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The Journal of Neuroscience, August 1, 1999, 19(15):6405-6416
Roles of Rapsyn and Agrin in Interaction of Postsynaptic Proteins
with Acetylcholine Receptors
Christian
Fuhrer1,
Medha
Gautam2,
Janice E.
Sugiyama1, and
Zach W.
Hall1
1 Section on Synaptic Mechanisms, Laboratory of
Cellular and Molecular Regulation, National Institute of Mental Health,
National Institutes of Health, Bethesda, Maryland 20892, and
2 Department of Molecular Biology and Pharmacology,
Washington University Medical School, St. Louis, Missouri 63110
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ABSTRACT |
At the neuromuscular junction, aggregates of acetylcholine
receptors (AChRs) are anchored in the muscle membrane by association with rapsyn and other postsynaptic proteins. We have investigated the
interactions between the AChR and these proteins in cultured C2
myotubes before and after treatment with agrin, a nerve-derived protein
that induces AChRs to cluster. When AChRs were isolated from detergent
extracts of untreated C2 myotubes, they were associated with rapsyn
and, to a lesser degree, with utrophin, 
-dystroglycan, MuSK,
and src-related kinases, but not with syntrophin. Treatment with agrin
increased the association of AChRs with MuSK, a receptor tyrosine
kinase that forms part of the agrin receptor complex, without affecting
other interactions. Analysis of rapsyn-deficient myotubes, which do not
form protein clusters in response to agrin, revealed that rapsyn is
required for association of the AChR with utrophin and
-dystroglycan, and for the agrin-induced increase in association
with MuSK, but not for constitutive interactions with MuSK and
src-related kinases. In rapsyn 
/ myotubes, agrin caused normal
tyrosine phosphorylation of AChR-associated and total MuSK, whereas
phosphorylation of the AChR subunit, both constitutive and
agrin-induced, was strongly reduced. These results show first that
aneural myotubes contain preassembled AChR protein complexes that may
function in the assembly of the postsynaptic apparatus, and second that
rapsyn, in addition to its role in AChR phosphorylation, mediates
selected protein interactions with the AChR and serves as a link
between the AChR and the dystrophin/utrophin glycoprotein complex.
Key words:
synaptogenesis; acetylcholine receptor; agrin; rapsyn; neuromuscular junction; tyrosine phosphorylation; protein
interactions
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INTRODUCTION |
The accumulation of acetylcholine
receptors (AChRs) is a characteristic feature of the postsynaptic
membrane at the neuromuscular junction and is believed to arise through
interactions with the cytoskeleton (Froehner, 1991
; Hall and Sanes,
1993). Little information is available, however, about the interactions
of the AChR with other muscle proteins and how they change during
development (Colledge and Froehner, 1998a).
The specialized postsynaptic membrane is formed through the action of
agrin, a neurally released protein necessary for synapse formation
in vivo (McMahan, 1990; Gautam et al., 1996). When added to
cultured myotubes in vitro or supplied by injected myofibers in vivo, agrin also causes aggregation of AChRs,
acetylcholinesterase, and other synaptic proteins (Wallace, 1989; Ferns
et al., 1992; Reist et al., 1992; Cohen et al., 1997; Meier et al.,
1997).
Little is known about the signaling pathway of agrin, but the receptor
tyrosine kinase MuSK (Jennings et al., 1993; Valenzuela et al., 1995)
appears to be a key component of the agrin receptor (Glass et al.,
1996). MuSK is activated by agrin, and mice lacking MuSK are similar to
agrin-deficient animals in that they lack AChR clusters (DeChiara et
al., 1996). Agrin also induces tyrosine phosphorylation of the subunit of the AChR (Wallace et al., 1991; Meier et al., 1995; Ferns et
al., 1996), a modification whose significance for AChR clustering is
unclear at present (Meyer and Wallace, 1998). This phosphorylation
appears not to occur through the direct action of MuSK (Fuhrer et al.,
1997) but rather through one or several downstream kinases, possibly of
the src family (Swope and Huganir, 1993, 1994; Fuhrer and Hall,
1996).
One protein closely associated with AChRs is rapsyn, a 43 kDa
peripheral membrane protein (Burden et al., 1983; LaRochelle and
Froehner, 1986). Rapsyn is essential for AChR clustering: rapsyn-negative mice lack AChR clusters and their cultured myotubes fail to respond to agrin (Gautam et al., 1995), and rapsyn causes AChR
aggregation upon coexpression in heterologous cells (Froehner et al.,
1990; W. P. Phillips et al., 1991). When coexpressed in QT-6
fibroblasts, rapsyn is also able to aggregate MuSK and dystroglycan and
to activate MuSK (Apel et al., 1995, 1997; Gillespie et al., 1996). On
the basis of such indirect evidence, rapsyn has been proposed to link
the AChR to the dystrophin/utrophin glycoprotein complex (Apel and
Merlie, 1995); rapsyn has also been postulated to link the AChR to MuSK
and to mediate interaction of the AChR with src-family kinases (Apel et
al., 1997).
Although considerable evidence, based largely on expression in QT-6
fibroblasts, supports either the direct or indirect association of the
AChR with other synaptic proteins, direct biochemical evidence for
these interactions is lacking. To investigate these associations and
the effect of agrin on them, we have isolated the AChR from extracts of
C2 and rapsyn-negative myotubes and have used immunological methods to
detect proteins that are specifically associated with it. Our data
suggest that aneural myotubes contain preassembled AChR complexes, that
agrin increases selected protein interactions, and that rapsyn mediates
some but not all associations of other proteins with the AChR.
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MATERIALS AND METHODS |
Cell culture. Cell culture reagents were purchased
from Life Technologies (Gaithersburg, MD). COS and C2C12 muscle cells
were grown at 37°C in 8% CO2. COS cells were maintained
in DMEM with 4.5 gm/l D-glucose containing 10%
fetal bovine serum, 2 mM glutamine, and
penicillin/streptomycin. C2C12 mouse muscle cells were grown as
myoblasts on 10 or 15 cm tissue culture dishes (Nunc, Naperville, IL)
in DMEM supplemented with 20% fetal bovine serum, 0.5% chick embryo
extract, 2 mM glutamine, and penicillin/streptomycin. Cells were shifted to fusion medium containing DMEM, 5% horse serum, and 2 mM glutamine after reaching 90% confluence. Formation of myotubes was generally evident after 1 d in fusion medium.
Cultures were harvested for experiments on day 2 in fusion medium, by
which time contracting myotubes were detectable. Rapsyn / (clones 11-4 and 11-7) and rapsyn wild-type (clone 12-10) myoblasts were grown
in the same basic medium as C2 cells, with an additional 4 U/ml
-interferon (-IFN). These cells were grown on dishes coated with
0.2% gelatin (Sigma, St. Louis, MO) and maintained at 33°C with 5%
CO2. To induce fusion, confluent cultures were shifted to
C2 fusion medium and 37°C, 5% CO2; the medium was
replaced every 2 d. Myotubes started to form after ~1.5 d and
were used for experiments after 3 or 4 d in fusion medium.
Expression of agrin constructs. The C-terminal half of agrin
isoforms was expressed in transfected COS cells to obtain soluble forms
of agrin. Constructs encoding the most active, neural-specific isoform
(C-Ag12,4,8) or the predominant muscle isoform
(C-Ag12,0,0) (Ferns et al., 1993) were expressed in
COS cells using an adenovirus-mediated DEAE-dextran method of
transfection (Forsayeth and Garcia, 1994). Because these two splice
isoforms of agrin are most prominent and widely used, we refer to
neural agrin as "4,8" and muscle agrin as "0,0" (e.g., see
Figs. 2, 6). After transfection of agrin constructs, the medium was
collected and replaced each day for 3 d. The concentration of
agrin in the medium was determined by immunoblotting using an
agrin-specific antiserum and purified agrin of known concentration as a
standard, as described previously (Fuhrer et al., 1997).
Generation of rapsyn / cell lines. To isolate mutant
muscle cell lines, we made use of a transgenic mouse bearing a
-IFN-inducible, temperature-sensitive T-antigen transgene (Jat et
al., 1991) (Immortomouse, Charles River Laboratories, Wilmington, MA).
Cells from different tissues of this mouse, including muscle, can be
maintained for several passages in an undifferentiated state under
permissive conditions (33°C, with -IFN) in vitro
(Morgan et al., 1994). Mice heterozygous for both rapsyn and the
immortalizing transgene (double heterozygotes) were generated in
crosses between mice bearing the transgene and rapsyn +/ mice. Double
heterozygotes were then crossed with rapsyn +/ mice to generate
rapsyn mutants and controls bearing the transgene. Litters from such
crosses were dissected at embryonic day 18 (E18), and muscles from the forelimbs and hindlimbs of each pup were trypsinized. The resulting suspension of cells was diluted and plated on 3 cm plates and maintained at 33°C in F10 medium containing 20% fetal bovine serum, 3% chick embryo extract, and 4 U/ml -IFN. Pups from each litter were genotyped by PCR of tail DNA within 2 d of initial plating; cell cultures from mutants and controls that tested positive for the
transgene were then trypsinized, counted, and replated at clonal
density. Colonies were allowed to develop over the next 10 d, and
each colony was expanded. We picked three clonal cell lines for further
studies: 11-4 and 11-7 are rapsyn / lines and 12-10 is a wild-type
(rapsyn +/+) cell line. Analysis of 11-4 versus 11-7 cells revealed no
clonal variability in the association of AChRs with postsynaptic
proteins (data not shown). We used 11-7 for most experiments, however,
because they routinely formed myotubes more efficiently than 11-4.
Antibodies. Affinity-purified rabbit polyclonal antibodies
against rapsyn, designated 5943 (W. D. Phillips et al., 1991), were a generous gift from the late Dr. J. P. Merlie and from Dr. J. R. Sanes (Washington University, St. Louis, MO). In immunoblot experiments with cultured muscle cells, the antibody recognized a major
protein of ~43 kDa that was specifically absent from rapsyn /
myotubes and thus represents rapsyn (see Fig. 3). A rabbit polyclonal
antiserum, 
DRP, reactive with utrophin (Ohlendieck et al., 1991),
was a gift from Dr. K. P. Campbell (University of Iowa, Iowa City,
IA). In immunoblots made with extracts from cultured myotubes, this
antibody labeled a single protein band of ~400 kDa, whose mobility
was very similar to utrophin found in purified sarcolemma of mouse
skeletal muscle (Ohlendieck et al., 1991). The rabbit antiserum
recognizing -dystroglycan was made against a peptide corresponding
to the C-terminal 20 amino acid of mouse -dystroglycan and was
produced commercially by Research Genetics (Huntsville, AL).
Immunoblotting with preimmune serum and immunoprecipitations with an
excess of free -dystroglycan immunizing peptide established the
specificity of the antibody (J. E. Sugiyama and Z. W. Hall,
unpublished observations). In all cases, -dystroglycan was detected
as a band of ~46 kDa, similar to its molecular weight in
dystrophin/utrophin glycoprotein complexes purified from rabbit
skeletal muscle (Ibraghimov-Beskrovnaya et al., 1992). In addition, a
mouse monoclonal antibody against -dystroglycan, purchased from
Novocastra Labs (Burlingame, CA), reacted with a protein of the same
molecular weight in immunoblots made from C2 myotubes (data not shown),
showing that our antibody indeed recognizes -dystroglycan in
cultured muscle cells. mAb124, a rat monoclonal antibody against the
AChR subunit, was a kind gift from Dr. J. Lindstrom (University of
Pennsylvania, Philadelphia, PA). The mouse monoclonal antiserum mAb
88B, reactive with the AChR and 
subunits, and mAb SYN1351
(Froehner et al., 1987), a mouse monoclonal antibody reactive with all
syntrophin isoforms (1, 1, and 2) (Peters et al., 1994), were
generously provided by Dr. S. C. Froehner (University of North
Carolina, Chapel Hill, NC). In C2 myotube extracts, syntrophin isoforms
were detected as three major protein bands between 55 and 65 kDa (see
Fig. 2). A rat monoclonal antiserum recognizing the mouse transferrin
receptor RI7 217 (Lesley et al., 1989) was a generous gift from Dr. J. Lesley (The Salk Institute, San Diego, CA). To detect src-related kinases, we used src-CT, a rabbit polyclonal antibody reactive with at least src, fyn, and yes (Santa Cruz Biotechnology, Santa Cruz,
CA) (Fuhrer and Hall, 1996). The rabbit polyclonal antiserum recognizing MuSK was as described (Fuhrer et al., 1997). For
phosphotyrosine immunoblotting, a mixture of two commercially available
mouse monoclonal antibodies was used, 4G10 (Upstate Biotechnology, Lake Placid, NY) and PY20 (Transduction Labs, Lexington, KY).
Isolation of AChRs and co-precipitation. To examine
association of postsynaptic proteins with the AChR, myotubes were
rinsed on ice with PBS supplemented with 1 mM
Na-orthovanadate and 50 mM NaF and extracted at 4°C in
lysis buffer. For these assays, most of the chemicals were purchased
from Sigma. The conditions for lysis were mild, i.e., 1% digitonin, 50 mM NaCl, 30 mM triethanolamine, pH 7.5, 5 mM EGTA, 5 mM EDTA, 50 mM NaF, 2 mM Na-orthovanadate, 10 mM
p-nitrophenylphosphate, 50 µM
phenylarsine-oxide, 1 mM benzamidine, 1 mM
N-ethylmaleimide, 1 mM Na-tetrathionate, 1 mM PMSF, 25 µg/ml aprotinin, and 25 µg/ml leupeptin.
Cells were lysed for 30 min, and insoluble material, such as nuclei and
cytoskeletal elements, was removed by centrifugation at 18,000 × g for 5 min. To precipitate the AChR, the cleared lysates
were incubated with -bungarotoxin (-BT) coupled to Sepharose
beads (Gu and Hall, 1988). In the case of control samples, we added an
excess (10 µM) of free uncoupled -bungarotoxin to
lysates to compete with AChR binding to the resin. Other control
samples were precipitated with uncoupled Sepharose beads. Beads were
then washed twice with wash buffer 1 (0.4% digitonin, 470 mM NaCl, 30 mM triethanolamine, pH 7.5, 5 mM EGTA, 5 mM EDTA, 2 mM
Na-orthovanadate, 1 mM PMSF, 10 mM p-nitrophenylphosphate, 50 µM
phenylarsine-oxide, 50 mM NaF) and twice in wash buffer 2 (same as 1 but containing 120 mM NaCl) and boiled in
SDS-PAGE sample buffer. The proteins were separated by reducing
SDS-PAGE and transferred to nitrocellulose membranes. To detect
AChR-associated proteins, the nitrocellulose was probed with the
appropriate antibodies, and immunoreactive bands were visualized using
horseradish peroxidase-conjugated secondary antibodies and enhanced
chemiluminescence (ECL, Amersham Corporation, Arlington Heights, IL).
Some immunoblots were stripped by incubating them for 20 min in 200 mM glycine, 0.1% Tween 20, pH 2.5; the blots were then
reprobed with src-CT to examine src-related kinases or with mAb 124 or
mAb 88B to confirm that equal amounts of AChRs were present in all
lanes. Experiments were repeated at least four times, and
representative samples are shown in Figures 2 and 6. Quantitation of
ECL immunoblotting data was performed by scanning films containing
gray, nonsaturated signals with a computerized densitometer (LaCie
Silverscanner IV) and using the NIH Image 1.54 software. Scanning data
were evaluated by one-way ANOVA followed by pair-wise Bonferroni's
t tests.
To study tyrosine phosphorylation of AChRs, AChR-associated MuSK, and
total MuSK, myotubes were treated with agrin and extracted in lysis
buffer as described above. Cleared lysates were split into two parts
and either incubated with -bungarotoxin-Sepharose beads to isolate
the AChR and receptor-associated MuSK or with MuSK antibodies followed
by protein A-Sepharose to precipitate MuSK. Bead-precipitated proteins
were eluted into SDS sample buffer, subjected to SDS-PAGE, and analyzed
by phosphotyrosine immunoblotting. Because of the similarities in the
precipitation protocols using MuSK antibodies and toxin-Sepharose, we
refer to the latter as "Tox-IP" in Figures 7 and 5, although no
antibodies are used in this case. The experiments were performed three
times, and representative samples are shown in Figure 7. Evaluation was
performed by densitometric scanning of nonsaturated signals and
statistical analysis as described above.
These experiments revealed the presence of a protein complex in C2
myotubes containing AChRs in association with rapsyn, MuSK, src-related
kinases, -dystroglycan, and utrophin. To test the relative strength
of these protein interactions, we used a range of progressively more
stringent conditions. These were based on the lysis and wash buffers
detailed above, with the following substitutions: (1) digitonin was
replaced by Nonidet P-40 (NP-40) or Triton X-100 (Tx-100) in lysis and
wash buffers; (2) digitonin was replaced with a fixed concentration of
1% Tx-100 in both lysis and wash buffers; (3) digitonin was replaced
with fixed concentrations of 1% Tx-100 and 0.5% deoxycholate in both
lysis and wash buffers; (4) same as (3) but 470 mM NaCl was
included in the lysis buffer, i.e., cell lysis was performed in the
presence of high salt; and (5) in addition, an extremely mild procedure
was applied, in which the lysis buffer contained digitonin as
described, but all wash buffers contained only low salt, i.e., 120 mM NaCl. Over this range of conditions, few changes in
protein binding were observed. Substituting digitonin with any other
detergent abolished association of AChRs with -dystroglycan, whereas
under condition (4), no agrin-dependent increase of the AChR-MuSK
interaction was seen.
Fluorescence microscopy. To visualize the distribution
of AChR on the surface of rapsyn / and wild-type myotubes, these cells were grown and fused on gelatin-coated chamber slides (Nunc). Myotubes were treated with or without 0.1 nM neural agrin
for 16 hr and incubated with 0.5 µg/ml rhodamine-conjugated
-bungarotoxin. After washing and fixation in 2% paraformaldehyde,
cells were mounted with p-phenylenediamine and examined
under a Nikon Microphot-FXA fluorescence microscope at a final
magnification of 400×.
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RESULTS |
AChRs are associated with rapsyn in C2 myotubes
We examined the associations of AChRs with other proteins by
extracting the AChR from C2 myotubes in a buffer containing 1% digitonin, a mild detergent, precipitating it with -bungarotoxin (-BT) conjugated to Sepharose beads, and probing immunoblots of the
precipitated proteins using the relevant antibodies. To test whether
proteins were bound to the resin specifically through their association
with the AChR, free -BT was added to extracts before the
purification or unconjugated Sepharose was used, as described
previously (Fuhrer et al., 1997).
In our initial experiments, we looked for the presence of the protein
rapsyn, because it has been postulated to be associated with the AChR
as the result of cross-linking experiments (Burden et al., 1983) and
because of its ability to cluster AChRs in heterologous cells
(Froehner et al., 1990; W. P. Phillips et al., 1991; Gillespie et
al., 1996; Apel et al., 1997). Other observations that implied a
complex between rapsyn and the AChR include their 1:1 stoichiometry in
C2 myotubes (LaRochelle and Froehner, 1987) and structural investigations that revealed a protein, presumably rapsyn, associated with Torpedo AChR on its cytoplasmic surface (Unwin, 1993).
Our immunoblotting experiments with rapsyn-specific antibodies revealed that the precipitated AChR is indeed associated with an immunopositive band that migrates as a protein of 43 kDa and represents rapsyn (Fig.
1). The band was not present when an
excess of free toxin was added to the extract or when unconjugated
Sepharose was used, confirming the specificity of the interaction. By
comparing the amount of protein precipitated with the amount of rapsyn
in the original extract, we estimate that 1.13 ± 0.29% of the
total rapsyn is specifically bound to the toxin beads (mean ± SD;
data from six experiments). Because only 14.9% of the total AChR in C2
lysates is recovered on -BT-Sepharose (Fuhrer et al., 1997), we
calculate that at least 7.5% of the total rapsyn in the extract is
associated with the AChR (Table 1).
Because rapsyn is likely to be poorly extracted relative to the AChR
(Froehner, 1991), our results are consistent with the association of a
significant proportion of the total rapsyn in muscle cells with the
AChR.
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Figure 1.
Rapsyn is associated with the AChR. C2 myotubes
were treated with 5 nM neural agrin as indicated and lysed
in a buffer containing 1% digitonin. AChRs were isolated using -BT
coupled to Sepharose beads; as controls, an excess (10 µM) of free soluble toxin was added
(+T) or unconjugated Sepharose was used
(C). Samples were analyzed by SDS-PAGE and
immunoblotting with a rapsyn-specific antiserum. For the bottom
part, the blot was stripped and reprobed with mAb 88b, which
recognizes the AChR and subunits. L indicates a
fraction (0.3%) of the total lysate.
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To our knowledge, this is the first demonstration of a specific
co-precipitation of soluble rapsyn with the AChR in extracts of muscle
cells. Biochemical co-purification of these two proteins has proven to
be difficult (Froehner, 1991; Bezakova and Bloch, 1998; Colledge and
Froehner, 1998b), and a recent report showed protein co-precipitation
but did not test the specificity of the proposed interaction or mention
proteins that are excluded from the precipitation procedure (Mitsui et
al., 1996). Another recent study described weak binding between
recombinant AChR and rapsyn proteins using protein overlay assays
(Buckel et al., 1998). The difficulties in biochemical co-purification
of rapsyn and the AChR from extracts of muscle or Torpedo
most likely originate from difficulties in extracting rapsyn from the
cytoskeleton and its protease sensitivity. With respect to the latter,
we have observed that within hours after cell extraction, the amounts of rapsyn and the AChR in the cell lysate decrease substantially (C. Fuhrer and Z. W. Hall, unpublished observations). For these reasons, we assume that our observed interaction originates from a much
more pronounced in vivo association between rapsyn and the AChR.
Association of the AChR with other synaptic proteins
We used the same method to investigate the association of the AChR
with other postsynaptic proteins. Using specific antibodies, we looked
for the presence of three proteins that are part of the
dystrophin/utrophin glycoprotein complex, -dystroglycan, utrophin,
and syntrophin. Small amounts of the first two proteins were
specifically associated with the AChR (Fig.
2A,B). A 400 kDa band
was immunopositive for utrophin, and a protein of 46 kDa was reactive
with -dystroglycan antibodies. In both cases, some of the observed
binding was nonspecific, because it could not be competed with excess
toxin and it also occurred to unconjugated Sepharose. There was a
consistent and statistically significant difference between toxin beads
and these controls, however, indicating that utrophin, as well as
-dystroglycan, is specifically associated with the AChR (also see
Fig. 6). In both cases, the amount of bound protein was low; we
calculate that ~0.30% of total -dystroglycan and 0.48% of total
utrophin are bound to the AChR in the cell extracts (Table 1). No
evidence was found for binding of syntrophin, the third member of the
complex tested. Using a monoclonal antibody, mAb SYN1351, that
recognized all three major syntrophin isoforms, 1, 1, and 2
(Peters et al., 1994), in C2 myotube extracts, no immunopositive
protein was detected bound to toxin beads (Fig. 2D).
The failure to detect association with syntrophin indicates that the
binding of -dystroglycan and utrophin is specific and does not
reflect nonselective protein aggregation. As a further control, we also
tested for the presence of an unrelated membrane protein, the
transferrin receptor, which was not associated with AChRs under any of
the conditions tested (Table 1) (Fuhrer et al., 1997).
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Figure 2.
Protein associations with the AChR in C2
myotubes. Myotubes were incubated with 5 nM neural
(4,8) or muscle (0,0) agrin as indicated.
Cells were then extracted under mild conditions, using 1% digitonin,
and AChRs were precipitated with -BT-Sepharose. As controls, an
excess (10 µM) of free toxin was added
(+T), or in some experiments unconjugated
Sepharose was used (C). Precipitates were
analyzed by immunoblotting using specific antibodies as indicated. The
AChR content in each lane was visualized by reprobing stripped blots
with an antiserum, mAb 88b, reactive with the AChR and subunits. For comparison, a fraction (0.2%) of the total cell lysate
was analyzed (L). Solid arrowheads
indicate the proteins detected by the antibodies used for
immunoblotting; open arrowheads point to the AChR and subunits.
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The effect of agrin on the interaction of the AChR with rapsyn,
MuSK, src-family kinases, and other proteins
We then tested the effect of agrin treatment on the interaction of
the AChR with rapsyn and other proteins. Agrin did not change the
amount of rapsyn bound to the AChR. Although occasional variations were
seen, no consistent effect of agrin on the association was observed
(Fig. 1). Even after a 16 hr treatment with a high dose (5 nM) of neural agrin, conditions under which AChR cluster formation is maximal (Ferns et al., 1996), the association remained unchanged as compared with untreated cells, suggesting that the interaction of rapsyn with AChRs in muscle cells is independent of the
agrin signaling pathway.
We have reported previously that the AChR in C2 myotubes is associated
with both of the src-family kinases, src and fyn (Fuhrer and Hall,
1996), and with the receptor tyrosine kinase MuSK, and that agrin
treatment increases the association of MuSK with AChRs (Fuhrer et al.,
1997). When we examined the effect of agrin on the association of the
AChR with src-family kinases, however, we found results that were
similar to those seen for rapsyn. Using an antiserum, src-CT, that
reacts with several src-family members including src, fyn, and yes, we
found no effect of agrin treatment on the amount of these proteins
associated with the AChR (Fig. 2C). We also found no
consistent effect of agrin on the association of utrophin and
-dystroglycan with the AChR, although variations in the binding were
occasionally observed (Fig. 2A,B; also see Fig.
6C).
Characteristics of the AChR-protein interactions
To investigate the strength of the observed protein interactions
with the AChR, we used a range of progressively more stringent conditions to extract C2 myotubes, including up to 1% Triton X-100, 0.5% deoxycholate, and 470 mM NaCl (see Materials and
Methods). Although most associations were unchanged, we did find two
differences in these experiments. First, substituting digitonin with
any other detergent inevitably abolished binding of the AChR to
-dystroglycan without affecting other constitutive interactions
(Table 1). We were thus able to observe a complex of the AChR in
association with utrophin, kinases, and rapsyn, but without
-dystroglycan. Second, under the harshest conditions tested, agrin
failed to increase the association of the AChR with MuSK (Table 1).
These data indicate that some protein interactions are very fragile and
that we most likely disrupt protein complexes during even the mildest
extraction procedures. Our calculations on the stoichiometries of the
protein interactions are thus minimal estimates of the associations as
they occur in intact cells and are likely to be merely an indication of
much more pronounced interactions in vivo. Furthermore, MuSK
appears to interact with the AChR in two ways. The first association is
independent of agrin and resistant to harsh conditions, whereas the
second is induced by agrin and disrupted by stringent extraction
buffers. Thus, different mechanisms appear to underlie constitutive as
opposed to agrin-induced association of MuSK with the AChR. Finally,
the range of detergent conditions allowed us to partially dissect the
AChR protein complex. Because -dystroglycan is selectively lost
under harsh conditions, the observed binding of other proteins to the
AChR does not require its presence. Thus, in the assembled AChR complex
in myotubes, -dystroglycan does not appear to link other proteins,
in particular, utrophin, to the AChR; instead, these proteins interact
with the AChR independently of -dystroglycan.
Production and characterization of rapsyn / cell lines
Because rapsyn has been proposed as a primary adaptor and
anchoring protein of the AChR, linking it to other proteins, we wished
to investigate its role in the association of the AChR with the
proteins mentioned before. For this purpose we made use of
rapsyn-deficient muscle cell lines bearing an immortalizing SV40
T-antigen transgene (Jat et al., 1991; Morgan et al., 1994). Rapsyn
mutant mice bearing the transgene were obtained in crosses between
rapsyn heterozygotes positive for the SV40 T-antigen transgene and
rapsyn +/ mice (see Materials and Methods). Cells dissociated from
limb muscles of E18 embryos were plated at clonal density under
permissive conditions (33°C, -interferon) for the isolation of
muscle cell lines. Each colony was expanded and tested for fusion
(37°C, no -interferon). Two of the resulting rapsyn / cell
lines (11-4 and 11-7) formed myotubes that displayed spontaneous contractions and appeared healthy (see Fig. 4). No significant difference in overall morphology was seen as compared with the control
wild-type muscle cell line (12-10, rapsyn +/+) generated from a
wild-type littermate. However, for both mutant and wild-type cell
lines, the overall efficiency of fusion and myotube formation was lower
than that seen with C2 myotubes.
The overall amounts of the AChR, and the proteins associated with it,
were compared in extracts of the rapsyn / and rapsyn wild-type cell
lines (11-7 and 12-10) by immunoblotting (Fig. 3). The cellular levels of all proteins
examined, including the AChR, utrophin, -dystroglycan, syntrophin,
src-related kinases, and MuSK, were indistinguishable in the two cell
lines. Only rapsyn, which was undetectable in the mutant cells, was
different. These results show that rapsyn does not affect the overall
levels of these proteins, nor is it required for the formation of
healthy myotubes in vitro.
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Figure 3.
Analysis of postsynaptic proteins in rapsyn /
and wild-type myotubes. Cells (11-7 and 12-10) were lysed in a buffer
containing 1% NP-40, and protein-matches fractions, ~1% of the
extracts, were subjected to immunoblotting using utrophin-, MuSK-, or
-dystroglycan-specific antibodies. Blots were stripped and reprobed
with the src-CT antibody; in a second round of stripping and reprobing,
mAb 124 antibodies recognizing the AChR subunit were used. Parallel
lysates were analyzed by immunoblotting with rapsyn- or
syntrophin-specific antisera. Arrowheads indicate the
proteins recognized by the respective antibodies.
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When myotubes from the two cell lines were examined for the presence of
AChR clusters using rhodamine-conjugated -BT and fluorescence
microscopy, the wild-type myotubes showed occasional clusters, but the
mutant cells had none (Fig. 4). Moreover,
when treated with agrin, the wild-type myotubes formed many AChR
clusters, but the mutant cells failed to form any aggregates at all.
After treatment of myotubes with 0.1 nM neural agrin for 16 hr, clusters were absent in the mutant cells, whereas an abundance of
clusters was visible on the wild-type myotubes, similar to observations with C2 myotubes (Fig. 4; data not shown). The complete absence of AChR
aggregates, in both untreated and agrin-treated mutant cells, was
striking and is in agreement with results from cultured primary
myotubes derived from rapsyn / mice (Gautam et al., 1995).
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Figure 4.
Distribution of AChRs on the surface of
agrin-treated rapsyn / and wild-type myotubes. 11-7 and 12-10 cells
were grown and fused on chamber slides and incubated with or without
0.1 nM neural agrin for 16 hr. Surface AChRs were
visualized by incubating cells with 0.5 µg/ml rhodamine-conjugated
-BT, fixing in 2% paraformaldehyde, and examination by fluorescence
microscopy. In rapsyn / myotubes, both spontaneous and
agrin-induced AChR clusters are completely absent, whereas wild-type
cells show increased cluster formation in response to agrin. Scale bar,
20 µm.
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AChR-protein interactions in rapsyn / myotubes
We then used the rapsyn / myotubes in our standard
precipitation experiments to determine whether rapsyn is required for each of the other protein associations with the AChR that we detected. We first looked to determine whether rapsyn is required for MuSK association with the AChR, because experiments in transfected QT-6
fibroblasts had suggested that rapsyn associates independently with
both the AChR and MuSK (Gillespie et al., 1996; Apel et al., 1997).
When rapsyn / myotubes were examined, however, MuSK was found to be
associated with the AChR in a specific manner and with a stoichiometry
comparable to that found in C2 myotubes (Fig. 5) (Fuhrer et al., 1997). We calculate
that 1.46 ± 0.46% of the total MuSK in extracts is associated
with the AChR (mean ± SD from six experiments; see also Table 1)
under conditions in which the efficiency of AChR precipitation is the
same in the two cell lines. We conclude that the constitutive
association of MuSK with the AChR does not require rapsyn.
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Figure 5.
Association of the AChR with MuSK and src-related
kinases in rapsyn / myotubes. Myotubes lacking rapsyn were lysed in
a buffer containing 1% digitonin, and AChRs were precipitated with
-BT coupled to Sepharose beads (TS). As controls, an
excess (10 µM) of free toxin was added
(+T), or unconjugated Sepharose was used
(CS). AChR-associated proteins were detected by
immunoblotting using MuSK or src-CT antibodies, and the presence of the
AChR was confirmed by stripping the src-CT blot and reprobing it with
mAb 124 reactive with the AChR subunit. L represents
0.2% of the total lysate.
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We found similar results when we examined the association of the
src-related kinases with the AChR in rapsyn / myotubes (Fig. 5).
The proportion of src-family kinases in the total extract that was
associated with the AChR (0.30 ± 0.13% in six experiments) was
not significantly different from that found in C2 myotubes (Fuhrer and
Hall, 1996). Thus, in muscle cells, rapsyn is also dispensable for
binding of src-related kinases to the AChR, although in transfected
fibroblasts rapsyn is associated with cell-endogenous kinase activity
(Qu et al., 1996).
Different results were found when the isolated AChR from rapsyn /
myotubes was examined for the presence of proteins associated with the
dystrophin/utrophin glycoprotein complex (Fig.
6). In contrast to results obtained with
C2 or rapsyn wild-type myotubes, no evidence was found for specific
binding of utrophin or -dystroglycan to the AChR in the mutant cells
(Fig. 6A,C). Even under the mildest conditions of
extraction, 1% digitonin and a total salt concentration of 150 mM in lysis and wash buffers, we could not detect specific associations. Analysis of several experiments showed that the association of the AChR with utrophin and -dystroglycan is
statistically significant in the wild-type cells, although some
background binding of these proteins occurs to the Sepharose resin
(Fig. 6C). In rapsyn-deficient myotubes, in contrast, no
significant difference was found between -BT-Sepharose and control
samples including excess free toxin and uncoupled Sepharose, ruling out
a specific association of the AChR with utrophin and -dystroglycan
in these cells. Thus, rapsyn is required for the association of the
AChR with two proteins of the dystrophin/utrophin glycoprotein complex, utrophin and -dystroglycan.
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Figure 6.
Effect of agrin on association of proteins with
the AChR in rapsyn / myotubes. A, Myotubes lacking
rapsyn (11-7) and control wild-type cells (12-10) were treated for 16 hr with 5 nM of neural (4,8) or muscle
(0,0) agrin as indicated. Cells were extracted under
mild conditions, using 1% digitonin, and AChR-associated proteins were
detected by -BT-Sepharose precipitation and immunoblotting using the
indicated antibodies. As a standard, 0.2% of the total lysate was
analyzed (L). Arrowheads point to
the proteins recognized by the respective antibodies. B, Quantitation of the AChR-MuSK
interaction. MuSK immunoblots made from samples treated as described in
A were quantitated by densitometric scanning of films,
and values of untreated cells were set to 100%. Data represent
mean ± SD of at least five experiments. C,
Quantitation of the AChR-utrophin and AChR--dystroglycan
interaction. Immunoblots as shown in A were quantitated
by densitometric scanning, and values of agrin-treated control samples
containing excess toxin (+T) were set to 100%;
other values were calculated accordingly. Data represent mean ± SD of at least six experiments. *Differs significantly from control
(p < 0.05, by ANOVA followed by
Bonferroni's t test).
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Finally, we examined the effects of agrin treatment on the associations
of the various proteins with the AChR in rapsyn / myotubes. No
effect was seen in any case. In particular, agrin did not increase the
association of MuSK with the AChR. In contrast, in C2 and rapsyn
wild-type myotubes, agrin increased the association by ~2.5-fold, as
shown in Figure 6B and as reported previously (Fuhrer
et al., 1997). These results suggest that MuSK associates with the AChR
in two ways, in a rapsyn-independent and a rapsyn-dependent mode, and
that the latter occurs as a response to agrin. These two modes of
interaction are consistent with the observations from C2 myotubes using
mild and stringent extraction buffers, as described above.
These data indicate that in muscle cells, rapsyn mediates interactions
of the AChR with two components of the dystrophin/utrophin glycoprotein
complex, utrophin and -dystroglycan, but not with src-related
kinases. In the case of MuSK, it is not involved in the constitutive
interaction but is necessary for the increased association observed
after treatment with agrin. Because rapsyn / myotubes do not form
any AChR clusters, either spontaneously or in response to agrin, the
rapsyn-independent binding of src-related kinases and MuSK in these
cells must occur to diffusely distributed AChRs.
In summary, in myotubes, the AChR is specifically associated with
multiple other proteins, including rapsyn, utrophin, -dystroglycan, src-related kinases, and MuSK. The rapsyn dependence of some of these
interactions indicates a role of rapsyn in selected associations and
rules out general protein aggregation as a cause for the observed AChR
protein complex. Several additional observations point toward the
specificity of the protein interactions in the complex. First, neither
syntrophin, another postsynaptic component of the dystrophin/utrophin glycoprotein complex, nor the transferrin receptor, a membrane protein,
is associated with the AChR, although both are easily detected in
muscle extracts. Second, the observed interactions occur with different
stoichiometries, ruling out general protein aggregation (Table 1).
Finally, the proteins are dissociated from the AChR at different ionic
strengths, with most of them stable in stringent conditions as
described above.
Tyrosine phosphorylation of MuSK and the AChR in rapsyn
/ myotubes
Neural agrin causes tyrosine phosphorylation of two of the
proteins that we observe in the AChR complex, MuSK and the AChR itself
(Wallace et al., 1991; Glass et al., 1996). MuSK phosphorylation, of
both the total pool and the AChR-associated fraction, occurs within
minutes after agrin treatment and is paralleled by phosphorylation of
the AChR subunit (Glass et al., 1996; Fuhrer et al., 1997). We
examined the dependence of each of these phosphorylation events on
rapsyn by determining their occurrence in rapsyn / myotubes. To
examine the phosphorylation of total MuSK, the myotubes were treated
with neural or muscle agrin; MuSK was immunoprecipitated and examined
by phosphotyrosine immunoblotting. The results obtained with rapsyn
/ myotubes were similar to those seen in C2 and rapsyn wild-type
myotubes because MuSK was phosphorylated within 15 min of treatment
with neural but not muscle agrin (Fig.
7A), as shown previously by
Apel et al. (1997). In addition, neural but not muscle agrin induced
tyrosine phosphorylation of the pool of MuSK that is associated with
the AChR (Fig. 7A). A statistical evaluation of several
experiments showed no difference in the agrin-induced tyrosine
phosphorylation of AChR-bound MuSK between wild-type and
rapsyn-deficient myotubes (Fig. 7C). The lower relative intensity of the phosphorylated bands representing AChR-bound MuSK in
rapsyn / and wild-type myotubes relative to C2 myotubes (Fuhrer et
al., 1997) is presumably attributable to the lower proportion of
myotubes as compared with myoblasts in the cultures, which
explains the higher background in these cells. However, because
agrin-induced phosphorylation of total and AChR-bound MuSK occurs
indistinguishably and rapidly in both rapsyn / and wild-type
myotubes, we conclude from these experiments that rapsyn is not
required for the tyrosine phosphorylation of either the total pool of
MuSK or MuSK that is associated with the AChR.
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Figure 7.
Agrin causes efficient tyrosine phosphorylation of
AChR-bound MuSK, but not of the AChR subunit, in rapsyn /
myotubes. Rapsyn / (11-7) and wild-type myotubes (12-10) were
treated with 1 nM neural (4,8) or muscle
agrin (0,0) as indicated. A, Lysates were
split into two parts and incubated either with -BT-Sepharose beads
(Tox-IP) to isolate AChRs or with MuSK antibodies and
protein A-Sepharose beads to precipitate MuSK (MuSK-IP).
Samples were analyzed by phosphotyrosine immunoblotting, and proteins
were identified by their molecular weight and by stripping and
reprobing the blots with the appropriate antisera (data not shown).
Agrin causes phosphorylation of total and AChR-bound MuSK in both cell
types. Phosphorylation of the AChR subunit is much stronger in the
wild-type cells; a shorter exposure of the blots in the middle section
reveals a strong agrin-induced signal in the wild-type cells and no
signal at all in the mutant (data not shown). B, Levels
of AChR-associated MuSK and AChRs isolated on toxin beads are equal in
rapsyn / and wild-type cells. AChRs were precipitated from lysates
of mutant and wild-type cells, and samples were analyzed by
immunoblotting with MuSK or AChR /-specific antibodies.
L, 0.3% of the total lysate was analyzed as a standard.
C, Quantitation of tyrosine phosphorylation of
AChR-bound MuSK and the AChR subunit. Immunoblots made from samples
treated for 40 min as described in A were quantitated by
densitometric scanning of films. Short exposures of films were used
that contained nonsaturated signals. The intensities were normalized
for the amount of AChR-bound MuSK, as revealed in a toxin precipitation
followed by a MuSK blot (B). Values of untreated
wild-type cells were set to 100%, and other values were calculated
accordingly. Data represent mean ± SD of three experiments.
*Differ significantly from control (p < 0.05, by ANOVA followed by Bonferroni's t test), but
not from each other.
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In contrast, when phosphorylation of the AChR was examined, we saw a
striking and significant difference between rapsyn / and wild-type
myotubes. Unlike in wild-type cells, the AChR subunit was not
phosphorylated efficiently in agrin-treated rapsyn / myotubes (Fig.
7A,C), as shown previously by Apel et al. (1997), although
there was no difference in the amounts of -BT-precipitated AChRs and
MuSK between the two cell lines (Fig. 7B). In addition, we
found that the weak constitutive tyrosine phosphorylation of the AChR
subunit, which is observed independently of neural agrin in
wild-type cells, is also reduced in rapsyn / myotubes (Fig.
7A,C). These experiments show that rapsyn is essential for constitutive AChR phosphorylation, as well as the agrin-induced AChR
phosphorylation, one of the early events in agrin signaling, but not
for activation of total and AChR-bound MuSK.
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DISCUSSION |
In this study, we show that the AChR is associated with rapsyn
and, to a lesser degree, utrophin and -dystroglycan. Because it also
interacts with src, fyn, and MuSK (Fuhrer and Hall, 1996; Fuhrer et
al., 1997), the AChR has multiple specific associations with other
proteins in myotubes. The rapsyn dependence of the utrophin and
-dystroglycan associations, together with the interaction between
these two proteins (James et al., 1996), suggests a complex containing
at least four proteins
AChR, rapsyn, utrophin, and -dystroglycan.
Although we do not know whether this same complex also contains
src-family kinases and MuSK, the occurrence of these associations in
aneural myotubes shows that the AChRs are part of one or more complexes
that are preformed before synaptogenesis; these complexes may thus play
a role in the initiation and growth of the postsynaptic specialization.
Molecular organization of the proteins associated with
the AChR
Of the proteins associated with the AChR, rapsyn is the most
abundant. Rapsyn is known to be in close contact with the AChR and is
present in a 1:1 stoichiometry with the AChR (Burden et al., 1983;
LaRochelle and Froehner, 1987). Despite these and other long-standing
indications that the two proteins are complexed, to our knowledge
rapsyn has never been previously co-purified or co-precipitated
specifically with the AChR, probably because of the difficulty of
extracting rapsyn from the insoluble cytoskeleton and its sensitivity
to proteases (Froehner, 1991). For these and other reasons, our
calculation on the stoichiometry of the AChR-rapsyn interaction is
likely to be merely an indication of a more pronounced interaction
occurring in vivo. One possibility, consistent with the 1:1
stoichiometry, is that all surface AChR molecules, both clustered and
unclustered, are complexed to rapsyn in C2 myotubes. In support of this
notion, both rapsyn and the AChR are diffusely distributed in mutant
myotubes lacking AChR clusters (Gordon et al., 1993). Their association
may occur initially intracellularly before surface transport, because
both rapsyn and AChRs are associated with subpopulations of post-Golgi
vesicles in Torpedo electrocytes (Bignami et al., 1998).
AChRs also interact in a rapsyn-dependent manner with
-dystroglycan and utrophin, two components of the
dystrophin/utrophin glycoprotein complex. On the basis of the
interaction between these two proteins (James et al., 1996), the
-dystroglycan could be bound to the AChR complex through
utrophin. Utrophin, however, does not appear to be bound through
-dystroglycan, because under high salt conditions, -dystroglycan
is selectively lost from the complex. Our experiments are thus
consistent with a novel model in which the AChR interacts directly with
rapsyn, which is bound, either directly or through an intermediary
protein, to utrophin, which is bound to -dystroglycan (Fig.
8). We cannot exclude the possibility
that -dystroglycan is necessary for recruitment of other proteins
(such as utrophin) during formation of this AChR protein complex. Once
these proteins are bound, however, -dystroglycan is no longer
required for their continued association with the complex.
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Figure 8.
Model showing protein interactions with the AChR
and steps in agrin-induced clustering of postsynaptic proteins.
A, In the absence of nerves and neural agrin, diffusely
distributed protein complexes exist in which rapsyn, -dystroglycan,
utrophin, MuSK, and src-related kinases interact with AChRs. Rapsyn
mediates interaction of the AChR with utrophin and -dystroglycan but
not with MuSK and src-related kinases. We assume that all AChRs are
complexed with rapsyn and that sites of nascent AChR clusters are
marked by MuSK, perhaps in association with some AChR.
B, Neural agrin leads to a link of the pre-existing
protein aggregates, using MuSK and rapsyn as effectors, thereby
increasing the interaction between MuSK and AChRs in a rapsyn-dependent
way. For more details, see Results. p, Phosphotyrosine;
src, src-related kinases; -DG,
-dystroglycan; -DG, -dystroglycan.
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These findings define a role for rapsyn as the link between the AChR
and the dystrophin/utrophin glycoprotein complex. At endplates,
utrophin colocalizes precisely with AChRs at the crests of the
postsynaptic folds (Colledge and Froehner, 1998a); our observed
association of utrophin with the AChR complex may underlie this correct
localization. By providing a cytoskeletal link early in development,
utrophin may thus hold the AChR complex in its proper subcellular
localization during postsynaptic differentiation. Indeed, in
utrophin-deficient mice, the density of AChRs and the number of
postsynaptic folds are reduced (Deconinck et al., 1997; Grady et al.,
1997). Because these effects are subtle, however, other molecules and
links are also important for postsynaptic differentiation and might
compensate in the absence of utrophin.
In addition to linking the AChR to utrophin, rapsyn may also form
a link to -dystroglycan, based on its ability to cluster this
protein in transfected fibroblasts and to interact with
-dystroglycan in biochemical assays (Apel et al., 1995; Cartaud et
al., 1998). Thus, at the adult endplate, multiple rapsyn-dependent
links between the AChR and the utrophin glycoprotein complex may exist
to ensure maximal stabilization of AChRs.
We have shown previously that src and fyn are bound to the AChR (Fuhrer
and Hall, 1996). As aggregated rapsyn is associated with an endogenous
tyrosine kinase in transfected QT-6 fibroblasts (Qu et al., 1996), Apel
et al. (1997) have suggested that src-related kinases bind to the AChR
via rapsyn, but our experiments demonstrate that in myotubes these
kinases interact with AChRs independently of rapsyn. Together, these
results suggest that interactions between rapsyn and src-related
kinases may also occur independently of the AChR. A similar situation
occurs in the case of MuSK; we show that MuSK is associated
independently of rapsyn with AChRs in untreated rapsyn / myotubes,
whereas MuSK colocalizes with rapsyn in co-transfected fibroblasts
(Gillespie et al., 1996; Apel et al., 1997). Thus, both MuSK and
src-family kinases may have an AChR-independent interaction with rapsyn
as well as a rapsyn-independent interaction with the AChR.
In summary, our work on the rapsyn / myotubes shows that the AChR
has two types of interactions with postsynaptic proteins: those that
depend on rapsyn and those that do not. These findings modify and
extend previously proposed rapsyn-mediated protein interactions that
were largely based on protein colocalization in transfected QT-6 fibroblasts.
Agrin signaling and AChR-protein interactions
As postsynaptic AChR clusters are induced by agrin during
synaptogenesis, we were particularly interested in examining agrin's effects on protein associations with the AChR. To our surprise, agrin
had no effect on most associations, except an increase in the case of
MuSK (Fuhrer et al., 1997). This increase appears to be a biochemical
correlate of the co-extensive clustering of both proteins, consistent
with its absence in rapsyn / myotubes. Furthermore, our results
reveal two types of interactions of MuSK with the AChR: a
rapsyn-independent interaction that occurs in the absence of agrin and
a rapsyn-dependent interaction that is induced by agrin. The latter has
an interesting parallel in transfected QT-6 cells, where coclustering
of MuSK with AChRs depends on rapsyn (Gillespie et al., 1996; Apel et
al., 1997). Provided that this represents biochemical interactions,
QT-6 cells thus appear as a "short-cut" model for agrin- and
rapsyn-mediated protein clustering. They allow protein aggregations to
occur that in myotubes are regulated in a more complex way by agrin,
emphasizing the need to analyze AChR-protein interactions in myotubes.
Agrin causes tyrosine phosphorylation of the AChR subunit, which
may result from a cascade in which agrin activates MuSK, leading to
activation of AChR-bound src-related kinases, which phosphorylate the
AChR (Fuhrer et al., 1997). In rapsyn / myotubes, as in
staurosporine-treated C2 myotubes (Fuhrer et al., 1997), agrin
activates total and AChR-bound MuSK, but the AChR is not phosphorylated
efficiently, suggesting that rapsyn is required for a step downstream
of MuSK. Because src-related kinases bind to AChRs independently of
rapsyn, presumably their activation or the activation of an unknown
intermediary protein must require rapsyn. Rapsyn could thus hold
AChR-bound MuSK and src-related kinases in a conformation that allows
activation of one kinase by the other, or it may itself interact with a
kinase or other protein that mediates activation. In both scenarios,
rapsyn appears as a scaffolding molecule involved in signal
transduction. These roles may be mediated by the various protein
domains of rapsyn (for review, see Colledge and Froehner, 1998b) and
are reminiscent of ion channel-binding proteins at central synapses,
e.g., PSD-95 at glutamatergic synapses (Kennedy, 1997; O'Brien et al.,
1998).
Most of our observed AChR-protein interactions are unchanged by agrin,
implying that they can occur with diffusely distributed AChRs. We show
this explicitly for src-related kinases and a fraction of MuSK, which
interact with AChRs in rapsyn-negative myotubes lacking spontaneous
AChR clusters. For rapsyn, in particular, its interaction with
unclustered AChRs is a novel idea and contrasts with earlier models
that postulated that rapsyn forms a scaffold for the recruitment of
AChRs and other proteins (W. P. Phillips et al., 1991). Our
observations rather suggest that conformational changes and/or
additional proteins are involved in docking diffusely distributed
pre-existing AChR-rapsyn complexes at sites of nascent AChR clusters.
Such sites may initially be marked by MuSK, perhaps in association with
some of the AChRs, consistent with the localization of MuSK at mutant
endplates in rapsyn / mice (Fig. 8) (Apel et al., 1997).
These observations raise the new concept that the AChR, while
still diffusely distributed, serves as a scaffold to which kinases as
well as anchoring proteins are attached. Indeed, these preassembled complexes of AChRs and other proteins may form the functional building
blocks for agrin-induced AChR clusters. Several observations support
this notion indirectly. For example, expression of a chimeric membrane-bound, activated MuSK kinase domain throughout myotubes causes
phosphorylation but not clustering of AChRs, consistent with the
assumption that MuSK's ectodomain and its association with AChRs are
crucial for cluster formation (Glass et al., 1997). Furthermore, in
AChR 
subunit-deficient recombinant mice, many postsynaptic
components are gradually lost along with AChRs during the short life
span of these mice, demonstrating a crucial stabilizing effect of AChRs
on other proteins at endplates (Witzemann et al., 1996; Missias et al.,
1997).
Our data thus imply that neural agrin acts to attach preassembled,
diffusely distributed AChR complexes to the sites of nascent clusters
(marked by MuSK) underlying nerve terminals (Fig. 8). This attachment
appears to occur by direct or indirect binding of the rapsyn in the
AChR-protein complexes to MuSK, leading to the observed increase in
the AChR-MuSK interaction. The clustered complex containing proteins
required for synaptogenesis (agrin, MuSK, rapsyn, and AChRs) then acts
as the scaffold that supports the growth and stabilization of the
cluster and the further elaboration of the postsynaptic membrane. A
crucial aim of future experiments will be to describe more fully the
molecular interactions and the proteins that underlie these events.
| |
FOOTNOTES |
Received Feb. 9, 1999; revised May 17, 1999; accepted May 21, 1999.
This work was supported intramurally by the National Institute for
Mental Health and the National Institute for Neurological Diseases and
Stroke and extramurally by a grant from National Institutes of Health
to M.G. C.F. was supported by a post-doctoral fellowship and a
grant from the Swiss National Science Foundation. We are very grateful
to Mia Nichol for assistance and to Dr. Joshua R. Sanes for advice.
Correspondence should be addressed to Dr. Christian Fuhrer at his
present address: Brain Research Institute, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Dr. Gautam's present address: Department of Pharmacological and
Physiological Science, St. Louis University Medical School, St. Louis,
MO 63104.
Dr. Sugiyama's present address: Hexos Inc., Bothell, WA 98021.
Dr. Hall's present address: Department of Physiology, University of
California San Francisco School of Medicine, San Francisco, CA 94143.
| |
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Campbell KP,
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Glass DJ,
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TOP
ABSTRACT
INTRODUCTION
