acetylcholine receptors at the neuromuscular junction [Côté et al. (1999) Nature Genet., 3, 338--342]. Dystroglycan is part of a multimolecular complex, either associated with dystrophin (the dystrophin-associated protein complex) at the sarcolemma or with utrophin (the utrophin-associated protein complex) at the neuromuscular junction. Understanding the assembly of this complex at the developing synapse led us to investigate, in Torpedo electrocyte, the intracellular routing and the targeting of several of its components, including dystroglycan, syntrophin, dystrophin and dystrobrevin. We previously demonstrated that acetylcholine receptors and rapsyn, the 43-kDa receptor-associated protein at the synapse, are cotargeted to the postsynaptic membrane via the exocytic pathway [Marchand et al. (2000) J. Neurosci., 20, 521--528]. Using cell fractionation, immunopurification and immuno-electron microscope techniques, we show that beta-dystroglycan, an integral glycoprotein that constitutes the core of the dystrophin-associated protein complex localized at the innervated membrane, is transported together with acetylcholine receptor and rapsyn in post-Golgi vesicles en route to the postsynaptic membrane. Syntrophin, a peripheral cytoplasmic protein of the complex, associates initially with these exocytic vesicles. Conversely, dystrophin and dystrobrevin were absent from these post-Golgi vesicles and associate directly with the postsynaptic membrane. This study provides the first evidence for a separate targeting of the various components of the dystrophin-associated protein complex and a step-by-step assembly at the postsynaptic membrane.

Formation of Postsynaptic-Like Membranes during Differentiation of Embryonic Stem Cellsin Vitro

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, α1subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the γ- and ϵ-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR ϵ-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiationin vitroneuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by “loss of function” analysisin vitro.

The Synapse-Associated Protein Rapsyn Regulates Tyrosine Phosphorylation of Proteins Colocalized at Nicotinic Acetylcholine Receptor Clusters

Protein tyrosine phosphorylation has been suggested to play an important role in the clustering of the nicotinic acetylcholine receptor (AChR) at the developing neuromuscular junction. Recent studies have shown that the 43-kDa synapse-associated protein rapsyn induces clustering of the AChR in heterologous expression systems. In this study we examined whether tyrosine phosphorylation is involved in this rapsyn-induced AChR clustering. Rapsyn-induced AChR clusters in fibroblasts contain phosphotyrosine, as detected using immunofluorescent labeling with anti-phosphotyrosine antibodies. No anti-phosphotyrosine staining of rapsyn clusters is seen in the absence of AChR expression, indicating that the AChR is required for the appearance of phosphotyrosine at clusters. In addition, coexpression of rapsyn with the AChR induces the tyrosine phosphorylation of the β amd δ subunits of the AChR. Surprisingly, mutation of the tyrosine phosphorylation sites in the AChR did not inhibit rapsyn-induced clustering of the AChR and clusters of the mutant AChRs still contained high levels of phosphotyrosine. Experiments with single AChR subunits demonstrate that the α subunit of the AChR appears to be necessary and sufficient for codistribution of phosphotyrosine with rapsyn-induced clusters of AChR subunits. Finally, transfection of cells with rapsyn activates cellular protein tyrosine kinase activity, resulting in the tyrosine phosphorylation of several membrane-associated proteins. These results suggest that rapsyn may therefore regulate clustering at least in part by regulating the tyrosine phosphorylation of cellular proteins.

Role of HB-GAM (Heparin-Binding Growth-Associated Molecule) in Proliferation Arrest in Cells of the Developing Rat Limb and Its Expression in the Differentiating Neuromuscular System

HB-GAM (heparin-binding growth-associated molecule) is a secretory, extracellular matrix-associated protein that was isolated by screening for proteins that enhance neurite outgrowth in perinatal rat brain neurons. In the present study we have investigated the possible role of HB-GAM in cell proliferation in the developing rat limb. Exogenously added recombinant HB-GAM was found to inhibit the proliferation of mesenchymal and epithelial cells in cultured limb buds, as demonstrated by bromodeoxyuridine incorporation and by staining for PCNA (proliferating cell nuclear antigen). The inhibitory effect of HB-GAM on cell proliferation was reversed by heparin, suggesting that HB-GAM may bind to a heparin-type carbohydrate epitope that is required for cell proliferation in the developing limb. Endogenous HB-GAM of the developing limb was found to be expressed in a proximal-to-distal pattern, in agreement with the putative role in proliferation arrest and cell differentiation. In addition, double immunostaining of HB-GAM with PCNA showed that in early (Embryonic Day 12) limb mesenchyme HB-GAM was associated mainly with the surface of growth-arrested cells. Furthermore, HB-GAM was associated with the muscle surface, as demonstrated in double immunostaining of HB-GAM with desmin and myosin heavy chain proteins. Coinciding with the onset of synapse formation (Embryonic Day 16), HB-GAM was found in patches on the muscle cell surface in close proximity to nicotinic acetylcholine receptor clusters. This finding is in agreement with a previous study that has suggested a role for HB-GAM in the differentiation of the neuromuscular junction in Xenopus muscle.