From the Department of Neurochemistry, Brain Research
Institute, University of Zürich, CH-8057 Zürich,
Switzerland, the § Department of Neurology & Neurosurgery,
McGill University, Montreal, Quebec H3A 2T5, Canada, and the
¶ Department of Physiology, University of Basel, CH-4051 Basel,
Switzerland
Received for publication, October 23, 2002
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ABSTRACT |
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The acetylcholine receptor
(AChR)-associated protein rapsyn is essential for neuromuscular synapse
formation and clustering of AChRs, but its mode of action remains
unclear. We have investigated whether agrin, a key nerve-derived
synaptogenic factor, influences rapsyn-AChR interactions and how this
affects clustering and cytoskeletal linkage of AChRs. By precipitating
AChRs and probing for associated rapsyn, we found that in denervated
diaphragm rapsyn associates with synaptic as well as with extrasynaptic
AChRs showing that rapsyn interacts with unclustered AChRs in
vivo. Interestingly, synaptic AChRs are associated with more
rapsyn suggesting that clustering of AChRs may require increased
interaction with rapsyn. In similar experiments in cultured myotubes,
rapsyn interacted with intracellular AChRs and with unclustered AChRs
at the cell surface, although surface interactions are much more
prominent. Remarkably, agrin induces recruitment of additional rapsyn
to surface AChRs and clustering of AChRs independently of the secretory pathway. This agrin-induced increase in rapsyn-AChR interaction strongly correlates with clustering, because staurosporine and herbimycin blocked both the increase and clustering. Conversely, laminin and calcium induced both increased rapsyn-AChR interaction and
AChR clustering. Finally, time course experiments revealed that the
agrin-induced increase occurs with AChRs that become cytoskeletally
linked, and that this precedes receptor clustering. Thus, we
propose that neural agrin controls postsynaptic aggregation of the AChR
by enhancing rapsyn interaction with surface AChRs and inducing
cytoskeletal anchoring and that this is an important precursor step for
AChR clustering.
Clustering of neurotransmitter receptors in the postsynaptic
membrane is a fundamental aspect of synapses and is thought to originate from receptor interactions with scaffolding proteins that
mediate binding to the cytoskeleton. At the neuromuscular junction
(NMJ),1 the 43-kDa
scaffolding protein rapsyn plays a pivotal role in clustering of
acetylcholine receptors (AChRs). This is best illustrated by rapsyn
Rapsyn's mode of action remains unclear, although rapsyn is sufficient
to drive clustering of AChRs upon expression in heterologous cells. In
these cells rapsyn forms aggregates in the absence of AChRs and, upon
coexpression, colocalizes with AChRs, MuSK, and In muscle cells, however, several observations indicate that AChR
aggregation is a precisely regulated process. First, AChR clustering in
muscle is regulated by the motor nerve in a process that requires
neural agrin (9). Agrin acts via the receptor tyrosine kinase MuSK, and
initiates a signaling mechanism that leads to
rapsyn-dependent AChR clustering (13, 14). Some spontaneous AChR clusters are still observed in aneural muscle in vivo
(10, 12) and in cultured myotubes in vitro, however (11).
Rapsyn colocalizes with these spontaneous clusters in myotubes (11) and, consistent with this, can also be detected in complexes with the
AChR (15). Secondly, unlike in heterologous cells, rapsyn does not
cluster in the absence of AChRs in muscle but needs some form of
association with the AChR in order to form aggregates (16, 17).
Thirdly, in myotubes, the relative expression levels of rapsyn and
AChRs are critical parameters for clustering. Transfected myotubes that
slightly overexpress rapsyn form more (although smaller) AChR clusters,
whereas strong overexpression of rapsyn abolishes clustering (18, 19).
Although these studies establish the importance of the general rapsyn
to AChR expression ratio, they do not address the ratio and regulation
of rapsyn-AChR interaction and the possible importance of this in
receptor clustering.
Regulation of AChR-rapsyn interactions could involve secretory
mechanisms, because in Torpedo electric organ, rapsyn and AChRs are
co-transported in post-Golgi vesicles suggesting that the secretory
pathway may deliver rapsyn with AChRs to the plasma membrane (20). It
remains unknown, however, whether rapsyn actually interacts with these
intracellular AChRs and whether the post-Golgi vesicles deliver rapsyn
and AChRs directly into the postsynaptic membrane or rather into
extrasynaptic membrane areas.
Thus, it is currently unclear where rapsyn interacts with AChRs
within developing mammalian muscle, and whether rapsyn binds only to
clustered receptors or also to unclustered surface AChRs. It is further
unclear how rapsyn-AChR interactions are modulated by agrin in the
process of clustering and how such modulation may contribute to the
formation of a cluster.
We have therefore investigated the interaction of rapsyn with
unclustered AChRs at the muscle surface and its regulation by agrin. We
find that rapsyn associates with extrasynaptic, unclustered surface
AChRs, that synaptic AChRs are associated with more rapsyn, and that
these interactions are much more prominent than intracellular interactions. Interestingly, we find that agrin increases the amount of
rapsyn associated with surface AChRs in myotubes in vitro.
The agrin-induced increase in the rapsyn-AChR interaction requires
tyrosine kinase activity, occurs independently of the secretory
pathway, parallels the rapid cytoskeletal linkage of AChRs, and
correlates highly with the subsequent clustering of the AChR. Thus, our
data suggest that the enhanced interaction of rapsyn with surface AChRs
is a crucial precursor step in the clustering process triggered by agrin.
Cell Cultures--
C2 (C2C12), S26, S27, and Sol8 cell lines
were propagated and fused as previously described (21, 22). Constructs
encoding the C-terminal half of neural and muscle agrin isoforms
(C-Ag12,4,8 and C-Ag12,0,0, respectively) were
expressed in COS cells as described earlier (23). To achieve the
20 °C block, myotubes were shifted to Dulbecco's modified Eagle's
medium containing 20 mM Hepes, pH 7.5, and incubated at
20 °C either in a cooled water bath or in a cooling incubator. To
inhibit tyrosine kinases, C2 myotubes were preincubated for 5 h
with 15 nM staurosporine or 2 µM herbimycin A, followed by agrin treatment in the presence of inhibitors (23).
Rapsyn Antibodies--
Two novel antibodies, Rap1 and Rap2, were
produced by Research Genetics (Huntsville, AL) in rabbits against
peptides corresponding to amino acids 133-153 (Rap1) and to the
C-terminal 11 amino acids (Rap2) of mouse rapsyn. Rap1 and Rap2, but
not their preimmune sera, recognize rapsyn as a single band of 43 kDa
in immunoblots made from biotinylated Preparation of Diaphragms--
Diaphragms were removed from
decapitated adult Wistar rats of at least 150 g body weight. For
denervation, rats (6 in total) were anesthetized with isoflurane and
the left phrenic nerve innervating the left hemi-diaphragm was cut.
5-6 days later, diaphragms were removed. From both denervated and
control animals, synaptic areas of the left hemi-diaphragm were
microscopically dissected from extrasynaptic areas, by observing the
location of the nerve and by using AChEsterase staining of parallel
control samples. From each preparation a control piece (4-5 mm wide)
was analyzed by serial 8-µm thick transverse or longitudinal
cryosectioning followed by rhodamine- Immunocytochemical Staining--
Secondary antibodies were
purchased from Jackson ImmunoResearch Laboratories (La Roche,
Switzerland) unless stated otherwise. C2 or S27 myotubes were fixed in
2% paraformaldehyde and 11% sucrose or in methanol at
Diaphragm sections were blocked by 5% NGS in phosphate-buffered
saline, followed by incubation with affinity-purified Rap2 antibodies
and Alexa 488-conjugated Isolation of AChR Complexes and Immunoblotting--
Total AChRs
(i.e. receptors representing the total cellular pool of
AChRs) were isolated from myotube lysates using
To isolate total AChRs from diaphragms, tissue from two animals was
pooled and homogenized in lysis buffer without detergent, using a tight
fitting Dounce homogenizer. After adding detergent, samples were
processed as detailed for myotubes above.
Blotting procedures and detection of precipitated AChR Extractability Assays--
Cytoskeletal linkage of surface
AChRs in Sol8 cells was assayed exactly as described previously (22).
Briefly, cells were scraped off in buffer lacking detergent and
pelleted by centrifugation. Pellets were then first extracted with low
detergent buffer (0.5% Triton X-100 for 10 min at 4 °C), followed
by centrifugation and re-extraction of the insoluble pellet in high
detergent buffer (1% Triton X-100 for another 10 min). From the
supernatant of each extraction, surface AChRs labeled with
biotin-
This method was adapted for differential extraction of C2 myotubes.
Here, a first extraction buffer (15), containing 0.05% Nonidet P-40,
was added directly to cells in the culture dishes, and cells were
extracted at 4 °C for 21 min. After centrifugation at 18,000 × g for 3 min at 4 °C, the insoluble pellet was
re-extracted in a second extraction buffer containing 1% Nonidet P-40
for 15 min at 4 °C, followed by re-centrifugation. From the
supernatant of each extraction, total AChRs were isolated using
biotin-
In both methods, the sum of AChRs from the two fractions was
constant irrespective of agrin treatment (100%, Fig.
8b, left), whereas the sum of AChR-bound rapsyn
was increased due to agrin (171%, Fig. 8b,
right). Rapsyn was predominantly associated with AChRs
isolated from the 1% detergent fraction in both methods (compare Fig.
8, a versus c).
Characterization of Rapsyn Antibodies--
To investigate
the association of rapsyn with the AChR, we first aimed at developing
sensitive tools to visualize rapsyn-AChR interactions and therefore
raised two polyclonal antibodies against different domains of rapsyn
(see "Experimental Procedures"), Rap1 and Rap2. We first tested the
specificity of these antibodies by precipitating total AChRs from
lysates of C2 myotubes using
Thus, Rap1 and Rap2 antibodies allow specific and efficient detection,
by immunoblotting, of AChR-associated rapsyn and total cellular rapsyn
in cultured myotubes. The sensitivity of this detection appears much
higher than in our previous studies using other rapsyn antibodies (15),
enabling us now to detect subtle changes in the rapsyn-AChR interaction.
Synaptic AChRs Interact with More Rapsyn than Extrasynaptic AChRs
in Vivo--
Rapsyn interacts with the receptor in cultured myotubes
not treated with agrin (Fig. 1 and Ref. 15), but it is unclear whether this association occurs only with AChR in spontaneous clusters or also
with unclustered AChR (15). To investigate this question, we first
studied rapsyn-AChR interaction in extrasynaptic versus synaptic areas of diaphragms of adult rats. Extrasynaptic and synaptic
areas were microscopically dissected and divided into two parts. The
smaller control parts were cryostat sectioned and serial transverse or
longitudinal sections throughout the piece of tissue were
double-labeled for AChRs and rapsyn using the affinity-purified Rap2
anti-rapsyn antiserum. This showed that synaptic preparations are rich
in co-localizing clusters of AChRs and rapsyn (Fig.
2a). Extrasynaptic
preparations, in contrast, were completely devoid of clustered AChRs
and rapsyn along the whole length of these preparations, stretching
from the medial to the lateral edges of the diaphragm muscle (Fig.
2a).
The major parts of the synaptic and extrasynaptic preparations were
lysed and AChRs precipitated using biotin-
Precipitation of total AChRs from these denervated diaphragms
showed that rapsyn was associated with AChRs in both extrasynaptic and
synaptic areas (Fig. 2d). Interestingly, significantly more rapsyn (73.9 ± 2.8% more) was co-precipitated with AChRs from synaptic than from extrasynaptic areas, although the amounts of isolated AChR were equal (Fig. 2e). These data demonstrate
that rapsyn interacts with unclustered AChRs in extrasynaptic areas of
the diaphragm muscle in vivo. However, synaptic AChR-rapsyn interactions are more pronounced, because AChRs isolated from muscle
areas that contain synaptically clustered AChRs are bound to more rapsyn.
Rapsyn Interacts with Unclustered Surface AChRs in Cultured
Myotubes--
The observed interaction of rapsyn with extrasynaptic
AChRs in vivo may be due to interactions of rapsyn with
intracellular AChRs and/or unclustered receptors at the cell surface.
To investigate this question, we first tested whether rapsyn interacts
with unclustered AChRs at the cell surface using myotube cultures of
non-agrin-treated C2 cells, as well as two proteoglycan mutants derived
from C2, S27, and S26. In contrast to C2, S27 and S26 cells completely lack spontaneous clusters of rapsyn and AChRs, and these proteins are
therefore distributed in a diffuse, unclustered manner in these two
cell lines (Fig. 3a and Ref.
11). We isolated total AChRs from myotube extracts using
Second, we tested whether rapsyn interacts with intracellular and/or
surface AChRs. For this purpose, we separately precipitated either
surface or intracellular AChRs (22), and analyzed bound rapsyn by
Rap1-immunoblotting. In order to precipitate surface AChRs, intact
myotube cultures were incubated with biotinylated
We next found that, although rapsyn co-precipitated with both surface
and intracellular AChRs, it was preferentially associated with the
surface receptor pool. Thus in C2, the size of the intracellular AChR
pool is ~40% of the surface receptor pool, but the amount of rapsyn
bound to intracellular AChRs is only ~10% of the amount of rapsyn
bound to surface AChRs (Fig. 3, d and e). S27
yielded similar results (Fig. 3, d and e). Rapsyn
therefore interacts with intracellular receptors, but the average
amount of rapsyn bound per AChR is much lower intracellularly than at
the surface.
Taken together, these findings indicate that rapsyn predominantly
associates with surface AChRs. In addition, the S27 data indicate that
rapsyn interacts efficiently with unclustered AChRs. Since the level of
rapsyn-AChR interaction is very similar for S27 and C2, this suggests
that in non-agrin-treated C2, the majority of rapsyn-AChR-complexes are unclustered.
Neural Agrin Increases the Interaction of Rapsyn with the AChR, and
This Precedes and Correlates with Clustering--
We show that, in
diaphragm, synaptic AChRs interact with more rapsyn than unclustered
extrasynaptic AChRs (Fig. 2). To investigate whether this difference is
caused by neurally derived agrin, which is concentrated at NMJs (25),
we treated C2 myotubes with recombinant neural agrin and determined the
amount of rapsyn co-precipitated with total AChRs by Western blotting
with the Rap1 antiserum. Neural agrin indeed caused an increase in the
amount of rapsyn bound to AChRs (Fig.
4a). A similar increase in the
rapsyn-AChR interaction was seen when
We next quantitated this increased interaction in response to neural
agrin and determined its dose-dependence and time course. Treatment of
C2 myotubes with a low concentration (100 pM) of neural
agrin for 1 h was sufficient to cause a maximal increase, ~70%,
in the amount of rapsyn bound to total AChRs (Fig.
5a). In time course
experiments, this increase occurred rapidly, reached its maximum within
40 min of neural agrin treatment and stayed elevated thereafter (Fig.
5b). Taken together, these data show that neural agrin
rapidly increases the amount of rapsyn associated with AChRs, and that
this precedes clustering of the AChR which is first detectable after
~4 h of agrin treatment (26).
To investigate the significance of the increase in rapsyn-AChR
interaction for clustering of the AChR, we treated C2 myotube cultures
with herbimycin and staurosporine, two tyrosine kinase inhibitors that
prevent agrin-induced AChR clustering (26). Assessment of the amount of
rapsyn co-precipitated with total AChRs revealed that both inhibitors
attenuated the agrin-induced increase in rapsyn-AChR association
without affecting the pre-existing interaction (Fig.
6a). In addition, we used S27
myotubes, which are not able to form AChRs clusters in response to even
high concentrations of agrin (26). In these cells, agrin did not affect
rapsyn-AChR association, not even at 50 nM (Fig.
6b and data not shown).
If the increase in rapsyn-AChR interaction is required for clustering,
one can expect that induction of clustering by alternative agents and
pathways will induce a similar increase in the interaction. Therefore,
we treated C2 myotubes with Ca2+ or laminin-1. These
treatments induce AChR aggregation in myotubes although through
different mechanisms because Ca2+ causes phosphorylation of
MuSK while laminin does not (27-29). Laminin, nevertheless, requires
rapsyn and the activity of downstream tyrosine kinases to induce AChR
clustering in myotubes (30). In our experiments, both Ca2+
and laminin elicited a clear increase in the amount of rapsyn co-precipitated with total AChR, and this increase was comparable to
the increase triggered by agrin (Fig. 6c).
These results demonstrate a strong correlation between increased
rapsyn-AChR interaction and clustering of these proteins, suggesting
that this is a common required precursor step in formation of AChR
clusters in muscle.
Agrin-induced Increase in Rapsyn-AChR Interaction Occurs at the
Cell Surface and Is Independent of the Secretory Pathway--
Since
rapsyn interacts predominantly with AChRs at the surface, we next
examined whether the agrin-induced increase in interaction occurs with
surface AChRs. Agrin treatment of C2 myotubes followed by selective
precipitation of surface AChRs revealed that agrin indeed increases the
interaction of rapsyn with surface AChRs (Fig.
7b). The extent of this
increase was identical to the increase in association of rapsyn with
total AChRs (see Fig. 5). Furthermore, precipitating intracellular
AChRs after agrin treatment did not reveal an increase in
rapsyn-intracellular AChR interaction showing that agrin specifically
increases the association of rapsyn with surface AChRs (data not
shown).
It has been proposed that rapsyn and AChRs are co-transported through
the secretory pathway to the postsynaptic apparatus in Torpedo
electrocytes (20, 31). This, together with our observed intracellular
rapsyn-AChR complexes (Fig. 3), raised the question of whether the
secretory pathway may contribute to the agrin-induced increase in
rapsyn-surface AChR interaction and to AChR clustering. To assess the
role of the secretory pathway in these processes, we first established
the effect of 20 °C incubation on AChR surface delivery. This
temperature selectively blocks formation of post-Golgi transport
vesicles, results in accumulation of newly made plasma membrane
proteins in the trans-Golgi network (TGN) and therefore
prevents surface delivery of AChRs (32). C2 myotubes were incubated
with free
We found that application of agrin at 20 °C still increased the
interaction of rapsyn with surface AChRs as assessed by selective precipitation of surface AChRs, showing that this increase does not
depend on surface delivery via the secretory pathway (Fig. 7b). Furthermore, incubation at 20 °C did not affect the
amount of pre-existing surface AChR-rapsyn complexes, whose short time maintenance thus does not depend on an intact secretory pathway. At
20 °C, prolonged agrin treatment induced AChR clustering to a
comparable degree as observed in control cultures at 37 °C (Fig. 7,
c and d). However, clustering at 20 °C
proceeds slower than at 37 °C because 7 h of agrin treatment at
20 °C were not sufficient to induce clusters (data not shown). AChR
clusters formed at 20 °C were in general smaller, thinner and less
intense in appearance than at 37 °C, which is presumably due to a
loss of AChRs from the surface since only exocytosis but not
endocytosis is blocked at 20 °C.
These data demonstrate that the secretory pathway is not required for
agrin-induced clustering of pre-existing surface AChRs, and that the
events critical for clustering thus occur at the cell surface.
Together, the results show that agrin causes an increase in the
interaction of rapsyn with surface AChRs independently of the secretory
pathway, and this increase in itself appears sufficient for clustering
in the absence of secretion.
Rapsyn Interaction Correlates with Linkage of the AChR to the
Cytoskeleton--
In the clustering process triggered by agrin, AChRs
become linked to the cytoskeleton (33). We analyzed whether the
increased rapsyn-AChR interaction following agrin treatment might
mediate this anchoring. In cultured myotubes, cytoskeletal linkage of AChRs can be assayed by a differential extraction procedure in which
AChRs are first extracted in a low detergent buffer, followed by
re-extraction of insoluble receptors in a higher detergent buffer.
Surface AChRs are then precipitated separately from these extractions
using biotin-
These data demonstrate that rapsyn-associated AChRs are preferentially
linked to the cytoskeleton and that the agrin-induced increase in
rapsyn-AChR interaction correlates highly with decreased extractability
and increased cytoskeletal anchoring of the AChR. This strongly
suggests that increased rapsyn interaction with surface AChRs may
mediate the agrin-induced anchoring of the receptor that precedes clustering.
In this study, we have investigated how rapsyn-AChR interactions
relate to clustering. We show that, in vivo, extrasynaptic (unclustered) AChRs are associated with rapsyn and that synaptic AChRs
are associated with more rapsyn. We therefore investigated how
rapsyn-AChR interactions are regulated in myotube cultures, and find
that neural agrin rapidly induces an increase in the interaction of
rapsyn with surface AChRs. This increase requires tyrosine kinase
activity, parallels enhanced cytoskeletal linkage of the AChR,
correlates strongly with subsequent receptor clustering, and (along
with clustering) occurs independently of the secretory pathway. These
observations suggest that agrin-induced increases in rapsyn-AChR
interactions trigger cytoskeletal anchoring and clustering of AChRs.
Rapsyn and the AChR Form Pre-assembled Complexes--
A
long-standing question in synapse formation concerns the sequence of
events in which postsynaptic components are assembled. At the NMJ much
evidence suggests that MuSK forms a primary synaptic scaffold to which
rapsyn is recruited followed by AChRs and other proteins, implying that
rapsyn interacts with AChRs only in clusters at the synapse (7, 8).
However, we recently reported that, in cultured C2 myotubes that were
not treated with agrin, AChRs already interact with several
postsynaptic proteins including rapsyn (15). Such interactions may have
originated from spontaneously clustered AChRs or from diffusely
distributed receptors. We investigated this issue and show here that
rapsyn interacts with unclustered AChRs at the surface of S27 myotubes,
a mutant derivative of C2 that completely lacks clusters. The
comparable extent of the rapsyn-receptor interaction between C2, S26,
and S27 cells strongly suggests that the majority of surface
AChR-rapsyn complexes in non-agrin-treated C2 are unclustered. We also
find that rapsyn interacts with unclustered AChRs in diaphragm in
vivo. Based upon the predominant association of rapsyn with
surface AChRs in C2 and S27 myotubes, it seems likely that the
extrasynaptic rapsyn-AChR complexes in diaphragm reside at the plasma
membrane rather than intracellularly. Taken together, these data show
for the first time that rapsyn interacts with AChRs outside of clusters
in pre-assembled, diffusely distributed complexes, both in cultured
myotubes and in diaphragm muscle.
These unclustered, pre-assembled surface complexes appear to be
important for clustering for the following reasons: Firstly, rapsyn
does not aggregate in the absence of AChRs in ectopically injected
myofibers in vivo (17). Secondly, we recently estimated that
in cultured C2 myotubes ~50% of total cellular rapsyn are constitutively bound to AChRs while the residual 50% represent free
rapsyn (17), indicating that pre-assembled rapsyn-AChR complexes are
very prominent. The pool of free rapsyn, which remains after treating
C2 myotubes with AChR-antibodies to down-regulate surface AChRs and
surface AChR-rapsyn complexes, is unable to form aggregates in response
to agrin (17). This illustrates that rapsyn requires some kind of
constitutive association with the AChR for subsequent clustering driven
by agrin in C2 myotubes. Our experiments shown here demonstrate that
constitutive rapsyn-AChR interactions mostly occur with unclustered
AChRs at the surface. Thus, the antibody treatment by Ref. 17
down-regulated mostly unclustered AChRs and unclustered AChR-rapsyn
complexes. Therefore, our present results, combined with these earlier
studies, indicate that rapsyn is extensively pre-assembled with
unclustered surface AChRs in non-agrin-treated C2 myotubes (Fig.
9, step 1), and that this
pre-assembly is necessary for subsequent clustering of rapsyn and AChRs
in response to agrin.
We find that rapsyn also interacts with intracellular AChRs. The much
lower extent of the intracellular association implies that rapsyn
interacts with only a subpopulation of intracellular AChRs. In support
of this idea are the co-localization of rapsyn and AChRs in post-Golgi
transport vesicles, the association of some rapsyn with the Golgi in
Torpedo electrocytes (20), and our preliminary results that rapsyn is
preferentially associated with AChRs late in the secretory pathway, at
the level of late Golgi or
TGN.3 The
intracellular interaction may facilitate AChR and/or rapsyn exocytosis,
a hypothesis we are currently investigating.
Agrin Increases the Interaction of Rapsyn with Surface AChRs, and
This Correlates with AChR Clustering and Anchoring--
We show that
in denervated muscle fibers in vivo, in which agrin remains
concentrated at NMJs (25), AChRs isolated from synaptic areas have more
(~70%) rapsyn associated than extrasynaptic AChRs. The higher amount
of rapsyn bound to synaptic AChRs most likely stems from the action of
motorneuron-derived agrin, as we find that neural agrin elicits the
same increase in rapsyn-AChR interaction in cultured myotubes. The
agrin-induced increase in interaction occurred only on surface AChR and
was not blocked at 20 °C, indicating that it does not require the
secretory pathway. At 20 °C, AChRs were clustered in response to
agrin, albeit in less densely packed aggregates, showing that
clustering per se does not depend on continuous surface
delivery of rapsyn and AChRs. The functional relevance of co-transport
of rapsyn and AChR through the secretory pathway (20, 31) thus remains
to be established. While it may maintain a high density of
pre-assembled surface rapsyn-AChR complexes, it is as such not required
for agrin-induced clustering. Rather, our present data show that the
critical events for AChR clustering occur independently of secretion at
the plasma membrane, where agrin regulates the extent of interaction
between a free pool of rapsyn (17) and surface AChRs (Fig. 9).
The agrin-induced increase in rapsyn-AChR interaction could potentially
reflect de novo formation of more rapsyn-AChR complexes, higher affinity binding between rapsyn and the AChR, or more rapsyn molecules bound per individual AChR. The formation of additional 1:1
rapsyn-AChR complexes (or the stabilization of such complexes) seems
possible, as the AChR and rapsyn have been shown to be present in
approximately equimolar amounts in Torpedo electric organ and in
cultured muscle cells (34). Ultrastructural studies also support a 1:1
stoichiometry and a direct interaction between rapsyn and AChRs (35,
36). On the other hand, the precise ratio of rapsyn to AChR in clusters
remains to be determined and our increased rapsyn-AChR interaction may
reflect binding of more rapsyn molecules to each AChR. If this is the
case, the additional rapsyn would necessarily bind at a different site
on the AChR, creating a distinct form of rapsyn-AChR complex with a 2:1
stoichiometry (see Fig. 9). Interestingly, it has been shown that the
expression ratio of rapsyn to AChR is critical for clustering (19) and
that this expression ratio is lower extrasynaptically than at the NMJ
(37). Our data are consistent with these studies and indicate that it is an increase in the association ratio of rapsyn to AChR at the surface that is likely to trigger clustering.
This notion is supported by our observation that the agrin-induced
increase in rapsyn-AChR interaction correlated strongly with AChR
clustering in several different experimental conditions. First, in S27
cells, agrin, even at high concentrations, failed to induce increased
interaction as well as clustering. Second and third, herbimycin and
staurosporine blocked both the agrin-induced increase in the
rapsyn-AChR interaction and AChR clustering in C2 cells. Herbimycin
inhibits agrin-induced phosphorylation of MuSK, while staurosporine
blocks a kinase downstream of MuSK, possibly a member of the Src-family
(21, 23). Thus, our results suggest that such a kinase may regulate
rapsyn-AChR interactions downstream of MuSK activation. Fourth, the
agrin-induced increase in rapsyn interaction occurs rapidly (within 40 min in C2), and parallels early signaling events that are important for
the subsequent clustering of the AChR, such as tyrosine phosphorylation
of the AChR
Rapsyn has long been proposed to mediate a cytoskeletal link of the
AChR. For example, extraction of rapsyn from clusters on rat myotubes
or from Torpedo electrocytes increases AChR mobility in the membrane
(38, 39). Furthermore, rapsyn mediates interaction of AChRs with the
dystrophin/utrophin glycoprotein complex, which binds to F-actin (15).
We now demonstrate that rapsyn is preferentially associated with AChRs
that are bound to the cytoskeleton, and that the agrin-induced increase
in interaction of rapsyn with AChRs correlates strongly with increased
cytoskeletal linkage of the AChR. This increased cytoskeletal linkage
occurs in parallel with AChR-associated signaling events (such as
tyrosine phosphorylation of AChRs, of AChR-bound MuSK, and Src-type
kinases (21)), and precedes detectable clustering of the AChR. The
emerging picture is thus that the AChR acts as a scaffold onto which
anchoring proteins like rapsyn and several signaling proteins are
rapidly recruited by agrin. Within the resulting AChR-protein
complexes, rapsyn appears as the most abundant postsynaptic protein
based on its high expression level (34) and its much higher extent of
binding to the AChR relative to other postsynaptic components (15).
Together, these observations strongly imply that it is the increased
rapsyn-AChR interaction that enhances the cytoskeletal anchoring of the
AChR in the postsynaptic membrane, and so promotes AChR clustering.
In summary, the data presented here, together with our previous studies
(17), suggest a model for AChR clustering in which pre-assembled,
unclustered AChR-rapsyn complexes occur at the plasma membrane and are
required for subsequent clustering (Fig. 9, step 1). Neural
agrin, released by the nerve terminal, then signals through MuSK and
further increases the interaction of rapsyn and the AChR in the
postsynaptic membrane, either by increasing the number of AChR-rapsyn
complexes or by increasing the amount of rapsyn interacting with an
individual AChR. This occurs through a kinase-dependent
step, independently of the secretory pathway, and by recruitment of
rapsyn from a free pool. We propose that the increased rapsyn-AChR
association then leads to enhanced cytoskeletal linkage, and to the
progressive postsynaptic clustering of the AChR (Fig. 9, step
2).
Pre-assembly of postsynaptic complexes may also play a role in
formation of central synapses. At excitatory synapses in hippocampal neurons, clusters of PSD-95 first appear in dendritic shafts or dynamic
spine precursors before moving, through cytoskeletal rearrangement, to
their final position in the mature spine head (40). At inhibitory synapses, much like AChRs and rapsyn at the NMJ, gephyrin and
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice, which lack differentiated NMJs and fail to cluster both
AChRs and cytoskeleton-interacting components such as utrophin and
dystroglycan (1). Furthermore, mutations in rapsyn can lead to
congenital myasthenic syndrome in humans and gradual loss of synaptic
AChRs (2).
-dystroglycan in
clusters (3-6). These observations have led to the concept that rapsyn
recruits AChRs into cytoskeleton-bound clusters, and that rapsyn
interacts with AChRs only in clusters (reviewed in Refs. 7 and 8).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-bungarotoxin (
-BT) AChR
precipitations; no signal is seen when excess free
-BT was included
in the precipitation (Fig. 1 and data not shown). After affinity
purification using the immunizing peptides, both Rap1 and Rap2
recognize rapsyn as a major band of 43 kDa in immunoblots made from
whole cell extracts of C2 myotubes or rat diaphragm muscle (Figs. 1 and
2 and data not shown). In immunostainings, affinity-purified Rap2
specifically recognizes rapsyn in clusters in C2 but not in rapsyn
/
myotubes (15) and at NMJs in rat diaphragm sections (Fig. 2 and
data not shown). The reactivities of Rap1 and Rap2 in immunoblots
allowed detection with higher sensitivity than antibodies 5943p and
mAb1234, well characterized rapsyn antibodies that we obtained from Dr. J. S. Sanes (Washington University, St. Louis, MO) and Dr. S. C.
Froehner (University of Washington, Seattle), respectively.
-BT staining. The control piece
had the same width as the main piece and, in case of extrasynaptic
preparations, stretched from the medial to the lateral edges of the
diaphragm muscle. This procedure revealed many AChR clusters in
synaptic, but none in extrasynaptic areas both in normal and denervated animals. The main diaphragm pieces were extracted and subjected to immunoblotting or
-BT-precipitation.
20 °C.
Following permeabilization in blocking buffer (5% normal goat serum
(NGS) and 0.1% Triton X-100 in phosphate-buffered saline), 100 nM tetramethylrhodamine-conjugated
-BT (Molecular Probes, Eugene, OR), and antibodies were applied in blocking buffer. Rapsyn was detected with mAb1234 followed by fluorescein
isothiocyanate-conjugated goat anti-mouse secondary antibodies.
-BT (Molecular Probes) in the same buffer.
Sections were then incubated with Alexa 546-conjugated goat anti-rabbit
secondary antibodies (Molecular Probes) and Alexa 488-conjugated
-BT, all in 5% NGS/phosphate-buffered saline. Cells or sections
were mounted in a solution containing glycerol and
p-phenyldiamine to reduce fading and examined by
fluorescence microscopy as described earlier (17).
-BT-conjugated Sepharose beads or soluble biotin-conjugated
-BT followed by streptavidin-coupled agarose beads (Molecular Probes) as described previously (21). To precipitate surface AChRs, intact myotubes were
incubated with 200 nM biotin-
-BT for 1 h at
4 °C. After washing, cells were lysed and AChRs isolated by adding
streptavidin-coupled agarose beads. For precipitation of intracellular
receptors, surface AChRs were first blocked by incubation of intact
cells with 0.5 µM free
-BT for 1 h at 37 °C.
After washing, cells were lysed and incubated with biotin-conjugated
-BT followed by streptavidin-coupled agarose beads. In control
experiments we added an excess (10 µM) of free
-BT
prior to biotin-
-BT, or used unconjugated Sepharose. We also
determined that 0.5 µM free toxin is sufficient to block surface AChRs (data not shown).
and
subunits were as described earlier (15, 22). AChR-bound or total
cellular rapsyn was detected using Rap1 or occasionally 5943p
antibodies. Immunoblot quantitation was performed by scanning films
with a computerized densitometer (Nikon Scantouch 210, Japan) and using
the NIH Image J 1.04b software (NIH, Bethesda, MD). To quantitate the
amount of rapsyn associated with precipitated AChRs (rapsyn on AChRs,
Figs. 2-7) signals for rapsyn co-precipitated with AChRs (total,
surface or intracellular) were normalized for the amount of AChR
isolated (as revealed by AChR
immunoblotting).
-BT were isolated using streptavidin beads.
-BT as described in the previous paragraph.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-BT followed by streptavidin-agarose as
previously described (21). AChR-bound rapsyn was then analyzed by
Western blotting with Rap1 or Rap2 antisera. Both antibodies, but not
their preimmune sera, identified a single band corresponding to rapsyn
that was co-precipitated with AChRs (Fig.
1a; data for Rap2 not shown). Precipitation of the AChR was confirmed by reprobing the blots with
antibodies against the AChR
-subunit (Fig. 1a,
bottom panels). In addition, Western blotting with Rap1 antisera,
after precipitating AChRs with an antibody against the AChR
subunit
(mAb35), identified a similar prominent band corresponding to rapsyn
(see Fig. 4c). Immunoblot analysis of C2 lysates without
AChR precipitation showed that affinity-purified Rap1 and Rap2, but not
the crude sera, identify a single band at the molecular weight of
rapsyn (Fig. 1b).
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Fig. 1.
Characterization of rapsyn antibodies and
precipitation of AChRs. a, AChRs, reflecting the total
pool, were precipitated from lysates of C2 myotubes using biotin- -BT
(Tox-P) and AChR-bound rapsyn was visualized by
immunoblotting with Rap1 (Rap1 blot; left panel). In
controls, preimmune serum was used (right panel) or 10 µM free
-BT was added (+T) or biotin-
-BT
was omitted (SB). Blots were reprobed with AChR
-subunit-specific antisera (lower panels). Rap1
specifically detects AChR-bound rapsyn. b, lysates from C2
myotubes were analyzed by immunoblotting using Rap1 and Rap2 crude sera
(ser) or affinity-purified antibodies (pur).
Purified Rap1 and Rap2 recognize a single band corresponding to
rapsyn.
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Fig. 2.
Rapsyn is associated with extrasynaptic AChRs
in vivo, although to a lesser degree than with
synaptic receptors. a, cross-sections of synaptic
(Syn) and extrasynaptic (Xtra) areas of rat
diaphragm were double-labeled with Alexa 488- -BT (green)
and Rap2 antibodies followed by Alexa 546-conjugated secondary
antibodies (red). AChRs and rapsyn co-localize at
NMJs (yellow) but are not detectable extrasynaptically.
Scale bar, 30 µm. b, equal proteins amounts
from lysates of extrasynaptic areas of innervated (In) or
denervated (Den) diaphragms were analyzed by immunoblotting
using antibodies against rapsyn (Rap1) or the AChR
-subunit. c and d, total AChRs were
precipitated from extrasynaptic (Xtra) or synaptic
(Syn) areas of innervated (c) or denervated
(d) diaphragms using biotin-
-BT (Tox-P),
followed by rapsyn and AChR
immunoblotting. e,
quantitation of blots as in d shows that in denervated
diaphragm, more rapsyn is associated per AChR (~70% more) in
synaptic than in extrasynaptic areas. Extra. Den.,
extrasynaptic denervated. Data represent mean ± S.D. of three
experiments. **, differs significantly from Extra. Den.
p < 0.01 by two-tailed Student's paired t
test.
-BT. Immunoblotting with
Rap1 revealed that rapsyn interacts with synaptic AChRs (Fig. 2c). The low amounts of extrasynaptic AChR and rapsyn (see
Fig. 2b) prohibited assessment of a possible interaction of
the two proteins in these regions. Therefore, we denervated the left
hemi-diaphragm, leaving the right hemi-diaphragm intact. Western blot
analysis 5 days after denervation showed that rapsyn and AChRs were
up-regulated in lysates made from extrasynaptic areas of the denervated
left hemi-diaphragm but not in lysates from extrasynaptic innervated diaphragm (Fig. 2b). The extent of up-regulation was higher
for AChRs than for rapsyn, in agreement with the different degree of
up-regulation of their respective mRNAs after denervation (24). This up-regulation, however, was not sufficient to allow
immunohistochemical detection of AChRs and rapsyn in extrasynaptic
areas of denervated hemi-diaphragm (data not shown). Importantly,
clusters of AChRs and rapsyn were never observed in these denervated
extrasynaptic preparations, either. In fact, immunostainings for rapsyn
and AChRs of denervated extrasynaptic areas yielded results
indistinguishable from those of innervated extrasynaptic areas as shown
in Fig. 2a.
-BT, and
then immunoblotted for bound rapsyn. We found that comparable amounts
of rapsyn were co-precipitated with receptors in non-agrin-treated C2,
S26, and S27 cells (Fig. 3, b and c). These
results demonstrate that rapsyn interacts with unclustered AChRs in
cultured myotubes.
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Fig. 3.
Intracellular and unclustered AChRs at the
surface of myotubes interact with rapsyn. a, double
labeling with rhodamine- -BT and rapsyn antibodies (mAb1234) shows
that spontaneous clusters of AChRs and rapsyn are absent in S27 but
present in C2 myotubes. Photographic exposure times for S27 were 3-fold
longer than for C2. Scale bar, 20 µm. b and
c,
-BT-Sepharose precipitation of total AChRs, followed
by rapsyn (5943p antibodies) and AChR immunoblotting, shows that rapsyn
is equally associated with AChRs in C2, S26, and S27 myotubes. As
controls, 10 µM free
-BT was added (+T),
unconjugated Sepharose was used (CS), and a fraction of the
starting lysate was analyzed (L). Quantitation represents
mean ± S.E. of six experiments; values for S26 and S27 were
combined. d, surface and intracellular AChRs were isolated
from C2 using biotin-
-BT, followed by rapsyn and AChR
immunoblotting; one-fifth of the surface precipitation was loaded to
allow comparison. e, for quantitation of blots as in
d, values of surface AChRs and of rapsyn bound to surface
AChRs were set to 100%, and intracellular values were calculated
accordingly. Data represent mean ± S.D. of four experiments. *,
differs significantly from intracellular AChRs (p < 0.05 by two-tailed Student's paired t test), indicating
that rapsyn associates to a higher degree with surface than with
intracellular AChRs.
-BT. After washing
and lysis of the cells, surface AChRs were isolated by adding
streptavidin-agarose. Intracellular AChRs, in contrast, were isolated
with biotinylated
-BT and streptavidin-agarose after surface AChRs
were first blocked with free
-BT. In each case, precipitated AChRs
were detected by immunoblotting for the AChR
-subunit, and
AChR-bound rapsyn was detected using Rap1 antibodies. Probing for the
AChR revealed that the majority of the receptor is located at the
plasma membrane with only a relatively small fraction of all AChRs
located intracellularly. Furthermore, the surface and intracellular
AChRs pools were additive to yield total AChRs (data not shown).
-BT-precipitated total AChRs
were probed with another rapsyn antibody (5943p) to detect AChR-bound
rapsyn (Fig. 4b). Furthermore, isolation of total AChRs by
immunoprecipitation with an antibody against the AChR
-subunit
(mAb35), followed by Rap1 immunoblotting, revealed a comparable
agrin-induced increase in the rapsyn-AChR interaction (Fig.
4c). Finally, this increase is specific for neural agrin
since treatment with muscle agrin isoforms had no effect (Fig.
4d).
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Fig. 4.
Neural agrin increases the interaction of
rapsyn with AChRs in C2 myotubes. a and b,
total AChRs were precipitated from myotube cultures treated with neural
agrin (0.25 nM) for 1 h or from control cultures.
AChR-bound rapsyn was visualized by immunoblotting with Rap1
(a) or 5943p (b) followed by reprobing with AChR
-subunit-specific antisera. c, alternatively, total AChRs
were immunoprecipitated with antibodies against the AChR
subunit
(mAb35) followed by immunoblotting with Rap1 and reprobing with AChR
/
-subunit-specific antisera (mAb 88b). As controls, the mAb35
antibody was omitted from the precipitation (No Ab) or the
myotube lysate was omitted (LB). The amount of rapsyn per
AChR in neural agrin-treated cultures is higher than in control
cultures. d, muscle agrin (0.25 nM) was applied
overnight, followed by the same procedure as in a. Muscle
agrin does not increase the rapsyn-AChR interaction. Data represent
mean ± S.D. of at least three experiments.
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Fig. 5.
Dose and time-dependence of the agrin-induced
increase in rapsyn-AChR interaction. a, myotubes were
treated with increasing concentrations of agrin (neural
isoform) for 1 h. Precipitation of total AChRs was followed
by rapsyn and AChR immunoblotting. The amount of rapsyn bound to total
AChRs was quantitated (lower panel). 100 pM of
agrin is sufficient to elicit a ~70% increase in the rapsyn-receptor
interaction. b, neural agrin (0.25 nM) was
applied for increasing times, and AChR-bound rapsyn was analyzed as in
a. Neural agrin causes maximal increase in rapsyn-receptor
interaction within 40 min. This increase does not decline thereafter.
Data represent mean ± S.D. of at least three experiments.
Significant differences from controls: *, p < 0.05;
**, p < 0.01, by two-tailed Student's paired
t test.
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Fig. 6.
Increased rapsyn-AChR interaction correlates
with clustering. Total AChRs were precipitated from C2 myotubes,
and receptor-bound rapsyn detected by Rap1-immunoblotting.
a, herbimycin (Herb) and staurosporine
(Stau) inhibit the agrin-induced increase in rapsyn-AChR
association in C2 cells (0.25 nM agrin for 1 h).
b, in S27 myotubes, 0.25 nM agrin does not
affect rapsyn-AChR interaction. c, increased rapsyn-receptor
associations are observed after treatment of C2 with Ca2+
(5 mM overnight) or laminin (100 nM overnight,
Lm), conditions that induce AChR clustering (not shown).
Data are mean ± S.D. from at least three experiments. Significant
differences from controls: *, p < 0.05; **,
p < 0.01; by two-tailed Student's paired t
test.
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Fig. 7.
Agrin increases the interaction of rapsyn
with surface AChRs and causes AChR clustering independently of the
secretory pathway. a, C2 myotubes were incubated for
1 h at 20 °C or 37 °C with free -BT (Tx),
followed by 1 h with biotin-
-BT (biotin Tx) at the
indicated temperature. Surface AChRs were isolated with
streptavidin-Sepharose and visualized by AChR
immunoblotting. At
20 °C, surface transport of newly synthesized AChRs is blocked
within 1 h. b, C2 myotubes were incubated with 0.25 nM agrin at 37 °C or 20 °C for the indicated times;
agrin was added 1 h after the shift to 20 °C. Surface AChRs
were isolated, subjected to rapsyn and AChR-immunoblotting, and rapsyn
bound to AChRs quantified, using untreated cells (black bar)
as controls. Agrin increases this interaction at the surface, even when
surface delivery of AChRs is blocked by shifting the myotubes to
20 °C. c, C2 myotubes were treated with 0.25 nM agrin for 20 h at 20 °C or 37 °C, and AChRs
were visualized with rhodamine-
-BT. d, the average number
of AChR clusters per myotube was determined by counting clusters (as
shown in c) in 20 visual fields per treatment. Agrin
increases the number of AChR clusters significantly even at 20 °C,
although these clusters have a thinner and less bright appearance. Data
represent mean ± S.D. from at least three experiments.
Significant difference from control: *, p < 0.05; **,
p < 0.01; ***, p < 0.001; by
two-tailed Student's paired t test.
-BT to block pre-existing surface AChRs, followed by
biotin-
-BT to identify newly inserted surface receptors (Fig.
7a). At 37 °C new AChRs were readily detected while at
20 °C insertion of receptors was inhibited (Fig.
7a, lane 1), even when this temperature was only
applied during the biotin-
-BT incubation. These data show that a 1-h
incubation at 20 °C is sufficient to block transport of the AChR
from the TGN to the surface in our myotube cultures. This temperature
is thus a useful tool to specifically block the secretory pathway at
the level of the TGN.
-BT (22). In cultured Sol8 myotubes, a 1-h agrin
treatment induced a decrease in the amount of surface AChR that was
extracted in low detergent, with more receptor being shifted to the
less extractable, cytoskeletal-associated fraction (Fig.
8, a and b).
Strikingly, rapsyn was predominantly associated with AChRs in the less
extractable fraction, and the agrin-induced increase (71%; see also
Fig. 5) in rapsyn-AChR interaction occurred predominantly with the less
extractable AChR pool (Fig. 8, a and b). Similar
results were obtained following agrin treatment of C2 myotubes, in
which the majority of newly recruited rapsyn molecules were also bound
to less extractable AChRs (Fig. 8, c and d).
Finally, time course experiments in Sol8 myotubes showed that the shift in extractability of the AChR occurring after 1 h of agrin
treatment (Fig. 8a) was maintained at 6 and 24 h of
agrin treatment.2 The
agrin-induced decrease in extractability of the AChR therefore parallels the kinetics of the increase in rapsyn-AChR interaction (see
Fig. 5b).
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Fig. 8.
Rapsyn interaction correlates with
cytoskeletal linkage of the AChR. a, Sol8 myotubes were
treated for 1 h with 0.5 nM agrin and extracted first
in 0.5% and then in 1% Triton X-100. Surface AChRs were isolated
separately from these extractions using biotin- -BT and analyzed by
AChR
subunit- and rapsyn-immunoblotting. b, quantitation
of the proportion of AChR recovered in each extraction (a)
shows that agrin causes a shift of receptors from 0.5 to 1% Triton
X-100, indicative of a cytoskeletal link (left). Rapsyn,
before and after agrin treatment, is predominantly associated with the
less extractable AChRs (1% Triton X-100; right). Data
represent mean ± S.E. of five experiments. Significant
differences: *, p < 0.02 by paired Student's
t test. c, differential extraction of AChRs from
agrin-treated and untreated C2 myotubes shows that rapsyn is
predominantly associated with less extractable AChRs. d, In
C2 cells, the total agrin-induced increase in rapsyn binding to all
isolated AChRs (72%), was set to 100%, and the percentages of newly
recruited rapsyn molecules were calculated for the more extractable
(0.05% detergent) and less extractable (1% detergent) AChRs. Thus,
agrin-induced rapsyn-AChR complexes are preferentially linked to the
cytoskeleton. Data are mean ± S.D. of five experiments. *,
differs significantly from low-detergent extraction (p < 0.02, by two-tailed Student's paired t test).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Model for AChR clustering driven by
agrin-induced changes in rapsyn-AChR interaction that increase
cytoskeletal linkage. In the absence of agrin, pre-assembled
rapsyn-AChR complexes at the plasma membrane are presumably the
result of insertion of intracellular complexes (open
arrows). Such complexes are mostly unclustered (step
1). Agrin treatment (black arrows) induces an increase
in the interaction of rapsyn with surface AChRs, which strengthens
linkage of AChRs to the cytoskeleton. This increase appears to be
necessary for clustering and originates from a non-secretory mechanism,
most likely by recruitment of rapsyn from a non-receptor-associated
free pool. Both the increased rapsyn-surface AChR interaction and
cytoskeletal linkage precede clustering. For simplicity, possible
membrane association of free rapsyn is neglected. For further details,
see "Discussion."
subunit (22). This temporal correlation raises the
possibility that rapsyn binding to the AChR is regulated by
subunit
phosphorylation, and that they are linked precursor steps in the
clustering pathway, a hypothesis we are currently investigating. Fifth,
laminin-1, which is thought to act independently of MuSK in AChR
clustering (27, 28), also leads to increased rapsyn-AChR interaction. Sixth, extracellular Ca2+, which acts via MuSK to induce
AChR
-subunit phosphorylation and receptor clustering (29), induces
a similar increase in rapsyn-AChR binding. Finally, agrin-induced
rapsyn-AChR interactions and clustering still occur at 20 °C, when
secretion and new AChR insertion into the plasma membrane are blocked.
This shows that the events critical for clustering occur at the cell
surface and suggests that increased interaction of rapsyn with surface
AChRs may be sufficient to drive clustering in the absence of secretion.
-aminobutyric acid type A (GABAA) receptors are mutually
important for clustering, most likely by forming interdependent
components of a protein complex (41). Our data substantiate the
possibility that postsynaptic specializations of synapses are in
general constructed from pre-assembled complexes of their components.
Such pre-assembly may ensure that postsynaptic membranes can be formed
and modified rapidly according to physiological needs.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Susanne Erb-Vögtli and Maja Enz for excellent technical assistance, Drs. Matthias Gesemann and Omolara O. Ogunshola for comments on the manuscript, and Roland Schöb for excellent help with photography.
![]() |
FOOTNOTES |
---|
* This work was supported by the Dr. Eric Slack-Gyr Foundation, the National Center of Competence in Research "Neural Plasticity and Repair," by grants from the Swiss National Science Foundation and the Swiss Foundation for Research on Muscle Diseases (to C. F.), and by a Canadian Institute of Health Research grant (to M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Brain Research
Inst., University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Tel.: 41-1-635-33-10; Fax:
41-1-635-33-03; E-mail: chfuhrer@hifo.unizh.ch.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M210865200
2 L. S. Borges and M. J. Ferns, unpublished data.
3 M. Moransard and C. Fuhrer, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NMJ, neuromuscular
junction;
AChR, acetylcholine receptor;
-BT,
-bungarotoxin;
TGN, trans-Golgi network;
NGS, normal goat serum;
mAb, monoclonal
antibody.
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REFERENCES |
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