From Biologie Cellulaire des Membranes,
Département de Biologie Supramoléculaire et Cellulaire,
Institut Jacques Monod, UMR 9922, CNRS et Université Paris VII,
2 Place Jussieu, 75251 Paris Cédex 05, France and
§ Biologia Cellulare, Istituto Superiore di Sanità,
V. le Regina Elena 299, Roma 00161, Italy
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ABSTRACT |
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The accumulation of dystrophin and associated
proteins at the postsynaptic membrane of the neuromuscular junction and
their co-distribution with nicotinic acetylcholine receptor (AChR)
clusters in vitro suggested a role for the dystrophin
complex in synaptogenesis. Co-transfection experiments in which -
and
-dystroglycan form a complex with AChR and rapsyn, a peripheral
protein required for AChR clustering (Apel, D. A., Roberds,
S. L., Campbell, K. P., and Merlie, J. P. (1995)
Neuron 15, 115-126), suggested that rapsyn functions as a
link between AChR and the dystrophin complex. We have investigated the
interaction between rapsyn and
-dystroglycan in Torpedo
AChR-rich membranes using in situ and in vitro
approaches. Cross-linking experiments were carried out to study the
topography of postsynaptic membrane polypeptides. A cross-linked
product of 90 kDa was labeled by antibodies to rapsyn and
-dystroglycan; this demonstrates that these polypeptides are in
close proximity to one another. Affinity chromatography experiments and
ligand blot assays using rapsyn solubilized from Torpedo
AChR-rich membranes and constructs containing
-dystroglycan
C-terminal fragments show that a rapsyn-binding site is present in the
juxtamembranous region of the cytoplasmic tail of
-dystroglycan.
These data point out that rapsyn and dystroglycan interact in the
postsynaptic membrane and thus reinforce the notion that dystroglycan
could be involved in synaptogenesis.
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INTRODUCTION |
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The accumulation and maintenance of high concentrations of acetylcholine receptors (AChRs)1 at the postsynaptic membrane of the neuromuscular junction (NMJ) involve several levels of regulatory mechanisms including transcriptional regulation of AChR subunits genes, clustering, and anchoring of the AChR to the cytoskeleton. Clustering of AChR is a complex process involving several partners being triggered by the nerve-derived trophic factor agrin (for a review, see Ref. 1). A major step in the understanding of how agrin works was provided recently by the discovery of MuSK, a synaptic muscle-specific tyrosine kinase (2, 3). Once activated by agrin, MuSK-receptor complex sparks a cascade of events that leads ultimately to the building of the NMJ (4).
In addition, several extrinsic proteins associated with the postsynaptic membrane in muscle or electrocyte have been implicated in the formation and/or the maintenance of AChR clusters. Extraction of these proteins from AChR-rich membrane induces the mobility of the AChR in the plane of the membrane (5-8). These proteins thus participate in the anchoring of AChRs in the membrane. Among them, the 43-kDa AChR-associated protein rapsyn (9) plays a direct role in the clustering of AChR and all other postsynaptic membrane components as deduced from co-transfection experiments (10, 11) and from rapsyn-deficient mice (12). In addition to its structural role, rapsyn is required in an early step of MuSK signaling, i.e. AChR phosphorylation (13). From these studies, it could be proposed that rapsyn interacts directly or indirectly with several components of the postsynaptic membrane including MuSK and AChR to fulfill both structural and signaling functions in synaptic differentiation.
Several other peripheral proteins, Mr 58,000 syntrophin(s) (14) and Mr 87,000 dystrobrevin
(15), have been identified in Torpedo electromotor synapse
and co-localize with AChRs at the NMJ. These latter two proteins are
part of the dystrophin-glycoprotein complex (DGC) present at the
sarcolemma (16) and of the utrophin complex at the synapse (for a
review, see Ref. 17). Since dystrophin and dystrophin-associated
proteins are found at large AChR clusters in cultured muscle cells as
well as at synapses in vivo, they may also participate in
synaptogenesis (11, 18, 19). Major components of the DGC are
dystroglycans. Dystroglycans consist of a 156-kDa extracellular
laminin-binding glycosylated protein (-dystroglycan) and a
46-50-kDa transmembrane glycoprotein (
-dystroglycan) that result
from proteolytic cleavage of a single transmembrane precursor (20).
Dystroglycans are believed to form a continuous link between the
extracellular matrix and the submembranous skeleton at the sarcolemma
(for a review, see Ref. 21).
-Dystroglycan has been identified as an
agrin-binding site and, as such, was postulated to represent a major
actor in synapse formation (for a review, see Ref. 22). However,
several observations have weakened the case for
-dystroglycan as the
signal-transducing agrin receptor (23-25). Nevertheless, it is likely
to play a structural role in AChR clustering, perhaps as part of a
diffusion trap for AChRs (18, 19). By using the quail fibroblast
expression system, Apel et al. (26) reported that upon
co-transfection with
- and
-dystroglycan along with rapsyn and
AChR subunits cDNAs, dystroglycans co-localize with rapsyn-induced
clusters both in the presence or in the absence of co-expressed AChRs.
These experiments highlighted a possible role for rapsyn as a link
between AChR and the dystroglycans (probably
-dystroglycan).
In order to explore the function of dystroglycans in AChR clustering,
we examine in situ and in vitro the molecular
interactions between rapsyn and -dystroglycan using chemical
cross-linking and binding experiments. In cross-linking experiments,
these two polypeptides are found in close proximity from one another in the native AChR-rich membrane. Affinity chromatography and ligand blot
in vitro assays allowed us to identify a rapsyn-binding site in the juxtamembranous region (corresponding to amino acids 787-819) of the cytoplasmic tail of
-dystroglycan. These new data support the
notion that rapsyn may function as a molecular link connecting AChRs
and DGC at the postsynaptic membrane of the neuromuscular junction. The
ability of
-dystroglycan and rapsyn to establish multiple
interactions with partners from both inside and outside the cell places
these molecules at a key position in the differentiation of the
postsynaptic membrane during synaptogenesis.
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EXPERIMENTAL PROCEDURES |
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Materials--
The following primary antibodies were used.
Anti-rapsyn mouse monoclonal antibody mAb 1234 A was a generous gift
from Dr. Froehner (27). Rabbit polyclonal anti--dystroglycan
antibody was generated against a synthetic peptide of the last 20 amino acids of
-dystroglycan C terminus (28). Anti-GST rabbit polyclonal antibody and glutathione-Sepharose 4B were from Amersham Pharmacia Biotech. Peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were from Promega, Madison, WI. The protein concentrations were
estimated using BCA protein reagent (Pierce). Succinimidyl 4-(p-maleimidophenyl) butyrate (SMPB) was purchased from
Pierce. All other reagents were from the highest grade available.
Purification of Acetylcholine Receptor-rich Membranes-- Acetylcholine receptor-rich membranes were purified according to Saitoh and Changeux (29) from freshly dissected Torpedo electric tissue obtained from the Institut de Biologie Marine, Arcachon, France. Proteolysis was prevented by the addition of the protease inhibitors leupeptin (Sigma, 5 µg/ml), pepstatin A (Sigma, 5 µg/ml), EDTA (5 mM), EGTA (5 mM), 4-(2-aminoethyl)benzenesulfonyl fluoride (Calbiochem, 0.2 mM), and N-ethylmaleimide (Sigma, 10 mM) in all buffers. Typically 10-20 mg of membrane proteins were obtained from 100-200 g of fresh tissue.
Cross-linking Experiments--
Cross-linking experiments were
performed according to the protocol described by Burden et
al. (30) using an heterobifunctional cross-linking reagent that
contains N-ethylmaleimide and
N-hydroxysuccinimide as reactive groups. Briefly, AChR-rich
membranes were washed with 10 mM sodium phosphate buffer, 1 mM EDTA, 1 mM EGTA, 0.3 mM
phenylmethylsulfonyl fluoride, 0.02% sodium azide, pH 7.4, pelleted by
centrifugation, and resuspended in 10 mM sodium phosphate
buffer, 1 mM EDTA, pH 8.0, at a final concentration of 4 mg
of protein/ml. SMPB (0.12 nm between reactive groups) in
Me2SO (2% v/v stock solution) was added to the membranes
at concentrations of 107 to 10
4
M and incubated for 30 min at room temperature in the dark.
Membranes were then pelleted and washed in 10 mM sodium
phosphate buffer, 1 mM EDTA, pH 8, before solubilization in
SDS-PAGE sample buffer. Forty µg of membrane proteins were loaded in
each lane. Analysis of the cross-linked products were subsequently
performed by Western blotting followed by ECL detection.
Solubilization of Rapsyn from AChR-rich Membranes by Alkali and LiS-- For affinity chromatography, rapsyn was solubilized by alkali treatment for 10 min at 4 °C according to Neubig et al. (31). The alkali extract (S11) was neutralized to pH 7.5 with 2 M Tris-HCl, pH 7.0. Stripped membrane fragments and insoluble aggregates were then removed by centrifugation for 40 min at 100,000 × g. For two-dimensional SDS-PAGE separation, rapsyn was extracted by lithium diiodosalicylate (LiS) according to Carr et al. (32) as follows. AChR-rich membranes were suspended at 15 mg of protein/ml in 10 mM Tris, pH 8.1. A 1/9 volume of 0.1 M LiS in 10 mM Tris, pH 8.1, was added; the suspension was incubated on ice for 60 min; and the membranes were then removed by centrifugation for 40 min at 100,000 × g. The final supernatant containing most of the solubilized rapsyn called LiS extract was further processed for two-dimensional analysis.
GST Fusion Protein Constructs of Dystroglycans--
Cloning of
rabbit brain dystroglycan and construction of plasmids encoding
-dystroglycan cytoplasmic fragments was achieved as described by
Rosa et al. (28). The constructs
-C2 and
-C3 corresponding to residues 821-895 and 787-819 of the cytoplasmic tail
of dystroglycan and a construct corresponding to residues 467-500 of
-dystroglycan (ectodomain) were expressed as glutathione S-transferase (GST) fusion proteins in BL21 strain of
Escherichia coli. Recombinant proteins were purified
following the procedure described by Kennedy et al.
(33).
Affinity Chromatography-- GST and GST fusion proteins were immobilized on glutathione-Sepharose 4B according to the supplier's instructions. Control GST and GST fusion proteins were equilibrated in phosphate-buffered saline and incubated 2.5 h at 4 °C with freshly prepared alkali extract (S11) neutralized to pH 7.5. After incubation, the glutathione-Sepharose 4B beads were washed five times with phosphate-buffered saline by centrifugation. For immunoblot analysis, Laemmli's sample buffer (34) was directly added to Sepharose beads before separation by SDS-PAGE and transfer onto nitrocellulose.
SDS-Gel Electrophoresis and Immunoblotting--
8 or 10%
SDS-polyacrylamide gel electrophoresis was performed using a Bio-Rad
Mini-Protean II slab cell (Bio-Rad). Two-dimensional gel
electrophoresis was accomplished essentially as described by O'Farell
(35) on LiS extract following the protocol described by Carr et
al. (32). Sample buffer was composed of 9.95 M urea, 2% Nonidet P-40, 100 mM dithiothreitol, 0.02% SDS, 0.1%
Triton X-100, and 2% Ampholines (1 part pH 3.5-10, 2.5 parts pH 5-8, and 2.5 parts pH7-9, Amersham Pharmacia Biotech, Bromma, Sweden). Isoelectrofocusing gels were made in 0.4% Ampholines pH 3.5-10, in
1% Ampholines pH 5-8, in 1% Ampholines pH 7-9 for 30 min at 150 V,
4 h at 200 V, and 10 min at 800 V in a Bio-Rad Mini-Protean II
slab cell, as described by Bollag and Edelstein (36). This method
allowed highly reproducible isoelectric focusing and easy comparison of
several protein samples. The second dimension was carried out on strips
of gels from the first dimension fitted over 10% SDS-PAGE. Proteins
separated by one- or two-dimensional gel electrophoresis were
electrotransferred to nitrocellulose membranes (Schleicher & Schuell)
according to Towbin et al. (37) and stained with Ponceau
Red. Western blots were performed as described previously (38),
revealed using enhanced chemiluminescent detection (ECL, Amersham
Pharmacia Biotech) and exposed to Fuji X-Ray films (Fuji, Tokyo,
Japan). In some cases, blots were stripped for 1 h at 55 °C in
2% SDS, 0.1 M -mercaptoethanol, 60 mM Tris, pH 6.7, and reprobed with antibodies as described (38).
Ligand Blot Overlay Assay--
-Dystroglycan-GST fusion
protein binding assay was achieved on blots as described by Carr and
Scott (39). GST control, dystroglycan constructs
-C2,
-C3, and
-ectodomain (1 µM) were incubated for 2 h on the
nitrocellulose strips on which proteins from purified AChR-rich
membranes or LiS extract were electrotransferred after separation by
one- or two-dimensional SDS-PAGE, respectively. Bound GST fusion
proteins were revealed using anti-GST antibody followed by ECL
detection.
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RESULTS |
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Cross-linking of Rapsyn and -Dystroglycan in Native AChR-rich
Membranes by Succinimidyl 4-(p-Maleimidophenyl) Butyrate
(SMPB)--
To study the topography of postsynaptic membrane
polypeptides toward rapsyn, we have used the cross-linking protocol
developed by Burden et al. (30) for the study of the
association between AChR subunits and rapsyn. These authors have taken
advantage of the fact that rapsyn contains most (up to 90%) of the
available free sulfhydryls in AChR-rich membranes. Accordingly, they
selected a heterobifunctional cross-linker, SMPB, containing
N-ethylmaleimide and N-hydroxysuccinimide as
reactive groups, to selectively tether rapsyn to neighboring
polypeptides. This reagent thus allows the exploration of the close
environment of rapsyn in the membrane. In these conditions, most
cross-linked products included rapsyn and extensive cross-linking among
other membrane polypeptides was minimized. As the reagent was not
cleavable, the composition of the polypeptide bands that appear
following cross-linking was analyzed by Western blotting using
antibodies against rapsyn and
-dystroglycan. At SMPB concentrations
ranging from 10
6 to 10
5 M, a
few cross-linked products containing rapsyn did appear in immunoblots
of purified AChR-rich membranes (Fig. 1).
A major band at 110 kDa was previously identified as the
rapsyn/AChR-
-subunit cross-linked product (Ref. 30 and Fig.
1A). Moreover, an additional 90-kDa band appeared following
cross-linking on Western blots probed with anti-rapsyn antibody and
revealed by ECL. This band was also immunolabeled by antibodies to
-dystroglycan and, therefore, represents a rapsyn/
-dystroglycan
cross-linked product (Fig. 1A, lanes 3 and 3' and
Fig. 1B). Interestingly, the apparent mass of this
cross-linked product (=90 kDa) corresponds almost exactly to the sum of
individual polypeptide masses of rapsyn and
-dystroglycan (43 and
46-50 kDa, respectively). The specificity of the immunodetections in
these experiments was attested by the fact that the major cross-linked product at 110 kDa did not cross-react with the anti-
-dystroglycan antibodies and that only one additional cross-linked product at about
140 kDa was observed upon probing with anti-
-dystroglycan. This
140-kDa product did not cross-react with anti-rapsyn antibodies (Fig.
1A, lanes 3 and 3'). The 140-kDa product is
presently still unidentified. To ascertain that the 90-kDa product
indeed resulted from cross-linking between rapsyn and
-dystroglycan,
the blots previously probed with the anti-
-dystroglycan antibody
were stripped and reprobed with the anti-rapsyn antibody (Fig.
1C). This experiment demonstrated that the 90-kDa product
cross-reacted with both antibodies. Cross-linking with higher SMPB
concentrations, i.e. with 10
4 M,
results in the complete disappearance of rapsyn at its usual position
in SDS-PAGE, whereas there is a corresponding increase in a high
molecular weight material at the top of the resolving and stacking gels
(Fig. 1A, lane 4'). In the same conditions only part of the
-dystroglycan was cross-linked (Fig. 1A, lane 4). This
indicates that only a fraction of dystroglycan undergoes cross-linking
with rapsyn. Although cross-linking experiments do not provide the
demonstration that the two proteins are indeed interacting, these
experiments provide support for a close proximity between rapsyn and
-dystroglycan in the postsynaptic membrane.
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Rapsyn Binds to the Juxtamembranous Domain of the Cytoplasmic Tail
of -Dystroglycan in Vitro--
In an attempt to investigate
in vitro the interaction between rapsyn and
-dystroglycan, we developed an affinity chromatography assay. Rabbit
brain dystroglycan cDNA which showed high homology with
Torpedo sequences (40) was digested, and two constructs containing
-dystroglycan C-terminal fragments corresponding to the
juxtamembranous and distal cytoplasmic tail domains have been generated. The schematic representation of these two
-dystroglycan constructs (
-C2 and
-C3) is shown in Fig.
2A. The two constructs cover
almost the entire
-dystroglycan tail without redundancy. The
purified fusion proteins have already been checked by SDS-PAGE and
their molecular weights corresponded to that expected (see Ref. 28). A
few lower size bands were observed for each fusion protein. These
products might result from incomplete translation or from a proteolytic
cleavage occurring near the C terminus, even though fusion proteins
have been produced in protease-deficient E. coli strain (see
Ref. 28 for discussion). Binding of rapsyn to
-C2,
-C3 constructs
and GST control was carried out using freshly solubilized rapsyn. The
rapsyn from AChR-rich membranes was solubilized by an alkali treatment
that efficiently dissociates rapsyn from the membrane as well as other
components of the submembranous cytoskeleton (31). Following adjustment
at pH 7.6 and centrifugation, the alkali extract (S11) was incubated
for 2.5 h at 4 °C with the two constructs and with GST control
immobilized on glutathione-Sepharose 4B column. After extensive
washings, the bound rapsyn was monitored by immunoblotting using mAb
1234 A anti-rapsyn antibody. As shown in Fig. 2B, rapsyn was
only recovered with the GST-
-C3-conjugated column. At variance, very
low rapsyn recovery was obtained with GST-
-C2 or GST columns.
However, as revealed by silver staining, several polypeptides were
trapped together with rapsyn on the GST-
-C3 column (data not shown).
This could eventually be attributed to the aggregation of
alkali-extracted polypeptides with rapsyn as a consequence of
adjustment to pH 7.6 before application to the column.
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DISCUSSION |
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In this work, we have shown that rapsyn is localized close to
-dystroglycan at the cytoplasmic aspect of the postsynaptic membrane
of Torpedo electrocytes in situ and that it binds
to
-dystroglycan in vitro. The cross-linker selected for
this study which reacts both with available free sulfhydryls,
essentially on rapsyn, and with primary amines on neighboring
polypeptides is therefore particularly suitable for the exploration of
the close environment of rapsyn in the postsynaptic membrane in
situ. The observation that only one product (the 90-kDa) among a
very few cross-linked products cross-reacted with both anti-rapsyn and
anti-
-dystroglycan antibodies strongly favors the hypothesis that
rapsyn and dystroglycan are intimately associated within the membrane.
In good agreement with this in situ approach, in vitro binding experiments pointed out a domain of
-dystroglycan (the juxtamembranous cytoplasmic 787-819 residues) which specifically binds to rapsyn at micromolar concentration. Interestingly, as deduced
from its primary sequence (see Ref. 21 and references therein), the
juxtamembranous domain of
-dystroglycan contains two clusters of
lysine residues that likely account for the efficient cross-linking to
rapsyn by SMPB. Our experiments thus suggests that the interaction
between rapsyn and dystroglycan observed in vitro also
occurs in situ. This is in full agreement with the co-distribution of the two proteins at the cytoplasmic side of the
postsynaptic membrane in Torpedo electrocyte (19).
At variance with rapsyn which is totally cross-linked at a high
concentration of SMPB, only part of dystroglycan can be cross-linked in
the same conditions. This may indicate that only a fraction of
dystroglycan molecules are engaged in interaction with rapsyn. Very few
cross-linking products containing -dystroglycan were observed in the
resolving gels. Thus, our cross-linking experiments do not allow us to
assess whether
-dystroglycan, dystrophin, or Grb2 were cross-linked
with
-dystroglycan. In fact, the cross-linking agent used in this
study was not designed to explore the environment of
-dystroglycan
but rather that of rapsyn.
Taken together, our results raise the possibility that distinct dystroglycan complexes may exist in the postsynaptic membrane. Moreover, since rapsyn is absent from extrasynaptic sarcolemma, one could postulate that several dystroglycan complexes, differing with their association with various partners, are confined to different domains of the sarcolemma. At the synapse, the rapsyn dystroglycan interaction could likely be involved in the clustering of AChR.
Various Functions for Distinct Dystroglycan Complexes at the Sarcolemma-- The ability of dystroglycans to establish multiple interactions with partners from both inside and outside the cell places these molecules at a central position in sarcolemmal organization. Dystroglycans directly contribute to the connection of the extracellular matrix with the actin-based cytoskeleton. Several signal transducing molecules, such as Grb2 (45) or nitric-oxide synthase (46), have been reported to associate with the DGC. However neither Grb2 nor NOS are part of the DGC sensu stricto. The present data further emphasize the property of the DGC to associate weakly and/or transiently with a variety of physiological partners in order to mediate various signals. In the synaptic membrane, the present experiments show that DGC associates with AChR via rapsyn. However, these two proteins do not co-purify with DGC after detergent extraction (38, 47), perhaps pointing to a loose association. Thus, the DGC is likely to play many more functions than previously thought.
Furthermore, dedicated regions of the cytoplasmic tail of
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Functional Implication for Rapsyn-Dystroglycan Interaction in
Synaptogenesis--
Several lines of evidence point to a role of the
DGC in AChR clustering. Dystrophin/utrophin and components of the DGC,
in particular /
dystroglycan, co-distribute with AChR clusters both in vivo and in vitro. In co-cultured nerve
and muscle cells derived from Xenopus embryos, Cohen
et al. (18) reported that dystroglycan associated with
synaptic as well as nonsynaptic AChR clusters and that it underwent the
same nerve-induced changes in distribution as AChR. Also, during early
Torpedo embryonic development, we observed that AChR
accumulates concomitantly with dystrophin and dystroglycan in the
ventral plasma membrane domain of developing electrocytes (19).
Finally, co-transfection experiments in quail fibroblasts with
recombinant AChR, rapsyn, and dystroglycan show that these proteins
form stable complexes in the membrane and that rapsyn can cluster AChR
and dystroglycan independently (26). However,
utrophin-dystrophin-deficient mice that display severe progressive
muscular dystrophy have moderately affected neuromuscular junctions
(51, 52). Thus, synaptic differentiation could occur in the absence of
dystrophin and utrophin. In these double knock-out mice, as reported by
Deconinck et al. (51), dystrophin-associated proteins
2-syntrophin and
-dystroglycan are retained at the
NMJ, suggesting that these components might be required for AChR
clustering. In this context, the present data support the notion that
rapsyn-
-dystroglycan interaction is involved in synaptic
differentiation.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Froehner for the generous gift of anti-rapsyn antibody; Professor J. P. Changeux for advice during the realization of this work; and L. Guillon for critical reading of the manuscript. M. Zini is acknowledged for expert technical assistance.
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FOOTNOTES |
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* This work was supported by the CNRS, the Université Denis Diderot, the Association Française Contre les Myopathies (to J. C.), and by the Italian Telethon Grant 187 (to T. C. P.).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: Biologie Cellulaire des Membranes, Institut Jacques Monod, CNRS, Université Paris VII, 2, Place Jussieu, 75251 Paris Cédex 05, France. Tel.: 33-1-44 27 69 40; Fax: 33-1-44 27 59 94; E-mail: cartaud{at}ijm.jussieu.fr.
1 The abbreviations used are: AChR, nicotinic acetylcholine receptor; DGC, dystrophin-glycoprotein complex; ECL, enhanced chemiluminescence detection; GST, glutathione S-transferase; LiS, lithium diiodosalicylate; NMJ, neuromuscular junction; PAGE, polyacrylamide gel electrophoresis; SMPB, succinimidyl 4-(p-maleimidophenyl) butyrate; mAb, monoclonal antibody.
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REFERENCES |
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