From the Max-Planck-Institut für Entwicklungsbiologie, Abteilung Biochemie, Spemannstrasse 35, D-72076 Tübingen, Germany
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ABSTRACT |
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During development of the neuromuscular junction,
neuronal splice variants of agrin initiate the aggregation of
acetylcholine receptors on the myotube surface. The muscle-specific
kinase is thought to be part of an agrin receptor complex, although the recombinant protein does not bind agrin with high affinity. To specify
its function, we induced phosphorylation and activation of this kinase
in the absence of agrin by incubating myotubes with antibodies directed
against its N-terminal sequence. Antibody-induced dimerization of the
muscle-specific kinase but not treatment with Fab fragments was
sufficient to trigger two key events of early postsynaptic development:
acetylcholine receptors accumulated into aggregates, and their
-subunits became phosphorylated on tyrosine residues. Heparin
partially inhibited receptor aggregation induced by both agrin and
anti-muscle-specific kinase antibodies. In contrast, it did not affect
kinase or acetylcholine receptor phosphorylation. These data indicate
that agrin induces postsynaptic differentiation by dimerizing the
muscle-specific kinase. They also suggest that activation of the kinase
domain can account for only part of agrin's effects. Dimerization of
this molecule appears to activate an additional signal, most likely by
organizing a scaffold for other postsynaptic proteins.
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INTRODUCTION |
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The basal membrane protein agrin plays a central role during the
early phase of synaptic differentiation at the neuromuscular junction
(1-3). Neuron-specific agrin isoforms containing an eight-amino acid
insertion generated by alternative splicing (4-6) are able to induce
the aggregation of AChRs1 and
other synaptic proteins on the surface of myotubes (7-10). Deletion of
the exon sequence encoding this insert in the agrin gene by homologous
recombination in mice results in malformed and misplaced AChR clusters.
Agrin(/
) mice die due to respiratory failure (11).
The mechanism of agrin-induced AChR aggregation is not completely
understood. Rapsyn, a peripheral membrane protein closely associated
with AChRs (12-15), is an essential component of this pathway. In
rapsyn-deficient mice, agrin is not able to induce the concentration of
AChRs and other synaptic components (16). Inhibitor studies suggest an
important role of tyrosine phosphorylation in this pathway (17). Agrin
induces the tyrosine phosphorylation of the -subunit of the AChR
(18). It is unknown whether this modification is necessary for AChR
aggregation.
-Dystroglycan, a component of the dystrophin-associated
glycoprotein complex, has been identified as the most abundant
agrin-binding protein on the myotube surface (19, 20). However, the
analysis of a series of agrin fragments has revealed no
correlation between their binding to
-dystroglycan and their
capability of inducing AChR aggregation (20-22).
Genetic experiments have demonstrated an essential role in the agrin pathway for a muscle-specific receptor tyrosine kinase (MuSK), which has recently been identified in different species (23-27). In mice, deletion of this gene prevents the concentration of AChRs and other proteins at the contact site between motoneuron and muscle fiber and is therefore lethal (23). MuSK is highly expressed in rat embryonic muscle and in the C2C12 mouse muscle cell line and colocalizes with AChRs at the neuromuscular junction (25, 27). Several observations suggest an important role of MuSK in the agrin pathway (28); incubation of myotubes with agrin causes the rapid tyrosine phosphorylation of MuSK (28). This reaction, a characteristic response of receptor tyrosine kinases to binding of their ligand (29, 30), is exclusively induced by biologically active fragments and isoforms of agrin.2 In addition, agrin can be cross-linked to MuSK expressed on myotubes (28). Upon transfection into the quail cell line QT-6, MuSK is concentrated in microaggregates together with rapsyn (31). Remarkably, the extracellular domain of the MuSK molecule is required for this interaction, which therefore must be indirect. It has been suggested that a hypothetical rapsyn-associated transmembrane linker (RATL) bridges these proteins (32).
Agrin does not directly bind to recombinant MuSK (28) (data not shown).
Therefore, a MuSK-accessory specificity component (MASC) has been
postulated, mediating its activation by agrin (28). To assess the role
of MuSK in the agrin signaling pathway, it is important to activate
this molecule independent of agrin. In an earlier attempt, a chimeric
molecule consisting of the extracellular domain of the neurotrophin
receptor TrkC and the intracellular domain of MuSK has been expressed
in myotubes. The TrkC ligand neurotrophin 3 added to these myotubes
induces the tyrosine phosphorylation of the chimeric receptor as well
as AChRs, but not AChR aggregation (33). Here we took a different
approach to bypass agrin in activating MuSK. We artificially dimerized
MuSK by incubating myotubes with bivalent polyclonal antibodies
directed against its N terminus. We demonstrate that MuSK is sufficient
to trigger responses normally evoked by neuronal agrin isoforms;
antibody-induced activation of MuSK causes aggregation of AChRs and the
tyrosine phosphorylation of their -subunit with high efficiency. We
also show that AChR aggregation but not AChR-phosphorylation is
inhibited by heparin, suggesting the existence of multiple pathways
activated by MuSK.
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EXPERIMENTAL PROCEDURES |
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Expression Constructs and Transient Transfection-- The soluble rat agrin constructs s-agrin-(4,19) and s-agrin-(0,8) have been described previously (34). Generation of the full-length MuSK construct has been reported elsewhere,2 and an expression construct coding for the extracellular part of MuSK was assembled by the addition of sequence coding for a hexahistidine tag followed by a stop codon to the appropriate site of the MuSK cDNA.3 COS-7 cells were transiently transfected with plasmids encoding soluble agrin (30 µg of DNA/15-cm dish) according to the method of Chen and Okayama (35). The collection of serum-free agrin-conditioned media and calibration of agrin concentrations has been described (34).
Antibodies and Fab Fragments-- Polyclonal antibody (pAb) Cyt-MuSK against a bacterial fusion protein comprising the first half of the cytoplasmic domain of MuSK was affinity-purified by absorption to the antigen immobilized on Affi-Gel (Bio-Rad). pAb N-MuSK was purified from a crude antiserum against a peptide (N-peptide) corresponding to 14 amino acids of the putative N terminus of MuSK by affinity chromatography on an immobilized N-terminal bacterial fusion protein. Both pAbs specifically recognize MuSK in immunoprecipitation and Western blot experiments.2 For the antibody specificity analysis shown in Fig. 1, membrane proteins were extracted from COS cells,2 whereas a plasma membrane fraction of C2C12 cells differentiated for 5 days in fusion medium was prepared as described (36).
The phosphotyrosine antibody mAb 5E2 (37) was a kind gift from Dr. A. Ullrich (Max-Planck-Institute for Biochemistry). The phosphotyrosine antibodies mAb PY20 and mAb 4G10 were purchased from Transduction Laboratories and Upstate Biotechnology Inc., respectively. mAb 124 (rat monoclonal) directed against theAnalysis of MuSK and AChR Tyrosine Phosphorylation-- C2C12 myoblasts were propagated as described previously (7). Unless indicated otherwise, cells were allowed to differentiate in 2.5% horse serum, 2 mM glutamine in DMEM (fusion medium) for 4-5 days. They were switched to 0.25% horse serum in DMEM for 3-12 h prior to stimulation with agrin or antibodies in various concentrations.
Immunoprecipitation of MuSK with Cyt-MuSK antiserum and enrichment of AChRs by binding to biotin-Quantitation of Antibody-induced AChR Aggregation--
C2C12
myotubes were cultured on chamber slides (Nunc). After 21/2 days
in fusion medium, they were stimulated with antibody preparations or
agrin for 10-16 h. AChRs were visualized with rhodamine--bungarotoxin, and the number of AChR aggregates in at
least 12 microscopic fields was quantitated as described previously (34). Many small AChR clusters were observed when formation of
aggregates was induced with pAb N-MuSK. These were not included in our
quantitation, since only clusters >5 µm in length were counted. In
experiments with heparin or N-peptide, the cells were pretreated with
these agents as outlined above. All experiments were performed 2-6
times. The number of AChR aggregates is displayed as the mean of 3-5
determinations ± S.E. Statistical significance of the observed
differences was verified by t test analysis
(p < 0.05).
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RESULTS |
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MuSK Antibodies Induce Tyrosine Phosphorylation of the Kinase-- Ligand-induced dimerization is an essential step for activation of receptor tyrosine kinases and in many cases is sufficient to activate these kinases (29, 30, 39). We therefore attempted to artificially dimerize and activate MuSK in the absence of agrin using a peptide antiserum directed against the N terminus of the MuSK protein.2 Polyclonal antibodies (pAb N-MuSK) affinity-purified from this serum recognized a single band in detergent extracts from COS cells transfected with a MuSK expression construct (Fig. 1A). A band of corresponding size was recognized by these antibodies in a plasma membrane preparation of the muscle cell line C2C12 (Fig. 1B). At least a subset of these antibodies was able to react with undenatured MuSK protein, since intact antibodies as well as Fab fragments bound to MuSK-transfected unfixed COS cells but not to mock-transfected controls (Fig. 1C). MuSK was concentrated in small patches on the surface of transfected COS-cells. Similar immunoreactive patches were observed when cells were fixed by incubation with paraformaldehyde prior to exposure to antibodies or Fab fragments (data not shown). This suggests a tendency for MuSK to self-aggregate.
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Antibodies against MuSK Trigger Tyrosine Phosphorylation of the
-Subunit of the AChR--
Agrin induces the tyrosine
phosphorylation of the
-subunit of the AChR in chick and C2C12
myotubes cultures (18, 40). We therefore investigated whether
antibody-induced dimerization of MuSK had similar effects. We isolated
AChRs from detergent extracts of myotubes treated with pAb N-MuSK or
from control preparations. Antibody-induced dimerization of MuSK caused
a significant and dose-dependent increase in tyrosine
phosphorylation of the AChR
-subunit (Fig. 2B). We
conclude that dimerization of MuSK induced not only kinase
autophosphorylation but also the phosphorylation of a downstream
target. Only bivalent N-MuSK-antibodies were able to induce AChR
phosphorylation; Fab fragments or control antibodies had no effect.
Reprobing of the blot with a monoclonal antibody directed against the
-subunit showed that comparable amounts of AChR were precipitated
from the detergent extracts in all samples.
Antibodies against MuSK Induce Aggregation of AChRs--
Next, we
asked whether activation of MuSK alone is sufficient to induce not only
phosphorylation but also clustering of AChRs. We incubated C2C12
myotubes with pAb N-MuSK or with soluble nerve agrin (s-agrin (4, 19))
for 12 h, visualized AChRs with rhodamine-conjugated -bungarotoxin, and analyzed their distribution.
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MuSK Antibodies Induced MuSK Phosphorylation with Higher Efficiency
than AChR Phosphorylation and Aggregation--
The experiments
described above demonstrated that MuSK activation alone mimics effects
normally triggered by agrin. However, they could not exclude the
possibility that interactions of agrin with components of the myotube
surface not connected to MuSK (e.g. -dystroglycan) play a
synergistic role in initiating AChR aggregation. To set a limit for the
potential effects of such MuSK-independent effects of agrin, it was
important to compare the ability of agrin and anti-MuSK antibodies to
induce different effects more quantitatively. C2C12 myotubes were
stimulated with various concentrations of pAb N-MuSK and s-agrin (4,
19) or with DMEM (control). From one aliquot of the cell lysates, MuSK
was immunoprecipitated; from another, AChRs were affinity-purified. In
both preparations, tyrosine phosphorylation evoked by the two effectors
was measured by quantitative Western blot analysis. In the
concentration range used in this experiment, pAb N-MuSK induced a
higher degree of MuSK phosphorylation than agrin, whereas AChR
phosphorylation was triggered with reversed efficiencies (Fig.
5). For example, 40 pM agrin
induced a comparable level of MuSK phosphorylation as 24 nM
pAb N-MuSK, but 3-fold higher antibody concentrations were required to
match the ability of 40 pM agrin to cause AChR phosphorylation. Similarly, 3-fold higher concentrations of antibody were required to induce AChR aggregation in comparison with MuSK phosphorylation (data not shown). Thus, a potential activation of
MuSK-independent receptors by agrin can at best play a small synergistic role in the agrin signaling pathway.
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Antibody-induced AChR Aggregation, but Not Tyrosine Phosphorylation of MuSK or AChRs Is Inhibited by Heparin-- The possibility of activating AChR aggregation in the absence of agrin allowed us to further delineate a target for the action of heparin, a well known inhibitor of nerve- as well as agrin-induced clustering of AChRs (42, 43). This inhibitor could prove useful in studies aiming at an understanding of the mechanisms by which MuSK activation triggers AChR aggregation. While heparin directly binds to a subset of agrin isoforms (21, 22, 44), this direct binding to agrin only accounts for part of its inhibitory effects. Recently, we showed that heparin acts as an inhibitor at an additional step in the agrin pathway (34), which has not been identified.
To narrow down this second target of heparin, we investigated whether heparin differentially affects AChR aggregation induced by agrin or anti-MuSK antibodies. High concentrations of heparin reduce the amount of AChR aggregates induced by a non-heparin-binding agrin isoform by 55-75% (34). Upon inducing AChR aggregation by incubation of myotubes with anti-MuSK antibodies, we observed an ~80% reduction in the number of AChR clusters (Fig. 6A), demonstrating that the target mediating this heparin inhibition is not localized up-stream of MuSK.
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DISCUSSION |
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The goal of our study was to specify the role of MuSK in the
assembly of the postsynaptic apparatus induced by agrin. We have shown
that incubation of myotubes with antibodies against MuSK triggers the
tyrosine phosphorylation of MuSK. More importantly, we have
demonstrated that this antibody-induced cross-linking is sufficient to
induce similar effects as treatment of myotubes with agrin; AChRs
started to aggregate, and their -subunits became phosphorylated on
tyrosine residues.
Fab fragments of pAb N-MuSK did not trigger similar effects, although they bound to MuSK to a similar extent as bivalent antibodies in several control experiments. We found no evidence for binding of our antibodies to other cell surface proteins besides MuSK. Furthermore, the small size of the N-peptide (14 amino acids) against which N-MuSK antibodies are directed makes it very unlikely that more than one antibody at a time bound to MuSK's N terminus. Based on these considerations, we conclude that the observed AChR aggregation is caused by a selective dimerization of MuSK and cannot be attributed to extensive cross-linking of this molecule.
In comparison with agrin, anti-MuSK antibodies displayed slightly different efficiencies of MuSK phosphorylation versus AChR phosphorylation and aggregation. These differences could indicate the synergistic participation of a hypothetical MuSK-independent signal, which is triggered by agrin but not by the antibodies. However, in a detailed study of the ligand specificity of agrin-induced effects, no evidence was found for the existence of such a signal.2 Alternatively, the reduced level of AChR phosphorylation and aggregation in our experiments was due to a slightly altered conformation of the agrin receptor complex in response to antibody-induced but not agrin-induced MuSK dimerization and/or an incomplete activation of the kinase domain of MuSK (Fig. 7). While the mode of activation of MuSK has not been analyzed so far, a stepwise autophosphorylation and activation process has previously been described for other receptor tyrosine kinases, e.g. the insulin receptor (45).
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While this manuscript was in preparation, another report described the activation of MuSK by a single chain antibody (46). In agreement with our results, this activation was sufficient to trigger the phosphorylation and aggregation of AChRs, although these effects were not studied quantitatively. In contrast to our antibodies directed against the N terminus of MuSK, several monovalent antibodies directed against unknown regions of the extracellular domain of MuSK caused the activation of the kinase. Surprisingly, bivalency of these antibodies was not required, suggesting a different mode of activation (47).
The effects of antibody-induced activation of MuSK described here demonstrate that anti-MuSK antibodies are a useful tool for elucidating MuSK's role in the agrin pathway. The comparison of our results with those of other attempts to activate MuSK agrin-independently (33) highlights a major difference between MuSK and other receptor tyrosine kinases: the crucial role of the extracellular domain of MuSK. Glass et al. (33) stimulated a chimeric receptor consisting of the extracellular domain of TrkC and the intracellular domain of MuSK with neurotrophin 3 and thereby efficiently induced phosphorylation of the chimera and the AChR. However, activation of TrkC/MuSK did not lead to the aggregation of AChR on the surface of C2C12 myotubes (Fig. 7). Clearly, stimulating the kinase activity of MuSK alone cannot account for AChR aggregation. In addition to the MuSK/TrkC chimera, antibody-induced dimerization of the full-length MuSK molecule and concomitant redistribution of putative MuSK-associated proteins was able to induce not only the phosphorylation of AChRs but also their aggregation.
This functional difference directly points at an essential role of the extracellular domain of MuSK. An inherent organizing function has previously been suggested by cotransfection experiments in a quail fibroblast cell line (31, 32). In this system, a kinase-defective mutant of the Torpedo MuSK ortholog (31) and a MuSK fragment in which most of the cytoplasmic domain had been deleted (32) were aggregated by cotransfected rapsyn. In muscle cells, a kinase-defective mutant of rat MuSK suppressed AChR clustering (33), demonstrating the requirement of tyrosine kinase activity for this process.
Our data complement the suggestion that two signals are necessary to induce the aggregation of AChRs in myotubes (Fig. 7) (32, 48); the first signal is the kinase activity of MuSK, and the second signal appears to originate from the physical association of other proteins with MuSK. It is neither known in which way this scaffolding depends on MuSK activation nor which proteins associate with MuSK. The most likely candidate appears to be RATL, which might directly tether rapsyn and the stoichiometrically complexed AChRs to MuSK (32). Alternatively, the passive redistribution of MASC induced by MuSK dimerization could be important for AChR aggregation. This second possibility appears less likely, since our data showed that a direct activation of MASC by the binding of agrin is not essential for this pathway. Any signal that might be triggered by the binding of agrin to MASC can be bypassed by the dimerization of MuSK.
Two types of inhibitors of the agrin pathway have been characterized so far. Staurosporin, an inhibitor of tyrosine kinases, blocks both phosphorylation and aggregation of AChRs (17) and apparently inhibits the first signal in the pathway (Fig. 7); our data suggest that heparin represents a second type of inhibitor, which interferes with the second signal. Heparin treatment caused a MuSK/TrkC-like "phenotype"; it inhibited AChR aggregation induced by a non-heparin-binding agrin isoform (34) and by anti-MuSK antibodies by more than 80%. Strikingly, it did not affect the phosphorylation of either MuSK or AChRs. This selective interference with receptor aggregation would be expected from a reagent interfering exclusively with the second signal in the agrin pathway. The extracellular domain of MuSK, which is involved in this step, is accessible to heparin and other polyanions added into the medium. The protein directly interacting with heparin has not been identified so far, but RATL is an interesting candidate.
The availability of specific activators and inhibitors of the agrin signaling pathway should be useful in the future to identify the missing players and understand how they interact with the already identified components.
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ACKNOWLEDGEMENTS |
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We thank Sigrun Helms and Vicky Kastner for excellent technical assistance, Axel Ullrich and Jon Lindstrom for the generous gift of antibodies, and Rongxing Gan and Uli Schwarz for critical reading of the manuscript. We also express our gratitude to Uli Schwarz for support.
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FOOTNOTES |
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* This work was supported by the Max-Planck Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by the Graduiertenkolleg Neurobiologie
Tübingen.
§ To whom correspondence should be addressed: Max-Planck-Institut für Entwicklungsbiologie, Abteilung Biochemie, Spemannstr. 35, D-72076 Tübingen, Germany. Tel.: 07071-601415; Fax: 07071-601447; E-mail: werner.hoch{at}tuebingen.mpg.de.
1
The abbreviations used are: AChR, nicotinic
acetylcholine receptor; DMEM, Dulbecco's modified Eagle's medium;
MuSK, muscle-specific kinase; MASC, MuSK-accessory specificity
component; mAb, monoclonal antibody; pAb, polyclonal antibody; RATL,
rapsyn-associated transmembrane linker; s-agrin, soluble agrin; PAGE,
polyacrylamide gel electrophoresis; TGFR I, transforming growth
factor
receptor I.
2 C. Hopf and W. Hoch, submitted for publication.
3 C. Hopf and W. Hoch, unpublished results.
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REFERENCES |
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