* Department of Cell and Structural Biology, and Department of Biochemistry, University of Illinois, Urbana, Illinois 61801
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Abstract |
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The clustering of acetylcholine receptors
(AChR) on skeletal muscle fibers is an early event in
the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR
clustering. Since the 7
1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR
clustering. The alternative cytoplasmic domain variants,
7A and
7B, and the extracellular spliced forms,
7X1 and
7X2, were studied for their ability to engage
in AChR clustering. Immunofluorescence microscopy
of C2C12 myofibers shows that the
7
1 integrin colocalizes with laminin-induced AChR clusters and to a
much lesser extent with agrin-induced AChR clusters.
However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of
the integrin and AChR. High concentrations of anti-
7
antibodies inhibit colocalization of the integrin with
AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin
with low concentrations of anti-
7 antibody initiates
cluster formation in the absence of agrin or laminin.
Whereas both the
7A and
7B cytoplasmic domain
variants cluster with AChR, only those isoforms containing the
7X2 extracellular domain were active.
These results demonstrate that the
7
1 integrin has a
physiologic role in laminin-induced AChR clustering,
that alternative splicing is integral to this function of
the
7 chain, and that laminin, agrin, and the
7
1 integrin interact in a common or convergent pathway in the
formation of neuromuscular junctions.
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Introduction |
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THE postsynaptic membrane of the neuromuscular
junction is a specialized structure characterized by a
high density of acetylcholine receptors (AChRs)1
on the surface of myofibers (Fertuck and Salpeter, 1976;
Anderson and Cohen, 1997; Frank and Fischbach, 1979
).
These clusters mediate the effects of acetylcholine released from the nerve on muscle fibers. Clustering of
AChRs takes place during synaptogenesis and involves
the migration of pre-existing dispersed AChRs in the sarcolemma to the postsynaptic membrane and an increase at the synapse in receptor synthesis (Hall and Sanes, 1993
;
Bowe and Fallon, 1995
).
Migration of AChRs to the nerve terminal is mediated
by agrin, a component of the synaptic basal lamina. Agrin
isoforms are generated by alternative RNA splicing and
each appears to have different capacities for clustering.
Although agrin is synthesized by both muscle cells and
motor neurons, the isoforms that most actively cluster
AChRs are synthesized and released by motor neurons (McMahan, 1990; Ruegg and Bixby, 1998
). Neural agrin
plays a central role in synaptogenesis by binding to a receptor on the muscle cell surface. The
v
1 integrin has
been shown to bind agrin and participate in agrin-induced
AChR clustering (Martin and Sanes, 1997
), and MuSK, a
muscle-specific tyrosine kinase, is required for the formation of neuromuscular junctions in vivo (Ganju et al., 1995
;
Valenzuela et al., 1995
; DeChiara et al., 1996
). Neural agrin is unable to cluster AChRs in myofibers that develop
in vitro from MuSK-deficient myoblasts (Glass et al., 1996
;
Sugiyama et al., 1997
).
Recent studies show that laminin can induce AChR
clusters both in wild-type and in MuSK-deficient myotubes (Sugiyama et al., 1997; Montanaro et al., 1998
). This
induction is specific to laminin-1 and the effect of laminin
on clustering is additive to those of agrin. These observations led to the suggestion that two alternative pathways
participate in the formation of AChR clusters, one mediated by neural agrin and one by laminin.
The 7
1 integrin is a major laminin receptor in skeletal
muscle (von der Mark et al., 1991
; Song et al., 1992
;
George-Weinstein et al., 1993
; Martin et al., 1996
). Different isoforms of the
7 chain (
7AX1,
7AX2,
7BX1, and
7BX2) are produced by developmentally regulated alternative RNA splicing (Collo et al., 1993
; Song et al., 1993
;
Ziober et al., 1993
; Hodges and Kaufman, 1996
). The alternative cytoplasmic (A, B, and C) and extracellular (X1
and X2) domains are predicted to affect either signal
transduction and/or ligand binding. Evidence to date suggests that the
7 integrins play multiple roles in skeletal
muscle development and in adult fibers. During development, the
7B
1 isoform is believed to mediate myoblast
migration (George-Weinstein et al., 1993
; Echtemeyer et al.,
1996
; Yao et al., 1996
; Crawley et al., 1997
) and the maintenance of myoblast proliferation on laminin (Foster et al., 1987
). As a consequence, this population of precursor cells
expands and becomes localized at sites of myofiber formation. After birth, specific isoforms of the
7
1 integrin are
present at myotendinous and neuromuscular junctions,
and serve as a transmembrane link between laminin in the
basal lamina and the myofiber cytoskeleton (Hodges and
Kaufman, 1996
; Martin et al., 1996
). Structural variants of
the
1 integrin chain in skeletal muscle also contribute to the functional repertoire of this receptor (Van der Flier et
al., 1995
; Zhidkova et al., 1995
).
To further understand the mechanism of neuromuscular
junction formation, we have investigated whether the
7
1 integrin participates in the formation of AChR clusters. Our results demonstrate that the
7
1 integrin does
participate in laminin-induced AChR clustering, that specific alternative splice isoforms of the integrin are integral
to this process, and that laminin, agrin, and the
7
1 integrin interact through a common pathway in the formation
of neuromuscular junctions.
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Materials and Methods |
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MCK Promoter 7 Constructs
The 298-bp DNA sequence encoding the mouse 7 integrin 5' untranslated region (UTR) and signal peptide was amplified by reverse transcriptase (RT)-PCR using the proof reading enzyme Pfu polymerase (Stratagene, La Jolla, CA). The forward primer (5'-ggtcaccttagctggtcctggggcagc-3') was engineered to contain a BstEII restriction site and this
product was cloned into pCRScript (Stratagene). The reverse primer was 5'-ggcaccccatgacgtccagattgaagg-3'. The following amplification conditions
were used: 95°C for 4 min, followed by 35 cycles of 95°C for 1 min, 65°C
for 30 s, and 72°C for 1 min. The 3.3-kb mouse muscle creatine kinase
(MCK) promoter (Jaynes et al., 1986
) was subcloned 5' to the
7 insert using PstI and BstEII restriction sites. The MCK-5'UTR
7 was subcloned
into pBKRSV containing the neoR gene. cDNAs encoding the rat
7AX1,
AX2, BX1, and BX2 isoforms were subcloned into MCK-5'UTR
7 using
AatII and KpnI sites to produce the complete mouse MCK-rat
7 constructs. DNA sequence analysis of each construct verified these clones.
Cell Lines and Transfection
C2C12 mouse myoblasts were cultured in Dulbecco's medium (low glucose) containing 20% FCS, 0.5% chick embryo extract (GIBCO BRL,
Gaithersburg, MD), 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 10 µg/ml kanamycin (P/S/K). These cells were transfected with 5 µg of linearized MCK-7 plasmid using Superfect Reagent
(QIAGEN Inc., Valencia, CA), as per company directions for the production of stable cell lines. Cells were selected in growth medium containing
500 µg/ml G418 (GIBCO BRL), and analyzed by Western blots and immunofluorescence (Song et al., 1992
, 1993
). RMo rat myoblasts (Merrill,
1989
) were grown in Dulbecco's medium (high glucose) containing 15%
horse serum, 2 mM glutamine, 0.5% chick embryo extract, and P/S/K.
Immunoprecipitation and Immunoblot Analysis
Approximately 2 × 105 C2C12 myoblasts transfected with the MCK-rat
7BX2 construct were seeded on fibronectin-coated (20 µg/ml), 100-mm
tissue culture dishes. At confluence, differentiation media was added to
induce myotube formation. AChR clustering was induced with 100 nM
laminin-1, 0.5 nM agrin, or 100 nM laminin-1 and 0.5 nM agrin for 18 h. Cells
were washed once in cold PBS (without Ca+2 or Mg+2) and harvested in
PBS containing 2 mM PMSF. Cells were extracted at 4°C in 200 mM octyl-
-D-glucopyranoside, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM
MgCl2, 2 mM PMSF, 20 µg/ml aprotinin, and 12.5 µg/ml leupeptin. Extracts were incubated at 4°C with anti-AChR antibody K-20 (Santa Cruz
Biotechnology, Santa Cruz, CA) at 10 µg antibody/mg of total protein for
12 h. Protein G-agarose (Sigma, St. Louis, MO) was added at 1 mg protein G/100-mm plate of cells for 4 h at 4°C. The beads were then centrifuged at 4°C, at 12,000 rpm and washed three times in extraction buffer.
Beads were mixed with 1× SDS sample loading buffer and centrifuged.
The supernatants were collected and samples were separated on an 8%
SDS acrylamide gel at 40 mA for 50 min. The protein was transferred to
nitrocellulose filters. Blocked filters were probed with a 1:500 dilution of polyclonal anti-
7CDB (347) antibody that recognizes the
7B cytoplasmic domain (Song et al., 1993
). The filters were washed and incubated with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin. Specificity of the antibody was determined using the immunizing peptide
to block the antibody. Immunoreactive protein was visualized with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
AChR Cluster Assay
LAB-TEK eight-well chamber slides (No. 177445; Nalge Nunc International, Rochester, NY) were coated with purified fibronectin (20 µg/ml)
for 24 h, washed with PBS, seeded with 1.5 × 104 myoblasts in growth medium per well, and then incubated 3 d at 37°C, in 5% CO2. Differentiation
medium containing 2% horse serum and 2 mM glutamine was then added
to induce myotube formation (Sugiyama et al., 1997). 3 d later, mature
C2C12 myotubes were treated with 60 nM laminin-1 (GIBCO BRL) and/or
0.5 nM recombinant COOH-terminal (N2) agrin, for 18 or 5 h. RMo-8
myotubes were treated with 120 nM laminin and/or 1.0 nM agrin. To determine the effect of anti-
7 antibody on colocalization and cluster formation, 50-150 µg H36 mouse anti-rat
7 antibody or 026 mouse anti-rat
7
antibody (Kaufman et al., 1985
; Song et al., 1992
) was added at the time of
cluster induction. To determine if anti-
7 antibody induced cluster formation, 5-250 µg/ml H36-
7 antibody was added in the absence of exogenous agrin or laminin. AChR clusters and
7
1 integrin localization were
then determined by immunofluorescence. Agrin was prepared from transfected COS cells as described (Hoch et al., 1994
).
Immunofluorescence and Quantitation
To detect the exogenous rat 7
1 integrin, transfected C2C12 myotubes
were incubated with 10 µg/ml purified O26 anti-rat
7 mAb (Song et al.,
1992
) and a 1:1,000 dilution of rhodamine-
-bungarotoxin (Molecular
Probes, Inc., Eugene, OR) for 1 h at 37°C, washed three times, and incubated with a 1:100 dilution of FITC-donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 45 min, and then fixed
and mounted as described (Song et al., 1992
). RMo myotubes were processed as above to quantitate the effect of anti-
7 antibody on endogenous rat integrin-mediated AChR clustering. Alternatively, to identify the
endogenous mouse
7
1 integrin, C2C12 myotubes were incubated with
labeled
-bungarotoxin, the cells were then fixed in 70% methanol and reacted with either 10 µg/ml purified rabbit anti-
7B cytoplasmic domain
antibody (Song et al., 1993
), or with a 1:2,000 dilution of rabbit anti-
1 integrin chain antisera (Chemicon International, Inc., Temecula, CA), followed by a 1:100 dilution of FITC-goat anti-rabbit F(ab')2 (Jackson ImmunoResearch Laboratories), or with 10 µg/ml rat anti-mouse
1 integrin
mAb (MAB-1997; Chemicon International, Inc.), followed by a 1:50 dilution of FITC-donkey anti-rat IgG (Jackson ImmunoResearch Laboratories). Quantitation of
7
1 and AChR colocalization was performed in
triplicate cultures, using a Zeiss Photomicroscope III (Carl Zeiss, Inc.,
Thornwood, NY) and 63× Planapo objective. Between 266 and 437 clusters were scored per experimental group. Images of immunofluorescence localization were acquired using a Sony DXC9000 color video
CCD camera.
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Results |
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The 7
1 Integrin Localizes at Laminin-induced
AChR Aggregates
To further understand the process of neuromuscular junction formation, we have investigated whether the 7
1 integrin participates in the formation of AChR clusters.
AChR clustering was induced in C2C12 myotubes with
laminin-1. 18 h after the initiation of clustering, the myotubes were stained using rhodamine-labeled
-bungarotoxin to detect AChR clusters and the polyclonal antibodies specific for the
7B cytoplasmic domain and the
1
integrin chain. Both the
7 and
1 integrin chains were detected at laminin-induced AChR clusters (Fig. 1).
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Specific 7 Integrin Isoforms Colocalize with
AChR Clusters
To study the colocalization of individual 7 integrin isoforms with the laminin-induced AChR clusters, C2C12
mouse myoblasts were transfected with cDNAs encoding
rat
7AX1, AX2, BX1, and BX2 under control of the
mouse MCK promoter. This promoter is active in differentiated myotubes but not in myoblasts (Jaynes et al., 1986
).
The rat-specific mouse anti-
7 mAb O26 was used to detect the rat
7 isoforms expressed in mouse myotubes.
This antibody also enables more definitive localization
and quantitation of the integrin than afforded by conventional antisera. Western blot analysis (not shown) and immunofluorescence (Fig. 2) showed that the transfected cell
populations produced equivalent amounts of the appropriate isoforms of rat
7 protein with the exception that
7BX1 was ~1.5-2-fold higher. The rat integrin was not
detected by immunoblot or immunofluorescence in untransfected C2C12 cells or in control cells transfected with
the parent plasmid pBKRSV. All the cell populations
differentiated at approximately the same time and were
morphologically indistinct from the C2C12 control cells.
Likewise, all cell populations also formed approximately equivalent numbers of AChR clusters.
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The transfected and control cells were analyzed for
laminin- and agrin-induced AChR clustering to determine
which 7 integrin isoforms colocalized with AChR clusters. Fig. 2 shows the results of laminin-induced AChR
clustering in C2C12 and
7 integrin-transfected myotubes.
Both the
7A and
7B alternative cytoplasmic domain isoforms that contain the X2 extracellular domain colocalize with AChR clusters. In striking contrast,
7A and
7B
chains bearing the alternative X1 extracellular domain
showed little or no colocalization. Thus, there is a marked
functional difference in the capacity of the X1 and X2 extracellular domains of the
7 integrin to participate in
laminin-induced AChR clustering. This is consistent with
the predominance of endogenous X2 isoforms in myotubes (Ziober et al., 1993
; Hodges and Kaufman, 1996
)
that become localized at these sites (Martin et al., 1996
).
Quantitation of colocalization confirms our visual observations. The extent of colocalization of 7 isoforms and
AChR clusters promoted by laminin was analyzed in three
separate experiments by scoring AChR clusters and determining the number of clusters with
7 integrin. Clusters
with <50% coincident
7 or
1 integrin and AChR signals
were not scored as colocalized. By this stringent criterion,
>80% of the AChR clusters were colocalized with
7AX2 and
7BX2 integrins upon induction with 60 nM laminin
(Fig. 3). This increase was not observed in
7AX1 or
7BX1 transfected cell lines, again indicating an essential
role for the
7X2 extracellular domain in laminin-induced
AChR clustering. When antibody specific for the rat
7 integrin was added to cultures at the same time cluster formation was initiated with laminin, the anti-
7 antibody inhibited colocalization of the rat integrin with AChRs.
Furthermore, this inhibition was restricted to colocalization of the
7AX2 and
7BX2 isoforms.
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To examine whether the integrin is also present at agrin-induced AChR clusters, colocalization of 7 isoforms and
AChR clusters promoted by agrin, laminin, and by a combination of laminin and agrin, was analyzed. Induction
with 0.5 nM agrin resulted in relatively low levels of receptor colocalization (8-19%) in myotubes expressing any of
the
7 chain isoforms (Fig. 4). In contrast, more than 90%
of the AChR clusters were colocalized with
7AX2 and
7BX2 integrins upon induction with 60 nM laminin. The
numbers of clusters that formed upon incubation with
both 0.5 nM agrin and 60 nM laminin were 30-74%
greater than those induced separately by each protein. Anti-
7 antibody will also inhibit this enhancement (see
below). Moreover, upon induction with both laminin and
agrin virtually all clusters colocalize with the
7AX2 and
7BX2 integrin isoforms. Taken together, these results
suggest that laminin, agrin, and the
7
1 integrin interact
in a common or convergent pathway. In this pathway, either inducer can independently promote AChR clustering,
whereas together, they promote a more vigorous response, with the
7
1 integrin at all clusters.
|
Because the anti-7 mAbs react with rat but not mouse
integrin, it was necessary to use rat myofibers to determine
if the enhancement of AChR clustering fostered by induction with both laminin and agrin could be inhibited with
anti-
7 antibody. Myofibers that developed from the RMo
line of rat myoblasts were induced with agrin, laminin, or
agrin and laminin. O26 anti-
7 antibody was added at the
time of induction. In two separate experiments the antibody reduced the numbers of clusters to ~50% that of
control cultures induced with laminin. The number of
AChR aggregates promoted by agrin was not diminished
by antibody, whereas the enhancement of clustering promoted upon induction with both agrin and laminin was reduced to that promoted by agrin alone (Fig. 5).
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It was recently suggested that laminin and agrin initiate
clustering by alternative pathways that can be distinguished both temporally and biochemically: agrin induces
clustering more rapidly than laminin and requires MuSK
(Sugiyama et al., 1997). We therefore examined early on in
the induction process whether we could distinguish the
association of the
7
1 integrin with agrin- and laminin-induced AChR clusters. Myofibers were treated with
agrin, laminin, or with both agrin and laminin, and cluster
formation was determined 5 h later. Initiation of cluster
formation with both laminin and agrin results early on in a
quantitatively synergistic response compared with induction solely with laminin or agrin (Fig. 6 A). Furthermore,
the rat
7
1 integrin was localized at >60% of the AChR
clusters induced by laminin and agrin (Fig. 6 B). As the total numbers of clusters formed in response to agrin and
laminin were so enhanced, this extent of coclustering is
much greater than expected if agrin induced AChR aggregation independently of the integrin. By 18 h, the
7
1 integrin is detected at essentially all AChR clusters that
form in the presence of laminin and agrin (Fig. 4). These
results suggest that agrin, laminin, and the
7
1 integrin
can participate jointly in the formation of AChR clusters and neuromuscular junctions.
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1 Integrin Is Localized at Both Agrin- and
Laminin-induced AChR Clusters
To confirm the localization of 1 integrins at AChR clusters, an additional anti-
1 integrin mAb was used. As seen
in Fig. 7, the integrin
1 chain was localized to both agrin
and laminin-induced AChR clusters. The
1 integrin colocalized with ~82% of agrin-induced clusters and 92% of
laminin-induced clusters. This result is consistent with the
inhibition of agrin-induced clustering by anti-
1 antibody and the role of the
v
1 integrin in this process (Martin
and Sanes, 1997
). The
1 integrin has also been localized
at agrin-induced AChR clusters in chick myofibers (Bozyczko et al., 1989
).
|
Immunoprecipitation of AChR and the 7
1 Integrin
To further demonstrate a physiologic role for the 7
1 integrin in neuromuscular junction formation, immunoprecipitation was carried out to determine whether the AChR
and integrin were physically associated. C2C12
7BX2
myotubes were induced with agrin, laminin, or agrin and
laminin. Anti-AChR mAb and protein G-agarose were used to immunoprecipitate the AChR and its associated
molecules. Western blot analysis reveals that the
7
1 integrin is coprecipitated with the AChR and that their association in this complex is dependent on induction with
laminin. Whereas the integrin is not detected in uninduced
fibers or upon induction with agrin, it is associated with
AChR when laminin or laminin and agrin were used to
promote clustering (Fig. 8). In the presence of both agrin
and laminin, the
7 chain may undergo increased proteolytic processing (Song et al., 1992
). This laminin-dependent association of the
7
1 integrin with AChRs further
supports the conclusion that the integrin has a direct physiologic role in the clustering of AChR.
|
Anti-7 Integrin Antibody Promotes AChR Clustering
Lastly, engaging the 7
1 integrin with laminin or cross-linking the exogenous integrin with anti-
7 antibody promotes a detergent-stable association of the integrin with
the cell cytoskeleton (Lowrey and Kaufman, 1989
; Song
et al., 1993
) and an increase in free intracellular calcium
(Kwon, M.S., C.S. Park, K.R. Choi, S.J. Kaufman, and
W.S. Song, manuscript in preparation). In the absence of
added laminin or agrin, relatively low concentrations of
anti-
7 antibody also promotes AChR clustering on
7BX2
transfected C2 myofibers (Fig. 9). At higher antibody concentrations, where excess antibody fails to cross-link the
integrin, cluster formation is minimal. Thus, engaging the
7
1 integrin with either its ligand or with antibody is sufficient to promote AChR clustering.
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Discussion |
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It has become clear that at least two molecules in the skeletal muscle basal lamina can initiate clustering of AChRs,
the heparan sulfate proteoglycan agrin and laminin. In this
report we have shown that specific isoforms of the 7
1
integrin participate in laminin-induced AChR clustering in
C2C12 myotubes as well as clustering induced by both
laminin and agrin. Laminin was shown to induce colocalization of the integrin and AChR. In the presence of both
laminin and agrin, cluster formation and colocalization with the integrin are markedly enhanced. High concentrations of two different anti-
7 mAbs inhibit colocalization
of the integrin with AChR clusters as well as the enhanced
response promoted by both laminin and agrin. In addition
to the laminin-dependent colocalization of the
7
1 integrin with AChRs, laminin also promotes the physical association of these two receptors, further suggesting that the integrin has a direct physiologic role in AChR aggregation. Engaging the integrin with concentrations of anti-
7
antibody that cross-link the integrin also induces AChR
clusters. This is likely a consequence of association of
the integrin and cell cytoskeleton (Lowrey and Kaufman,
1989
; Song et al., 1993
), and an
7
1-mediated increase in
intracellular calcium (Kwon, M.S., C.S. Park, K.R. Choi,
S.J. Kaufman, and W.S. Song, manuscript in preparation)
that is essential to AChR clustering (Megeath et al., 1998).
Anti-laminin antiserum will also enhance receptor clustering induced by agrin (Montanaro et al., 1998
), presumably
by this same mechanism. Although laminin- and agrin-
induced clustering of AChR can be studied independently, we suggest that a unified mechanism may underlie receptor aggregation and differentiation of the postsynaptic membrane. The following evidence supports this: (a)
Laminin and agrin together produce more vigorous clustering than either protein induces alone and virtually all
clusters are associated with the integrin by 18 h. The
7
1
integrin is also localized in vivo at neuromuscular junctions (Martin et al., 1996
). As only 60% of clusters formed 5 h after induction are colocalized with the exogenous integrin, agrin and laminin may initiate clustering independently; a common aggregation mechanism predominates
thereafter. (b) The enhanced aggregation of AChR clustering induced by agrin and laminin is completely inhibited by anti-
7 integrin antibody. (c) AChR and the
7
1
integrin physically associate upon induction of cluster formation with laminin or with laminin and agrin. (d) AChR
aggregates do form in agrin null mice, although they are
reduced in number, size and density (Gautam et al., 1996
).
Thus, agrin appears to be essential, but not sufficient for
the normal development of postsynaptic sites in vivo. Our
data suggest that laminin and the
7
1 integrin are additional components required for normal synaptic organization. Laminin, agrin, and the
7
1 integrin are also all expressed during formation of the postsynaptic membrane in
vivo (Bayne et al., 1984
; McMahan, 1990
; Hall and Sanes,
1993
; Bowe and Fallon, 1995
; Martin et al., 1996
; Ruegg
and Bixby, 1998
). Although
7 null mice do form neuromuscular junctions, no detailed structural study of these
sites, nor the expression of other integrins at postsynaptic
sites, nor physiologic studies of these mice have been reported (Mayer et al., 1997
). (e) MuSK, a receptor protein tyrosine kinase (Valenzuela et al., 1995
; Ganju et al.,
1996), is required for AChR aggregation in vivo: MuSK
null mice do not form these clusters (DeChiara et al.,
1996
). Although laminin can induce limited AChR clustering in MuSK null myotubes in vitro (Sugiyama et al.,
1997
), MuSK appears to be essential to the rigorous combined laminin and agrin-induced AChR aggregation in
vitro and in vivo. (f) Since myotubes synthesize laminin,
it is questionable whether any in vitro experiments on
AChR clustering have been conducted in the complete absence of laminin. Therefore, it is not surprising that "spontaneous" clusters that form in the absence of exogenous
laminin or agrin do have localized
7 integrin, and that 10-
20% of AChR clusters formed in response to agrin do
have exogenous
7
1 integrin at those sites. (g) Anti-
1
integrin antibody completely inhibits agrin-induced cluster
formation, presumably by inhibiting
v
1 integrin (Martin
and Sanes, 1997
) as well as the
7
1 integrin mobilized by
endogenous laminin. This is consistent with our localization of the
1 integrin chain at agrin-induced AChR clusters and similar findings in the chick (Bozyzcko et al., 1989). Differences in antibody specificity, determinant accessibility, and expression of endogenous laminin may account for the lower level of
1 integrin detected by Montanaro et al. (1998)
at agrin-induced AChR clusters. (h)
Laminin and agrin have common structural motifs and
thus have the potential to interact with common elements. Furthermore, although the agrin used in many in vitro
studies lacks the domain that supports its binding to laminin, native neural and muscle agrin can directly interact
with laminin (Hall and Sanes, 1993
; Bowe and Fallon,
1995
; Sugiyama et al., 1997
; Denzer et al., 1998
; Ruegg and
Bixby, 1998
). Thus the two inducers may act in concert
both physically and mechanistically. The interaction of
laminin and agrin may enhance their association with the
MuSK receptor complex (Glass et al., 1996
). Our results
suggest that the
7
1 integrin may also be part of this
complex. (i) The
7
1 integrins provide the potential for
signal transduction and for the association with the cytoskeleton that may be significant to cluster formation and
the integrity of neuromuscular junctions (Song et al., 1993
;
Hodges and Kaufman, 1996
). (j) Engaging the integrin
with laminin or anti-
7 antibody induces AChR aggregation and an increase in intracellular calcium that is required for agrin-induced AChR clustering (Kwon, M.S.,
C.S. Park, K.R. Choi, S.J. Kaufman, and W.S. Song, manuscript in preparation; Megeath et al., 1998).
We believe these results can best be explained by a unified mechanism of laminin and agrin-induced clustering.
Additional molecules also appear important to the aggregation of AChRs. The v
1 integrin binds agrin and also
appears to be required for clustering (Martin and Sanes,
1997
).
-Dystroglycan can bind laminin and agrin and it
also may function in laminin and agrin-induced AChR
clustering (Bowe and Fallon, 1995
; Montanaro et al., 1998
; Ruegg and Bixby, 1998
). Although Montanaro et al. (1998)
suggest that integrins may not be involved in laminin-
induced AChR aggregation, our results demonstrate otherwise. It will be interesting to further define the molecular
orchestration of this concerted mechanism of receptor aggregation.
With respect to the integrin, both the 7A and
7B alternative cytoplasmic domains engage in AChR clustering,
and both are localized at neuromuscular junctions in vivo
(Martin et al., 1996
). In contrast, the alternative X1 and
X2 extracellular domains of the
7 chain have distinct
functions. Although both can bind laminin, only those isoforms containing the 39-amino acid X2 sequence engage in AChR clustering. In addition to a role in synaptogenesis, specific isoforms of the
7
1 integrin also appear essential to the maintenance of skeletal muscle and synaptic
function (Martin et al., 1996
; Hodges et al., 1997
). Lastly,
based on the involvement of the
7
1 integrin in the formation and integrity of neuromuscular junctions, mutations in the
7 integrin gene might be expected to underlie
muscle diseases that exhibit a neurologic component. In
this regard, we have recently identified three patients with mutations in the
7 gene that do not express the
7 integrin protein and who show congenital myopathies with delayed motor milestones (Hayashi et al., 1998
).
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Footnotes |
---|
Address correspondence to Stephen J. Kaufman, Ph.D., Department of Cell and Structural Biology, University of Illinois, B107 Chemical and Life Sciences Laboratory, Urbana, IL 61801. Tel.: (217) 333-3521. Fax: (217) 244-1648. E-mail: stephenk{at}uiuc.edu
Received for publication 24 June 1998 and in revised form 28 September 1998.
We thank Dr. S. Hauschka for providing the MCK promoter used in these experiments and Dr. Jean Buskin for her helpful advice. We also thank Mr. Gregory Wallace for his skillful technical assistance.
This work was supported by the Muscular Dystrophy Association of America and National Institutes of Health.
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Abbreviations used in this paper |
---|
AChR, acetylcholine receptor; MCK, muscle creatine kinase; MuSK, muscle-specific tyrosine kinase.
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