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The Journal of Neuroscience, August 15, 1998, 18(16):6340-6348
 -Dystroglycan Functions in Acetylcholine Receptor Aggregation
But Is Not a Coreceptor for Agrin-MuSK Signaling
Christian
Jacobson,
Federica
Montanaro,
Michael
Lindenbaum,
Salvatore
Carbonetto, and
Michael
Ferns
Departments of Biology and Neurology and Neurosurgery, McGill
University and the Centre for Research in Neuroscience,
Montreal, Quebec, H3G 1A4 Canada
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ABSTRACT |
 -dystroglycan (-DG) is an agrin-binding protein that
has been implicated in acetylcholine receptor (AChR) clustering, but it
is unclear whether it acts as a coreceptor involved in initial agrin
signaling or as a component involved in later events. To investigate
its role, we have generated antisense derivatives of the C2
mouse muscle cell line, which have reduced -DG expression. When
compared with wild-type cells, the -DG-deficient myotubes have a
dramatic reduction in the number of spontaneous and agrin-induced AChR
clusters. Several findings suggest that this decrease in AChR
clustering is likely not because of a defect in agrin signaling through
the MuSK receptor tyrosine kinase. Compared with wild-type cells, the -DG-deficient cell lines showed only a transient
reduction in the level of agrin-induced MuSK tyrosine phosphorylation
and no reduction in AChR  -subunit tyrosine phosphorylation.
Additionally, agrin-induced phosphorylation of MuSK in wild-type
myotubes was not decreased using agrin fragments that lack the domain
primarily responsible for binding to -DG. Finally, neural
agrin-induced phosphorylation of MuSK was unaffected by treatments such
as excess muscle agrin or anti--DG antibodies, both of which block
agrin--DG binding. Together, these results suggest that -DG is
not required for agrin-MuSK signaling but rather that it may play a
role elsewhere in the clustering pathway, such as in the downstream
consolidation or maintenance of AChR clusters.
Key words:
synaptogenesis; neuromuscular junction; dystroglycan; agrin; acetylcholine receptor; phosphorylation
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INTRODUCTION |
The development of the neuromuscular
synapse occurs via the interplay of signals between the innervating
motor nerve and the muscle cells that leads to a progressive, localized
specialization of the pre- and postsynaptic cells (Hall and Sanes,
1993 ). A motoneuron-derived factor called agrin plays a key organizing
role in this process and is required for the induction of many aspects
of both pre- and postsynaptic differentiation (for review, see
Bowe and Fallon, 1995; Kleiman and Reichardt, 1996). In particular,
neural agrin induces the postsynaptic clustering of the acetylcholine
receptor (Gautam et al., 1996), as well as the accumulation at these
clusters of other synaptic proteins such as rapsyn, dystroglycan, and
utrophin (Wallace, 1989; Campanelli et al., 1994; Gee et al., 1994;
Bowe and Fallon, 1995). Neural agrin seems to act via a receptor
tyrosine kinase, MuSK, that is localized to the end-plate
regions of skeletal muscle (Valenzuela et al., 1995). Neural agrin
induces a rapid tyrosine phosphorylation of that receptor (Glass et
al., 1996), and in MuSK-deficient mice, there is a complete absence of
postsynaptic differentiation, including clustering of acetylcholine
receptor (AChR) beneath the nerve terminal (DeChiara et al.,
1996).
Agrin also binds with relatively high affinity to -dystroglycan
(-DG) (Bowe et al., 1994; Campanelli et al., 1994; Gee et al., 1994; Sugiyama et al., 1994), a cell surface glycoprotein that is
part of a transmembrane complex of dystrophin-associated proteins (for
review, see Carbonetto and Lindenbaum, 1995). /-DG is
concentrated at AChR clusters in vivo and in
vitro, and antibodies to -DG can disrupt agrin-induced AChR
clustering (Campanelli et al., 1994; Gee et al., 1994; but see Sugiyama
et al., 1994). In addition, a minimal agrin fragment that lacks the
-DG-binding domain (Hopf and Hoch, 1996) is significantly less
active in inducing AChR clustering and phosphorylation than are larger
fragments that do bind -DG (Gesemann et al., 1995, 1996; Meier et
al., 1996). These findings implicate -DG in agrin-induced AChR
clustering; however the precise function of -DG is currently
unclear. -DG could act as a coreceptor involved in presenting agrin
to the receptor tyrosine kinase MuSK. Consistent with this idea, it has been proposed that a coreceptor is required for agrin-MuSK signaling because agrin does not bind or activate MuSK expressed in fibroblasts or myoblasts (Glass et al., 1996). Alternatively, -DG may act downstream of agrin-receptor signaling by contributing to the growth
and/or maintenance of AChR aggregates, or it may merely have a
structural role in stabilizing the neuromuscular junction via
interactions with the basal lamina.
To elucidate the role of -DG in agrin-induced AChR clustering,
we have generated antisense derivatives of the C2 mouse muscle cell line that have reduced -DG. These lines show a dramatic reduction in the number of spontaneous and agrin-induced AChR clusters
but little or no change in agrin-induced MuSK and AChR -subunit
phosphorylation. Additionally, the activation of MuSK by agrin in
wild-type C2 myotubes was not inhibited by treatments that block
agrin--DG binding or decreased using agrin fragments that do not
bind -DG. Together, these results suggest that -DG is not
required for agrin-MuSK signaling but that it may play a role in the
subsequent formation of AChR clusters.
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MATERIALS AND METHODS |
Generation of -DG antisense lines. An 1800 bp
fragment of the mouse dystroglycan cDNA, extending from approximately
 100 bp (5' to the translational start site) to the HindIII
site situated at +1725 bp, was removed by digestion of a mouse
dystroglycan cDNA subclone in bluescript SK() (M. Lindenbaum and S. Carbonetto, unpublished observations) with
NotI/HindIII. This
NotI/HindIII cDNA fragment was then subcloned in
the antisense orientation into the
NotI/HindIII-digested expression vector pRC
cytomegalovirus (Invitrogen, San Diego, CA) using standard
subcloning techniques. Before transfection, the plasmid was linearized
by digestion with BglII.
Stable transfections were performed on C2 myoblasts by the method of
calcium phosphate coprecipitation. Briefly, C2 myoblasts plated on
10-cm dishes were incubated with fresh media for 3 hr, and the
DNA/CaPO4 coprecipitate was prepared using 5 µg of
linearized plasmid per plate. The coprecipitate was added to culture
medium, and the cells were incubated for 16 hr. After incubation, the medium and precipitate were removed by aspiration, the cells were washed briefly with DPBS + 0.5 mM EDTA to remove excess
bound precipitate, and fresh medium was added. Twenty-four to
thirty-six hours after the start of transfection, the medium was
replaced with selection medium consisting of growth medium supplemented with 750 µg/ml G418 (active concentration; Life Technologies, Gaithersburg, MD). Selection was performed for up to 10 d or until all the cells on an untransfected control plate were killed. At that
point, drug-resistant C2 cell colonies could easily be seen on
transfected plates. Colonies were then picked and expanded for further
characterization. Once expanded, stable clones were maintained in 100 µg/ml G418.
Culture of muscle cell lines. Mouse C2 muscle cells were
cultured as described previously (Ferns et al., 1992). Briefly,
myoblasts were plated onto 100-mm tissue culture dishes and fed daily
with DMEM with high glucose (DMEM-HI) supplemented with 20%
fetal bovine serum, 5% chick embryo extract, and 100 U/ml
penicillin-streptomycin (Life Technologies). After reaching
confluence, cells were switched to fusion media (DMEM-HI supplemented
with 1-5% horse serum and 100 µg/ml L-glutamine) and
allowed to differentiate into myotubes for 3-4 d. For experiments
involving the -DG antisense lines, all cells (C2, IIF, and
IIE) were cultured in dishes coated with 0.1% gelatin.
Quantification of AChR clusters. For analysis of spontaneous
and agrin-induced clusters, wild-type C2 or -DG antisense lines were
grown within two compartments drawn with a wax pencil in Falcon 10-mm
tissue culture dishes. After 3 d of differentiation in fusion
media, myotube cultures were treated with recombinant agrin (C-Ag
4,8) at a range of concentrations for 16 hr, and the cultures
were then fixed in 2% paraformaldehyde in PBS for 15 min. To visualize
the AChR, we then incubated the myotubes in the compartments with
rhodamine-conjugated -bungarotoxin (1 µg/ml; Molecular Probes,
Eugene OR) for 20 min and coverslipped and viewed the myotubes with a
Zeiss fluorescence microscope at a final magnification of 400×. The
number of AChR clusters above 2.5 µm in diameter and the area of
myotube segments (Gee et al., 1994) were determined for 10-30
random fields for each compartment. The number of AChR clusters was
expressed per myotube area to take into account any difference in cell
density between the cultures. Statistical significance of differences
in the responsiveness of C2, IIF, and IIE cells to agrin treatment was
determined with an ANOVA and Fisher's test (using StatView).
Agrin fragments and treatment. The C-Ag constructs used in
these experiments have been described previously (Ferns et al., 1993),
and a truncated G1 or N4 4,19 construct was generously provided by Werner Hoch (Hoch et al., 1994). The 4,8 and 0,8 isoforms of the N4 fragment were generated by digesting the plasmid with KpnI and ligating in the corresponding fragment from C-Ag
4,8 or 0,8. The agrin fragments were expressed by transient
transfection of COS cells as described previously, and the
concentration of the fragments in the conditioned media was determined
by comparison with a standard of purified agrin on immunoblots (Ferns
et al., 1993).
For experiments assessing MuSK or AChR -subunit tyrosine
phosphorylation, agrin fragments were typically added to myotube cultures at 100 pM to 1 nM for 15-30 min. In
competition experiments, neural agrin was added at 50 pM
and muscle agrin at 5 nM. Anti--dystroglycan antibody
IIH6 was added at concentrations equivalent to those cited in
Gee et al. (1994).
Extraction and isolation of MuSK and AChR. In assays for
MuSK or AChR phosphorylation, myotube cultures were treated with agrin,
rinsed, scraped off in Ca- and Mg-free PBS containing 1 mM
sodium vanadate, and pelleted. Cell pellets were then extracted with
buffer containing 1% Triton X-100 and 25 mM Tris-glycine, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 50 mM sodium fluoride, and
the protease inhibitors 1 mM aprotinin, 1 mM
leupeptin, 1 mM pepstatin A, 1 mM
bisbenzamidine, 1 mM iodoacetamide, and 1 mM
PMSF. After incubation on ice, the insoluble material was removed by
centrifugation at 16,000 × g for 5 min at 4°C. The supernatant was retained, and the protein concentration of the extracts
was determined.
To immunoprecipitate MuSK, we incubated the extracts at 4°C with an
anti-MuSK peptide antibody for 1-2 hr and then with Protein G beads
for an additional 1 hr. The beads were pelleted and washed four times
in a 50 mM Tris buffer containing 0.5 M NaCl.
The proteins isolated on the beads were then eluted directly in 30 µl
of SDS-PAGE sample buffer.
To isolate the AChR, we incubated the extracts with
-bungarotoxin-conjugated beads for 1-2 hr and washed and eluted as
above. Alternatively, to isolate selectively surface AChR, we added
biotinylated -bungarotoxin (Molecular Probes) to myotube cultures 1 hr before harvesting. Cells were then extracted as above and incubated
with 100 µl of streptavidin-conjugated sepharose beads (Molecular
Probes) for 2 hr.
Western blot analysis. Samples were electrophoretically
separated on 8% SDS-PAGE gels and transferred onto nitrocellulose membranes. For phosphotyrosine blots, the proteins were probed with a
monoclonal anti-phosphotyrosine antibody (mAb 4G10; Upstate Biotechnology, Lake Placid, NY) in buffer containing 5% BSA, 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.5% NP40,
and 0.1% Tween-20. The blots were then incubated with a horseradish
peroxidase-conjugated anti-mouse Ig secondary antibody (Amersham,
Arlington Heights IL) and bound antibody was visualized by
chemiluminescence (ECL; Amersham). To probe for -DG, we incubated
the blots with mAb IIH6 in buffer with 5% dry milk and detected -DG
with an HRP-conjugated goat anti-mouse IgG-IgM secondary antibody. The
immunoblots were reprobed for the AChR -subunit with monoclonal
antibody 124 or for MuSK with an anti-MuSK polyclonal antibody.
To quantify levels of tyrosine phosphorylation, we performed
densitometric analysis of the relevant bands using Sci-Scan 5000 Bioanalysis software (United States Biochemicals, Cleveland, OH). To
average several independent experiments, we expressed all values as a
percentage of that for agrin-treated C2 cells at 60 min. We confirmed
the accuracy of this analysis by running a dilution series of
tyrosine-phosphorylated MuSK in which we found that the measured
densities closely matched the expected values. In addition, we always
performed the analysis on films with subsaturating exposure levels.
Antibodies. An anti-MuSK antisera was generated by injecting
a 20 amino acid peptide corresponding to the C terminal of MuSK into each of two rabbits. After several boosts, the resulting antisera
were tested for reaction against the immunizing peptides by ELISA. The
IgG fraction was purified from the antisera of highest titer, and this
antibody was found specifically to immunoprecipitate MuSK from C2
myotube extracts, as the immunoprecipitation could be competed by
addition of excess peptide.
The mAb 124 specific to the -subunit of the AChR was generously
provided by Dr. J. Lindstrom (University of Pennsylvania), and the
anti--dystroglycan antibody IIH6 was generously provided by Dr. K. Campbell (University of Iowa).
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RESULTS |
Acetylcholine receptor clustering is decreased in cell lines with
reduced -DG
We have tested whether -DG is required for agrin-induced
clustering by generating variants of the C2 mouse muscle cell line that
express reduced levels of -DG. Myoblasts were stably transfected with an antisense construct for -DG and a neomycin-resistance gene,
and clones were selected for G418 resistance. Myotube cultures of each
of the lines were then assayed for -DG protein levels, and the
results are shown in Figure 1,
A and B. The immunoblots shown were probed with
anti--DG antibody IIH6, but identical results were obtained with a
polyclonal antibody to the core protein (M. Lindenbaum, F. Montanaro,
and S. Carbonetto, unpublished observations). We found two lines
that had significantly reduced levels of -DG protein compared with
that in wild-type C2 cells; these lines are IIF and IIE, which have
~40 and 10% of the C2 level, respectively (Fig.
1A). Some other lines, such as 9B, 10C, and
11A, had levels of -DG similar to that of wild-type C2
myotubes (Fig. 1B) and were used as controls. Further
characterization of the cell lines indicated that the decrease in
-DG was specific, because several other proteins were still
expressed at levels equivalent to those seen in the wild-type C2
myotubes, including the MuSK receptor tyrosine kinase (Fig.
1A) and -sarcoglycan (Lindenbaum, Montanaro, and
Carbonetto, unpublished observations). In addition, we found that surface levels of AChR were similar in all the lines (Fig. 1A,B) and that all lines formed
extensive myotubes, suggesting that the -DG antisense lines reached
a similar level of differentiation to that of wild-type C2 myotubes. In
a separate study, we show that the decreased levels of -DG in these
lines reduce the amount of cell surface-associated laminin (Lindenbaum,
Montanaro, and Carbonetto, unpublished observations).
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Figure 1.
Acetylcholine receptor clustering in -DG
antisense cell lines. A, B,
-Dystroglycan antisense cell lines were assayed for the expression
of -DG, AChR -subunit, and MuSK. Immunoblots for -DG with
antibody IIH6 indicate that two antisense lines (IIF and
IIE) have a dramatic reduction in -DG levels compared
with that in wild-type C2 cells. Immunoblots of MuSK and
of AChR -subunit show that their levels are equivalent in all cell
lines assayed. Because the AChR was isolated after the labeling of live
cells with biotinylated -bungarotoxin, the amount of
-subunit reflects surface levels of AChR. C,
Agrin-induced AChR clustering was compared in wild-type and -DG
antisense cell lines by treating myotubes overnight with 1 nM neural agrin (C-Ag 4,8). AChR clusters visualized with
rhodamine -bungarotoxin were counted for each line and are expressed
as a percentage of the C2 level. The number of AChR
clusters was decreased in the antisense lines with reduced -DG
(IIF and IIE) but was unaffected in the
lines with near normal -DG levels (9B,
10C, and 11A). Black bars
represent spontaneous clusters; gray bars are
agrin-treated. Values shown represent the average of two to three
experiments.
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To test whether -DG is required for agrin-induced AChR clustering,
we initially treated the different muscle cell lines with neural agrin
at saturating concentrations (C-Ag 4,8 at 1 nM for 16 hr)
(Ferns et al., 1996) and assayed AChR cluster numbers. We found that
the two lines that have reduced levels of -DG (IIF and IIE) also
have significantly fewer agrin-induced AChR clusters compared with that
in wild-type C2 cells (Fig. 1C). In contrast, muscle lines
expressing near wild-type levels of -DG (9B, 10C, and 11A) showed
normal levels of AChR clustering. The level of agrin-induced receptor
clustering also appeared to correlate with the remaining level of
-DG in the two lines and did not correspond to AChR levels that were
approximately equivalent in all lines. Thus, these findings suggest
that -DG is required for some phase of agrin-induced AChR
clustering.
To define further the role of -DG, we compared levels of AChR
clustering in the IIF, IIE, and wild-type C2 lines in relation to agrin
concentration. We found that the -DG antisense lines have a reduced
ability to form both spontaneous and agrin-induced AChR clusters
compared with wild-type C2 myotubes. In control cultures (not treated
with agrin), C2 myotubes had an average of 923 spontaneous AChR
clusters/mm2, whereas IIE and IIF myotubes had only
187 and 34 clusters, respectively (p < 0.0001, ANOVA and Fisher's test). Similarly, we found that the number of
agrin-induced clusters is decreased in both IIF and IIE cells relative
to that in C2 cells at all agrin concentrations tested (Fig.
2). In particular, when agrin is applied
in saturating concentrations ( 1 nM), the peak cluster
number is 2708, 1018, and 566 per mm2 for C2, IIF,
and IIE cells, respectively. After correcting for the differing levels
of spontaneous clusters, we find that IIF and IIE cells form only 46 and 30% of the wild-type number of clusters, respectively
(p < 0.0001) (Fig. 2). The ability of the antisense cells to cluster AChRs is thus approximately proportional to
their expression level of -DG, that is, 40% for IIF and 10% for
IIE cells.
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Figure 2.
Agrin-induced AChR clustering is decreased in cell
lines with reduced -DG. The level of AChR clustering was determined
for C2, IIF, and IIE myotubes after overnight incubation with neural
agrin, at concentrations ranging from 1 pM to 100 nM. The IIF and IIE myotubes with reduced -DG show a
significant decrease in the number of spontaneous and agrin-induced
AChR clusters per myotube mm2 compared with that in
wild-type C2 cells (p < 0.05 at all
concentrations of agrin, by ANOVA and Fisher's test). All three muscle
lines respond to agrin at similar concentrations; however the IIF and
IIE cells form 2.7- and 4.8-fold fewer AChR clusters, respectively, at
saturating agrin concentrations than do wild-type cells. Each value
represents the mean ± SD for two to five experiments.
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Although spontaneous and peak cluster numbers differ between the three
lines, the agrin dose-response curves for IIE and IIF are not shifted
toward significantly higher effective concentrations relative to that
for wild-type C2 myotubes (Fig. 2). Indeed, the agrin concentration
required to induce half-maximal AChR clustering in each line is 20 pM for C2 and 30 pM for the IIF and IIE muscle lines. The -DG antisense lines therefore respond to agrin at approximately equivalent concentrations to that of wild-type C2 cells
but form fewer clusters. In addition, we find in preliminary experiments that these remaining clusters are the same size as those in
wild-type C2 myotubes and that they have -DG associated with them.
Together, these results suggest that -DG is required for
agrin-induced AChR clustering but that it may not act in the initial,
signaling phase of the pathway. In the following experiments, we
test further whether this is the case.
Agrin-induced phosphorylation of MuSK and AChR -subunit in C2
cell variants with reduced -dystroglycan
Agrin has been shown to induce AChR clustering by signaling
through the MuSK receptor tyrosine kinase (DeChiara et al., 1996; Glass
et al., 1996) and triggers a variety of intracellular events including
tyrosine phosphorylation of the AChR -subunit (Wallace et al., 1991;
Meier et al., 1995; Ferns et al., 1996). To test more directly whether
-DG might be involved in agrin signaling, we compared agrin-induced
MuSK and AChR phosphorylation in C2 and the -DG-deficient lines. The
C2 and IIE myotube cultures were treated with equal concentrations of
recombinant neural agrin (C-Ag 4,8; 1 nM) (Ferns et al.,
1993), and immunoprecipitates of MuSK or AChR were electrophoresed and
immunoblotted with an anti-phosphotyrosine antibody (mAb 4G10) (Fig.
3). Reprobes of the immunoblots with
antibodies to MuSK or to AChR subunits showed that equal levels of each
were present in the C2 and IIE samples (data not shown). A quantitative
comparison of the levels of MuSK and AChR -subunit phosphorylation
in C2 and IIE myotubes is shown in Figure
4 (n = 3-7). For these
experiments, densitometric analysis was always performed on exposures
that were within the linear range of the film (see Materials and
Methods). Comparing wild-type and IIE myotubes treated with agrin for
15 min to 3 hr, we find that on average the level of agrin-induced
phosphorylation of MuSK was comparable, with a reduction being evident
in IIE myotubes only at 15 min (p < 0.01, Student's t test; n = 7) (Figs.
3A, 4A). Thereafter, there was a trend
toward lower levels of MuSK phosphorylation in IIE cells, but these
differences were not statistically significant. Moreover, levels of
MuSK phosphorylation were not decreased in the IIF cell line at any
time point (data not shown). In all cell lines, agrin-induced
phosphorylation of MuSK began to decline after 1 hr and was only
one-third of the peak level by 3 hr (Figs. 3A,
4A). A similar, transient decrease in the level of
MuSK phosphorylation in IIE cells compared with that in wild-type C2
was also found using a lower agrin concentration of 200 pM (data not shown).
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Figure 3.
Agrin signaling is not significantly inhibited in
muscle lines with decreased -DG. Representative immunoblots
comparing agrin-induced tyrosine phosphorylation of MuSK and the AChR
in C2 and IIE myotubes after incubation with 1 nM agrin for
the indicated times are shown. MuSK and the AChR were isolated from
cell extracts by immunoprecipitation and then immunoblotted with the
anti-phosphotyrosine antibody 4G10. A, The level of
agrin-induced tyrosine phosphorylation of MuSK is similar in C2 and IIE
myotubes. B, Agrin-induced tyrosine phosphorylation of
the AChR -subunit is also equivalent in C2 and IIE cells. Reprobes
of these immunoblots with either anti-MuSK or anti--subunit
antibodies indicated that equal amounts of each protein were present in
all samples from the two lines (data not shown). Molecular mass markers
(kDa) are indicated on both the left and
right.
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Figure 4.
Quantitative comparison of MuSK and AChR
-subunit phosphorylation in C2 and IIE myotubes. A,
Agrin-induced phosphorylation of MuSK was quantified by densitometric
analysis of immunoblots and is shown averaged for five experiments. The
level of MuSK phosphorylation is decreased in IIE compared with C2
myotubes by ~60% at 15 min (p < 0.01, paired Student's t test). At longer time points, slight
differences are apparent, but they are not statistically significant.
B, Levels of AChR -subunit tyrosine phosphorylation
are shown averaged for three experiments. No difference in
agrin-induced phosphorylation of the -subunit is evident between C2
and IIE myotubes at any time point. Results represent the mean ± SEM. C2 cells are represented by shaded bars, and IIE
cells are represented by black bars.
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When we compared agrin-induced tyrosine phosphorylation of the AChR
-subunit, no significant difference was noted between C2 and IIE
myotubes at any time point (Figs. 3B, 4B;
n = 3). Thus, the transiently decreased level of MuSK
phosphorylation in IIE myotubes compared with that in C2 myotubes was
not reflected in the level of AChR tyrosine phosphorylation (Figs.
3B, 4B). Together, these findings show
that although there is a transient reduction in agrin-induced MuSK
phosphorylation in the IIE -DG-deficient myotubes, this does not
significantly affect downstream signaling events like phosphorylation
of the AChR. It seems unlikely, therefore, that the approximately
threefold decrease in the level of AChR clusters in IIE myotubes
compared with that in wild-type myotubes is caused by a defect in
initial agrin signaling.
Agrin-induced phosphorylation of MuSK does not require domains that
bind -DG or heparin
To investigate the possible role of -DG further, we tested
whether agrin--DG binding is required for agrin signaling in wild-type C2 myotubes. Agrin has been shown previously to bind to
-DG through defined domains in the C terminal of agrin (Gesemann et
al., 1996; Hopf and Hoch, 1996). We therefore tested whether -DG
acts as a coreceptor by deleting the primary -DG-binding domains
from agrin and then by assaying the ability of these agrin fragments to
induce tyrosine phosphorylation of the MuSK receptor. We first compared
agrin fragments that either contained or lacked the G1 domain that is
primarily responsible for binding to -DG (Fig.
5A) (Hopf and Hoch, 1996).
Neural agrin fragments that contained the G1 domain (C-Ag + G1 4,8 or
0,8) induced a rapid and dramatic increase in MuSK tyrosine
phosphorylation (Fig. 5B). These agrin forms were active in
inducing MuSK phosphorylation at concentrations of ~50 pM
or greater. When we then examined agrin forms in which the G1 domain
had been deleted (G1 4,8 or 0,8), we found that they were similarly
active in inducing MuSK phosphorylation. On average (n = 4), there was no quantitative difference in the concentrations at
which agrin forms plus and minus the first G1 domain were active. We
conclude that the deletion of the G1 domain has no effect on the
ability of agrin to induce MuSK phosphorylation (Fig. 5B), and an agrin--dystroglycan interaction via this domain is unlikely to be necessary in agrin signaling.
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Figure 5.
Agrin-induced phosphorylation of MuSK correlates
with heparin but not -DG binding. A, Schematic
depicting the agrin constructs used to test whether agrin-induced
phosphorylation of MuSK is dependent on the first G domain that binds
-DG with high affinity (horizontal solid line) or on
the 4 amino acid-splicing insert that confers binding to heparin
(horizontal solid line). The region responsible for
clustering activity (horizontal solid line) and its
dependence on the splicing inserts at sites x, y,
and z are also shown. Dashed lines denote regions
that make minor contributions to -DG binding or to AChR clustering
activity. Neural agrin forms contain the 4 and 8 amino acid inserts
(denoted 4,8), whereas muscle agrin forms lack both these inserts
(0,0). The domains of agrin are represented as follows:
ST, serine/threonine-rich domain; E1-E4,
EGF-like domains; and G1-G3, laminin globular domains.
B, Phosphotyrosine immunoblot of MuSK isolated from C2
myotubes that were incubated with the indicated agrin construct for 15 min at 1 nM. The presence or absence of the first G1 domain
has no effect on the ability of agrin to induce MuSK phosphorylation
(compare the +G1 and G1 lanes for each isoform). In
contrast, the 4 amino acid insert does affect the level of MuSK
phosphorylation (compare the 4,8 vs 0,8 lanes) but is
not absolutely required. The letter c indicates the
control with no agrin treatment, and 0,0 represents the muscle
isoform. Molecular mass markers are indicated to the
left.
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We also compared the activity of splice variants of agrin that contain
or lack a 4 amino acid (aa) insert (Fig. 5A) that has been
shown to confer agrin binding to heparin (Campanelli et al., 1996;
Gesemann et al., 1996; O'Toole et al., 1996). Potentially this
heparin-binding domain could mediate an interaction with -DG or with
some other proteoglycan coreceptor. We find that agrin isoforms that
contain the 4 aa insert (C-Ag 4,8) induce, on average
(n = 3), a fivefold higher level of MuSK
phosphorylation than do forms lacking this insert (C-Ag 0,8) (Fig.
5B). Absence of the 4 aa insert also resulted in decreased
activity in the truncated fragments that lack the G1 domain (G1 4,8 compared with G1 0,8). The presence of the 4 aa insert thus increases the ability of agrin to activate MuSK, possibly indicative of an
interaction with a heparan sulfate proteoglycan in the agrin-signaling complex.
Competition of agrin binding to -dystroglycan does not block
agrin-induced phosphorylation of MuSK
Although agrin signaling does not seem to involve an interaction
with -DG via the G1 domain, agrin could interact weakly with -DG
via other binding domains. We therefore tested whether treatments that
compete with neural agrin binding to -DG might also inhibit agrin
signaling. Muscle agrin has been shown to compete effectively with
neural agrin binding to -DG immobilized on nitrocellulose membranes
and, in fact, has a 10-fold greater affinity for -DG than does
neural agrin (Sugiyama et al., 1994; Campanelli et al., 1996; Gesemann
et al., 1996). Myotubes treated with neural agrin in the presence of a
100-fold excess of muscle agrin (C-Ag 0,0 or C-Ag 4,0 at 5 nM) showed no decrease in agrin-induced phosphorylation of
MuSK (Fig. 6A).
Although this excess of muscle agrin may not fully saturate all
-DG-binding sites (Bowen et al., 1996), we did not use higher
amounts because muscle agrin can be active at very high concentrations
(50 nM) (Ferns et al., 1993). The anti--DG antibody
IIH6 also blocks neural agrin binding to -DG immobilized on
immunoblots (Gee et al., 1994; Sugiyama et al., 1994) and expressed on
cells (Campanelli et al., 1994). Treatment with the IIH6 antibody
failed to block agrin-induced phosphorylation of MuSK in myotubes
treated with neural agrin either at 1 nM (Fig. 6B) or 200 pM (data not shown). Thus,
several treatments that specifically block the interaction of agrin
with -dystroglycan fail to inhibit agrin-induced phosphorylation of
MuSK.
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Figure 6.
Competition of agrin binding to -DG does not
inhibit MuSK phosphorylation. C2 myotube cultures were incubated with
neural agrin for 15 min in the presence or absence of several reagents
that block agrin binding to -DG. Neural agrin-induced
phosphorylation of MuSK was then assayed by immunoprecipitating MuSK
from cell extracts and immunoblotting with anti-phosphotyrosine
antibody 4G10. A, Competition with muscle agrin. C2
myotubes were incubated with no agrin (c), muscle
agrin alone (0,0) or (4,0), neural agrin alone (4,8), or neural agrin
in the presence of a 100-fold excess of either form of muscle agrin.
Neural agrin-induced phosphorylation of MuSK was not decreased in the
presence of an excess of muscle agrin (0,0) or (4,0). Pretreatment of
cultures with muscle agrin for 30 min (asterisk) before
neural agrin addition also failed to inhibit phosphorylation of MuSK.
B, Competition with the -DG-specific monoclonal
antibody IIH6. C2 myotubes were incubated with no agrin
(c), neural agrin alone (4,8), neural agrin in
the presence of IIH6 ascites at the indicated dilutions, or IIH6
antibody alone. The anti--DG antibody did not inhibit neural
agrin-induced phosphorylation of MuSK. C, Competition
with heparin. C2 myotubes were treated with no agrin
(c) or with the 4,8 or 0,8 agrin isoform in the
presence or absence of 100 µg/ml heparin
(H). Heparin inhibits MuSK phosphorylation
induced by the 4,8 agrin isoform, but not the 0,8 isoform, and is
therefore dependent on the presence of the 4 amino acid splice insert.
Molecular mass markers are indicated to the left.
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|
Finally, heparin has also been shown to block the agrin--DG
interaction (Campanelli et al., 1994; Gee et al., 1994; Sugiyama et
al., 1994; O'Toole et al., 1996) and to block agrin-induced AChR
clustering (Wallace, 1990). We find that agrin-induced phosphorylation of MuSK is blocked by soluble heparin in a dose-dependent manner. Moreover, the inhibition with heparin was only observed for agrin forms
that contain the 4 aa insert (Fig. 6C). Thus, C-Ag
4,8-induced MuSK phosphorylation was significantly decreased by heparin
at 100 µg/ml, but C-Ag 0,8-induced phosphorylation was unaffected. Heparin presumably inhibits agrin activity, therefore, by binding only
to the G2 domain of agrin forms that contain the 4 aa insert. It is
unclear whether the blocking effect of heparin is caused by competition
of a heparin-like interaction with -DG or another unidentified proteoglycan, or merely by steric hindrance of binding to
MuSK.
| |
DISCUSSION |
We have investigated the potential role of -dystroglycan in the
formation of AChR clusters. We find that both spontaneous and
agrin-induced AChR clustering are decreased in -DG-deficient muscle
cell lines compared with that in wild-type cells. This clustering
defect seems to be attributable specifically to the decrease in -DG
because levels of other proteins like MuSK and the AChR were equivalent
in all the muscle lines (Fig. 1A,B)
and the defects in clustering correlated with the residual level of -DG (Figs. 1C, 2). These observations suggest that -DG
is required for AChR clustering, and as a result we have examined
whether -DG acts in agrin signaling or elsewhere in the clustering
pathway.
-DG is not a coreceptor for MuSK
Several findings suggest that a coreceptor, denoted MASC (for
myotube-associated specificity component), is necessary for the
activation of the MuSK receptor tyrosine kinase by agrin (Glass et al.,
1996). -Dystroglycan is an obvious candidate for MASC, because
-DG has been shown to bind agrin (Campanelli et al., 1994) and to
cocluster together with the AChR (Campanelli et al., 1994; Gee et al.,
1994; Sugiyama et al., 1994; Cohen et al., 1995). Several of our
findings suggest, however, that -DG is not required for agrin-MuSK
signaling. First, we found that agrin acted at equivalent
concentrations in wild-type and -DG-deficient lines, indicating that
there is not a shift in the dose-response curve for agrin in the
-DG-deficient lines. Secondly, we found that agrin-MuSK signaling
was not substantially inhibited in the -DG-deficient lines compared
with that in wild-type cells. Although agrin-induced phosphorylation of
MuSK was slightly reduced in IIE compared with that in wild-type C2
cells (but only significantly so at 15 min), the downstream tyrosine
phosphorylation of the AChR -subunit (Glass et al., 1997) was
equivalent at all time points assayed. The levels of MuSK and AChR
-subunit phosphorylation are therefore equalized well in advance of
AChR clustering, which was assayed in these experiments at 18 hr. Thus,
there does not seem to be a major or long-lasting defect in agrin
signaling in the -DG-deficient lines that might account for their
dramatically decreased level of AChR clustering. We cannot entirely
discount a signaling role for -DG, however, because the residual
-DG in the antisense lines may be sufficient for function as a
coreceptor.
The findings from several, independent experiments on wild-type C2
cells provide further evidence against a signaling role for -DG.
Foremost, we find that the domain of agrin that binds -DG is not
required for agrin-MuSK signaling. When we deleted the primary
-DG-binding domain in agrin (laminin G1 domain; Hopf and Hoch,
1996), we found absolutely no effect on the ability of agrin to induce
tyrosine phosphorylation of MuSK (Fig. 5). This result is consistent
with previous studies that have shown that the ability of agrin
fragments to induce AChR clustering is not significantly affected by
the presence of the first G1 domain (Hoch et al., 1994; Gesemann et
al., 1995).
In addition, we found that treatments that are known to block
agrin--dystroglycan binding also failed to inhibit agrin signaling via MuSK. For example, muscle agrin binds -DG with 10-fold greater affinity than does neural agrin (Sugiyama et al., 1994; Gesemann et
al., 1996); yet excess muscle agrin failed to inhibit neural agrin-induced phosphorylation of MuSK, consistent with earlier work
showing that it does not block AChR clustering (Bowen et al., 1996).
Similarly, inhibition of agrin binding to -DG with the anti--DG
antibody IIH6 (Campanelli et al., 1994; Gee et al., 1994; Sugiyama et
al., 1994) had no effect on the activation of MuSK by agrin (Fig.
6B). The reported disruption of agrin-induced AChR
clustering by this anti--DG antibody (Campanelli et al., 1994; Gee
et al., 1994) thus does not stem from a block of signaling (see below).
These several independent lines of evidence argue strongly against
-DG acting as a coreceptor that is required for agrin-MuSK
signaling.
Although specific inhibitors of agrin--DG binding did not affect
signaling, we did observe a block of MuSK phosphorylation using
heparin. Heparin has been shown previously to inhibit nerve- and
agrin-induced receptor clustering (Hirano and Kidokoro, 1989; Wallace,
1990) and to block the agrin--DG interaction (Campanelli et al.,
1994; Gee et al., 1994; Sugiyama et al., 1994; O'Toole et al., 1996).
We found that heparin effectively inhibits agrin-induced phosphorylation of MuSK, but only for agrin forms containing the 4 amino acid splice insert in the second G domain (Fig. 6C).
Because this 4 aa insert has been shown to confer heparin binding
(Campanelli et al., 1996; Gesemann et al., 1996; O'Toole et al.,
1996), this indicates that heparin is acting by binding directly to
agrin rather than to some cell surface component. It is unclear whether this reflects the block of the interaction of agrin with a proteoglycan or is merely attributable to steric hindrance of agrin binding to MuSK.
We also found that agrin isoforms that contained the 4 aa insert
induced a fivefold higher level of MuSK phosphorylation than did forms
lacking this insert (Figs. 5B, 6C), consistent with earlier studies assaying AChR-clustering activity of different agrin isoforms (Ferns et al., 1993). These results allow the
possibility that agrin binding to a proteoglycan plays a small role in
agrin signaling, but one that is not absolutely required. The blocking experiments, discussed above, make it unlikely that -DG is involved in this putative heparin-binding interaction with agrin. Moreover, there is currently some debate as to whether -DG is in fact a proteoglycan, and some results suggest it may instead have a mucin-like structure (Smalheiser and Kim, 1995; Chiba et al., 1997).
The role of -DG in AChR clustering
We have found that muscle lines with reduced -DG exhibit
seemingly normal signaling through the MuSK receptor and yet form significantly fewer AChR clusters than do wild-type cells, suggesting that -DG is required at some other stage of the clustering pathway. Given that the activation of the MuSK receptor by agrin is thought to
be the initial event and occurs very rapidly, it seems most likely that
-DG acts downstream in the pathway, such as in the consolidation of
microclusters or in the growth and maintenance of AChR aggregates.
Consistent with this notion, previous studies have shown that treatment
with an anti--DG antibody disrupts both spontaneous and
agrin-induced AChR clustering (Campanelli et al., 1994; Gee et al.,
1994; Cohen et al., 1995; but see Sugiyama et al., 1994). In
particular, Campanelli et al. (1994) reported that the anti--DG
antibodies appeared to block the consolidation of microclusters into
larger aggregates, suggesting that -DG might act at a stage of
clustering subsequent to initial agrin signaling. In addition, heparin
has recently been shown to partially inhibit some downstream step in
AChR clustering (Hopf and Hoch, 1997), and because heparin binds to the
protein core of -DG (Bowe and Fallon, 1996), it may be acting
by blocking -DG function.
What kind of role might -DG play in AChR cluster formation? One
possibility is that it forms part of the scaffold that is postulated to
immobilize the AChR and other synaptic proteins in the postsynaptic
membrane (Froehner, 1991; Carbonetto and Lindenbaum, 1995; Apel et al.,
1997). Indeed, -DG becomes coclustered with the AChR at developing
synapses (Cohen et al., 1995) by a rapsyn-dependent mechanism (Apel et
al., 1995). The recruitment of -DG to this postsynaptic scaffold
might then help consolidate or stabilize developing clusters (Fig.
7), as -DG is known to interact with laminin (Ibraghimov-Beskrovnaya et al., 1992; Gee et al., 1993) and
both nerve and muscle agrin (Sugiyama et al., 1994) in the synaptic
basal lamina. In addition, the cytoplasmic tail of -dystroglycan has
been found to bind to dystrophin/utrophin that is selectively localized
in the subsynaptic cytoskeleton (Suzuki et al., 1994).
View larger version (20K):
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|
Figure 7.
Model showing where -DG might act in
agrin-induced AChR clustering. Our findings suggest that -DG
probably does not act as a coreceptor involved in initial agrin
signaling via the MuSK receptor complex (1). Rather, -DG might
function downstream of MuSK activation, in the initial formation of
AChR aggregates (2) or in the consolidation and maintenance of clusters
(3). Consistent with this idea, -DG becomes coclustered with MuSK
and the AChR and could be required for the further development of this
postsynaptic scaffold. For example, -DG might form part of the
scaffold that immobilizes and stabilizes AChR aggregates, possibly via
its links to the extracellular matrix and the cytoskeleton (3).
Rap, Rapsyn; , , - and -dystroglycan; and
P, tyrosine phosphorylation.
|
|
A potential function of -DG in this scaffold may be to help
immobilize the AChR (Fig. 7). Consistent with this, several studies have shown that exogenous laminin can induce aggregation of -DG (Cohen et al., 1997) and increase the number, size, and density of AChR
clusters in cultured myotubes (Sugiyama et al., 1997; Montanaro et al.,
1998). Laminin-induced aggregation of the AChR is not accompanied by
the phosphorylation of MuSK (Sugiyama et al., 1997) and is apparently
mediated by aggregation of -DG (Montanaro et al., 1998), indicating
that -DG can play a role in AChR immobilization. Laminin alone
probably does not act to initiate clustering of AChRs in
vivo, however, because there is a complete absence of clusters in
MuSK-deficient mice that presumably express normal levels of laminin
(DeChiara et al., 1996). The laminin or agrin interaction with -DG
may instead act downstream of agrin signaling via MuSK, in the
subsequent development of clusters.
In summary, we find that -DG is required for agrin-induced AChR
clustering. Our results suggest that -DG is unlikely to be a
required coreceptor for agrin signaling via MuSK but rather that it may
have a role downstream of MuSK activation, such as in the consolidation
and maintenance of AChR clusters.
| |
FOOTNOTES |
Received March 18, 1998; revised May 20, 1998; accepted May 29, 1998.
This work was supported by Canadian Medical Research Council
Grants MT-13237 to M.F. and MA-9000 and MA-10182 to S.C., the Canadian
Network Centres of Excellence, and the Muscular Dystrophy Association
of the United States. C.J. and F.M. are supported by studentships from
the Canadian Neuroscience Network Centres of Excellence. We
would like to thank Dr. Werner Hoch for his generous gift of the
truncated-agrin fragments.
C. Jacobson and F. Montanaro are equal coauthors of this manuscript.
Correspondence should be addressed to Dr. M. Ferns, Montreal General
Hospital, Research Institute, Rs1-133, 1650 Cedar Avenue, Montreal, Quebec, H3G 1A4 Canada.
| |
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Top
Abstract
Introduction
