From the Department of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7, Suita, Osaka 565, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The rat L6 skeletal muscle cell line was used to
study expression of the dystrophin-containing glycoprotein complex and
its interaction with the integrin system involved in the cell-matrix adhesion reaction. A complex of dystrophin and its associated proteins
was fully expressed in L6 myotubes, from which anti-dystrophin or
anti--sarcoglycan co-precipitated integrin
5
1 and other focal
adhesion-associated proteins vinculin, talin, paxillin, and focal
adhesion kinase. Immunostaining and confocal microscopy revealed that
dystrophin,
-sarcoglycan, integrin
5
1,
and vinculin exhibited overlapping distribution in the sarcolemma,
especially at focal adhesion-like, spotty structures. Adhesion of cells
to fibronectin- or collagen type I-coated dishes resulted in induction of tyrosine phosphorylation of
- and
-sarcoglycans but not
-sarcoglycan. The same proteins were also tyrosine-phosphorylated
when L6 cells in suspension were exposed to Arg-Gly-Asp-Ser peptide.
All of these tyrosine phosphorylations were inhibited by herbimycin A. On the other hand, treatment of L6 myotubes with
- and
-sarcoglycan antisense oligodeoxynucleotides resulted in complete
disappearance of
- and
-sarcoglycans and in significant reduction
of levels of the associated focal adhesion proteins, which caused about 50% reduction of cell adhesion. These results indicate the existence of bidirectional communication between the dystrophin-containing complex and the integrin adhesion system in cultured L6 myocytes.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several classes of cell adhesion receptors, including integrins,
dystroglycan, cadherins, and members of the immunoglobulin family, are
coexpressed by skeletal muscle and presumably play critical roles
during skeletal muscle differentiation and development (see Ref. 1 for
review). Integrins form a large family of heterodimeric transmembrane
proteins with different and
subunits (2). The ligands of the
integrins can be components of the extracellular matrix or other
integral membrane proteins, such as VCAM-1. Previous studies suggest
that different integrins may play specific roles in different
developmental and cellular phenomena. The
1 integrin subfamily, with its different
subunits, is a major group of integrins expressed in muscle cells and has been shown to be required for myoblast differentiation and myotube formation (1, 3). A large
number of different integrin
subunits are expressed in developing
muscle cells, and some of them localize in unique functional cellular
regions and seem to play distinct roles in regulation of myogenesis,
although their precise functions remain to be clarified (1, 4, 5).
Myogenic cells associate with extracellular matrix, which is
advantageous for cell terminal differentiation. The extracellular matrix-integrin interaction occurs at various cytoskeletal-sarcolemmal linkages, such as focal adhesion-like structures, myotendinous and
neuromuscular junctions, and the costameres (1, 4, 6). At the focal
adhesions present in cultured cells, integrins cluster and associate
with many cytoskeletal proteins, such as talin, vinculin, paxillin, and
-actinin, as well as FAK1
and other protein kinases (7-9). The focal adhesion complex is the
major site of actin filament attachment. Recent evidence indicates that
assembly of the focal complex requires both the engagement of integrins
with extracellular matrix and the integrin activation by intracellular
signaling, although the physiological mechanism of the latter is
largely unknown (9). Integrin occupancy with ligands and clustering
have been shown to trigger tyrosine phosphorylation of several
intracellular proteins, including FAK, paxillin, tensin, and
mitogen-activated protein kinase (2, 7, 9). Recent evidence suggests
that tyrosine phosphorylation of FAK and other focal adhesion proteins
is not required for focal adhesion formation but is important for the
other integrin-induced signalings, such as control of cell growth (10).
However, assembly of the focal adhesion complex can be inhibited by
tyrosine kinase inhibitors (8, 11).
Dystroglycan, which exists as a noncovalently linked complex of -
and
-subunits, is another important cell adhesion receptor that
links the extracellular matrix with the actin cytoskeleton (12-14). In
skeletal muscle cells, it forms a tight complex with dystrophin
together with other dystrophin-associated proteins (DAPs) including
-,
-,
- and
-sarcoglycans, the 25-kDa protein, and
syntrophins (13, 14, 16-20). Dystroglycan is expressed widely in
nonmuscle cells, as well as in myocytes, whereas sarcoglycans are
expressed predominantly in striated muscle cells (12, 15-20). The
integrity of the dystrophin-DAP complex seems to be essential for the
viability of muscle cells, because disruption of the complex due to a
defect in dystrophin or any one of the sarcoglycans has been reported
to cause various forms of inherited muscular dystrophy (13, 14, 16-18,
21). Sarcoglycans thus seem to be functionally and pathologically as
important as dystrophin. At present, however, like all other components
of the dystrophin-DAP complex, little is known about the functional and
structural roles of sarcoglycans, including their interaction with
other cellular proteins.
In our recent preliminary report (22), we have presented evidence that
the dystrophin-DAP complex is associated with the focal adhesion
assembly proteins, such as the integrin 1 subunit, vinculin, and FAK in serum-deprived, differentiated BC3H1
cells, a nonfusing muscle cell line. We have also shown that loss of
-sarcoglycan induced by the antisense ODN treatment results in a
significant inhibition of adhesion to the substrate by these cells. In
this communication, we report that muscle-specific
- or
-sarcoglycans were tyrosine-phosphorylated when differentiated rat
skeletal L6 cells were adhered to the extracellular matrix. On the
other hand, treatment of these cells with antisense ODN directed
against
- or
-sarcoglycan significantly decreased the associated
focal adhesion proteins and concomitantly inhibited cell adhesion. The
results indicate the existence of bidirectional communication between
the dystrophin-DAP complex and the integrin adhesion system in these
cultured cells.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies--
Monoclonal mouse antibodies against dystrophin
(VIA4-2 A3, Upstate Technology Inc.), -sarcoglycan (IVD3-1,
Upstate Technology Inc.),
-dystroglycan (NCL-43DAG, Novocastra),
laminin (anti-laminin monoclonal antibody, Sigma), integrin
1 (MAR4, Pharmingen), vinculin (VIN-11-5, Sigma), and
phosphotyrosine (PY20, Upstate Technology Inc.) were used. Anti-FAK,
anti-paxillin, anti-ezrin, and anti-cortactin monoclonal antibodies
were obtained from Transduction Laboratories. Anti-syntrophin
monoclonal antibody SYN1351 was a gift from Dr. Stanley C. Froehner
(23). Polyclonal antibodies against integrin
5
1 (VLA-5) and
V integrin
subunit (VNR139) were purchased from Chemicon and Life Technologies,
Inc., respectively. Anti-actin polyclonal antibody, which recognizes
all actin isoforms, was a gift from Dr. Keigi Fujiwara (24). A
polyclonal rabbit antibody against the cytoplasmic domain of
-sarcoglycan fused to glutathione S-transferase was
prepared as described previously (25). Preparation of polyclonal
chicken antibodies against glutathione S-transferase fusion
proteins containing amino acids 92-318 of rabbit skeletal muscle
-sarcoglycan and amino acids 64-291 of rabbit skeletal muscle
-sarcoglycan was also described (26). These chicken antibodies,
which were subsequently affinity purified using glutathione S-transferase and glutathione
S-transferase-sarcoglycan fusion proteins as described
previously (27), recognized single proteins of the expected molecular
weights for respective sarcoglycans in the immunoblot assay of L6 cell
lysates (see Fig. 3B).
Cell Culture and Cell Adhesion Assay-- L6 rat skeletal myoblasts (ATCC) were grown on 100-mm tissue culture dishes (Falcon) in DMEM supplemented with 10% heat-inactivated FCS, 100 units/ml penicillin and 100 µg/ml streptomycin. After 4-5 days, ~80% of cells were fused to form myotubes. We used these cells for all experiments in this study. For cell adhesion assay, these cells were detached from dishes by trypsinization, washed once with DMEM containing 2% FCS and twice with DMEM containing 0.5% BSA, and subsequently preincubated with DMEM containing 0.5% BSA for 15 min at 37 °C on a rotator. Cells (104 cells) were then plated onto dishes coated with fibronectin (4 µg/cm2), collagen (type I) (2 µg/cm2), or poly-L-lysine (3 µg/cm2) and incubated at 37 °C for the times indicated (see Fig. 4). After being washed twice with DMEM containing 0.5% BSA, attached cells were counted after staining with trypan blue. The cells referred to as "cells in suspension" were held in suspension for 30 min. In some experiments, cells in suspension were incubated with Arg-Gly-Asp-Ser peptide (RGDS peptide; Peptide Institute, Inc.).
Preparation of Cell Lysates, Immunoprecipitation, Immunoblotting,
and Laminin Overlay Assay--
Cells (1.5 × 106
cells) were washed three times with PBS (137 mM NaCl, 2.7 mM KCl, 9.3 mM Na2HPO4,
1.5 mM KH2PO4, pH 7.4) and lysed
for 5 min with immunoprecipitation buffer (50 mM Tris/HCl, pH 7.4 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 1 mM phenylmethanesulfonyl fluoride, 1 mM sodium
orthovanadate, 10 mM NaF, 10 mM sodium
pyrophosphate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). The
lysates were centrifuged at 12,000 × g for 15 min at
4 °C, and the resultant supernatants were precleared with 50 µl of
protein A-Sepharose beads for 1 h at 4 °C on a rotator. After
centrifugation, the supernatants were incubated with antibodies for
2 h at 4 °C and then with 100 µl of protein A-Sepharose beads
for 1 h. The resultant immunocomplexes were washed 7-10 times
with the ice-cold immunoprecipitation buffer devoid of SDS and
deoxycholate and finally boiled in 30 µl of SDS-polyacrylamide gel
electrophoresis sample buffer for 5 min. The samples (100 µg/lane)
were then subjected to SDS-polyacrylamide gel electrophoresis on a 7.5 or 10% gel as described previously (28). In some experiments, we used
an immunoprecipitation buffer that was similar except for containing
0.2% SDS and omitting the Triton X-100 and sodium deoxycholate for
immunoprecipitation and washing (see "Results"). For immunoblot
analysis, proteins were transferred to Immobilon membranes, reacted
with primary and then with secondary antibodies, and subsequently
visualized with the ECL immunoblotting detection system (Amersham
Corp.) as described previously (28). For detection of -dystroglycan,
laminin overlay assay was performed as described previously (29).
Antisense ODN Treatment--
The following 16-mer
phosphorothioate-modified ODNs were synthesized (Sawady Technology,
Inc.) and used: -sarcoglycan antisense ODN, 5
-GTGGTCTGCTGGGCCT-3
(nucleotides 64-79 of rat
-sarcoglycan cDNA);
-sarcoglycan
antisense ODN, 5
-AGCCTTCTCCCGCATGGA -3
(nucleotides 96-113 of rat
-sarcoglycan cDNA);
-sarcoglycan antisense ODN, 5
-
TTGGAGAAAACCACATCACTT-3
(nucleotides 228-248 of rat
-sarcoglycan
cDNA); and control ODN with a random nucleotide sequence. After
cultivation in 10% FCS for 5 days, L6 cells were treated with 300 nM antisense or control ODN in the presence of 10 µg/ml
Lipofectin (Life Technologies, Inc.) but in the absence of FCS for
6 h. Cells were then washed three times with DMEM and left in DMEM
containing 5% FCS and 300 nM ODN for an additional 48 h. The latter procedure was repeated to expose cells to ODN for a total
of 5 days. Cells were subsequently screened for expression of
-,
-, and
-sarcoglycans by immunoblotting.
Immunofluorescence--
L6 cells (1 × 104
cells) cultured on fibronectin-coated 60-mm plates were fixed in PBS
containing 4% paraformaldehyde for 5 min at room temperature and then
permeabilized in PBS containing 0.5% Triton X-100 for 5 min at room
temperature. After being washed three times with PBS, cells were
incubated with a blocking solution (Block ACE, Dainippon
Pharmaceuticals) containing 3% BSA for 1 h at room temperature
and then washed three times with PBS containing 0.1% Tween 20. Cells
were treated with one of the following primary antibodies for 4-6 h at
4 °C: anti--sarcoglycan (dilution, 1:500), anti-dystrophin
(1:100), anti-integrin
1 (1:200), anti-vinculin (1:500),
or anti-integrin
5
1 (1:500). After being
washed three times with PBS containing 0.1% Tween 20, cells were
incubated with either rhodamine-labeled donkey anti-rabbit IgG
(Chemicon) (1:1000) or fluorescein-labeled donkey anti-mouse IgG
(Cappel) (1:500) for 1-3 h at 4 °C and then washed three times with
PBS containing 0.1% Tween 20. For double staining with a combination of polyclonal (rabbit) and monoclonal (mouse) antibodies, fixed and
permeabilized cells were incubated with a mixture of two primary antibodies and then with a mixture of the fluorescein-labeled anti-mouse and rhodamine-labeled anti-rabbit IgGs. Cells were examined
by confocal laser scanning microscopy using a Bio-Rad MRC-1024 system
(Bio-Rad) mounted on an Olympus BX50WI epifluorescence microscope with
a plan-apochromat × 60 water immersion objective lens
(Olympus).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression and Localization of Dystrophin-DAP Complex in L6 Myotubes-- The rat L6 skeletal muscle cell line has widely been used as a model for studying myogenesis. Upon reaching subconfluency in 10% serum, L6 myoblasts spontaneously fuse with each other to form multinucleated myotubes and express skeletal muscle proteins (30, 31). Using specific antibodies, we examined the expression and distribution of the components of the dystrophin-DAP complex in L6 cells that had been cultured on fibronectin-coated dishes for 4-5 days.
The material immunoprecipitated from adherent L6 cells with anti-dystrophin contained proteins recognized by antibodies against dystrophin,
|
|
Cell Adhesion-induced Tyrosine Phosphorylation of
Sarcoglycans--
We investigated tyrosine phosphorylation of proteins
in L6 cells in response to cell adhesion. L6 cells plated on
fibronectin-coated dishes for 30 min exhibited a high level of tyrosine
phosphorylation of proteins, with Mr (in
thousands) 120~130, 100, 70~85, 50, and 30~45 (Fig.
3A, lane 3). We identified one
of the phosphoproteins in the Mr
120,000~130,000 range to be FAK (see below), which is consistent with
similar findings obtained with fibroblastic cells under comparable
conditions (11, 33). We further examined whether the proteins
precipitated from adherent L6 cells with anti-dystrophin or
anti--sarcoglycan were tyrosine-phosphorylated. In each
immunoprecipitate, proteins of Mr 50,000 and
35,000 were most prominently tyrosine-phosphorylated, suggesting that
they are
- and
-sarcoglycans (Fig. 3A, lanes 4-6).
These proteins, however, were not
tyrosine-phosphorylated in nonadherent L6 cells (Fig. 3A, lane
2, see also Figs. 4A and
5A). Tyrosine phosphorylation of 50- and 35-kDa proteins were not detected in the material
precipitated with anti-
V integrin subunit (Fig.
3A, lane 7).
|
|
|
|
Effect of Sarcoglycan Antisense ODNs on Adhesion of L6
Cells--
In the experiment shown in Fig.
7A, we compared adhesion
activity of L6 cells treated with each of sarcoglycan antisense ODNs. We found that adhesion to fibronectin-coated dishes was inhibited by
about 50% in cells pretreated with - or
-sarcoglycan antisense ODN for 5 days. In these cells, tyrosine phosphorylation of the protein
recognized with anti-FAK was also decreased to 64.5 ± 4.9 and
70.3 ± 5.5% (n = 3) of control, respectively.
Treatment with random ODN did not affect adhesion activity of L6 cells, whereas treatment with
-sarcoglycan antisense ODN decreased cell adhesion slightly (Fig. 7A). We found that these antisense
ODNs did not alter the phalloidin-stained actin cytoskeleton structure of L6 cells.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have provided several lines of evidence
suggesting that the dystrophin-DAP complex in cultured rat L6 myotubes has specific bidirectional interactions with the
5
1 integrin adhesion system: 1)
anti-dystrophin or anti-
-sarcoglycan co-precipitated integrin
5
1 and other focal adhesion-associated
proteins (vinculin, talin, paxillin, and FAK) together with the
components of the dystrophin-DAP complex (Figs. 1A and
5B). Anti-dystrophin did not co-precipitate other
cytoskeletal proteins, such as
V integrin subunit,
ezrin, cortactin, and actin (Fig. 1, A and C).
Conversely, anti-
1 integrin subunit or anti-FAK
co-precipitated the components of the dystrophin-DAP complex, although
antibodies against
V integrin subunit, ezrin, and
cortactin did not precipitate them (Figs. 1B and
5B and "Results"). 2) Immunofluorescence study using confocal microscopy revealed that
-sarcoglycan and vinculin
exhibited overlapping distribution in the sarcolemma, especially at the spotty structures in the basal cell area, when L6 cells were plated on
fibronectin-coated dishes (Fig. 2). 3) Adhesion of L6 cells to
fibronectin or collagen type I resulted in induction of tyrosine phosphorylation of
- and
-sarcoglycans (Figs. 3-5). Tyrosine
phosphorylation of these proteins was similarly induced when L6 cells
in suspension were exposed to RGDS peptide that interacts with
integrins (2, 34) (Fig. 6). 4) Finally, treatment of L6 cells with
-
and
-sarcoglycan antisense ODNs resulted in complete disappearance of
- and
-sarcoglycans and significant reduction of the
associated focal adhesion proteins (Figs. 3B and
7B), which caused about 50% reduction of cell adhesion
(Fig. 7A).
The integrin system involved in the bidirectional interaction with the
dystrophin-DAP complex in L6 cells seems to be mainly one that is
engaged in the cell-matrix adhesive reaction because adhesion-induced
physical association of the dystrophin-DAP complex with focal
adhesion-associated proteins is required for the tyrosine phosphorylation of - and
-sarcoglycans. In L6 cells in
suspension,
- and
-sarcoglycans and FAK were not
tyrosine-phosphorylated, and anti-dystrophin or anti-
-sarcoglycan
did not co-precipitate FAK, vinculin, and talin from these cells,
although they co-precipitated some integrin
5
1 (Figs. 1 and 5). However, when cells
in suspension had been treated with RGDS peptide,
- and
-sarcoglycans and FAK were co-precipitated, and their tyrosine
phosphorylation occurred as in cells plated on fibronectin (Fig. 6 and
"Results"). On the other hand, tyrosine phosphorylation of
- and
-sarcoglycans was not detectable in cells plated on
poly-L-lysine (Fig. 5A, lane 2), although a
small amount of phosphorylated FAK was found to be present in the
-sarcoglycan immunoprecipitate (Fig. 5B, lane 2).
Confocal double-labeling study revealed a marked reduction of the
overlap of signals from anti-
-sarcoglycan and anti-vinculin at the
basal portion of a cell plated on poly-L-lysine (Fig.
2E). Thus, adhesion-induced recruitment of relevant proteins
to the focal adhesion-like, spotty structures seems to be required for the phosphorylation of sarcoglycans. Of note, the amount of integrin
5
1, vinculin, talin, or FAK
co-precipitated with anti-dystrophin was about 15% of the amount of
each that was present in the total cell lysate (see "Results"),
suggesting that relatively small fractions of these proteins are
tightly associated with the dystrophin-DAP complex. This could be due
to existence in L6 cells of much larger amounts of integrin
5
1 and other focal adhesion proteins
compared with the dystrophin-DAP complex. Another possibility could be that colocalization of these focal adhesion proteins with the dystrophin complex occurs only at the limited cell area. By
immunofluorescence staining, dystrophin,
-sarcoglycan,
5
1, and vinculin were present at the
sarcolemma in both the basal and nonbasal portions of the cell (Fig.
2). Signals of
-sarcoglycan and vinculin were partially colocalized
in both of these membrane areas with much intense overlap at the basal
portion of the cell (Fig. 2C). At present, the quantitative
aspect of the interaction between these proteins is unclear.
In fibroblasts and other cells, formation of the focal adhesion contact
induces autophosphorylation and subsequent activation of FAK, a member
of a family of structurally distinct tyrosine kinases (9, 35). Once
phosphorylated on its tyrosine residue (Tyr397), FAK binds
to SH2 domain of pp60src, recruiting the latter to focal
adhesion contacts (36). In this study, we found that recombinant
pp60src is able to tyrosine phosphorylate - and
-sarcoglycans in
vitro.2 Thus,
phosphorylation of sarcoglycans in response to cell adhesion in L6
cells is likely to be catalyzed by either FAK or an FAK-stimulated tyrosine kinase, such as a member of the Src family of kinases.
Sarcoglycans are intrinsic membrane proteins of
Mr 35,000-50,000, forming a tight subcomplex,
which is associated with the dystroglycan subcomplex, dystrophin, and
other proteins to form the dystrophin-DAP complex (see
"Introduction"). Sarcoglycans possess large extracellular domains
and small cytoplasmic tails containing no obvious catalytic domains
(17-20, 37). We found that cell adhesion induces tyrosine
phosphorylation of - and
-sarcoglycans but not
-sarcoglycan
and that phosphorylated
-sarcoglycan co-precipitates with
phosphorylated
-sarcoglycan and vice versa (Fig. 3,
A and B).
-Sarcoglycan contains only one
tyrosine residue (Tyr310) in the cytoplasmic tail at a
region close to the transmembrane segment (37), whereas
-sarcoglycan
has four cytoplasmic tyrosine residues (18). Tyr310 in
-sarcoglycan would thus be phosphorylated in adherent L6 cells,
although no information is available for the phosphorylation site in
-sarcoglycan. There is also no information concerning the
significance of tyrosine phosphorylation of
- and
-sarcoglycans with respect to their structural or signaling roles.
The antisense ODN directed against - or
-sarcoglycan for 5 days
inhibited adhesion of L6 cells to fibronectin by about 50% (Fig.
7A). After each antisense ODN treatment,
- or
-sarcoglycan disappeared completely from these cells (Fig.
3B), although expression levels, as well as the ability of
dystrophin and other DAPs to form a tight complex, were not affected by
the treatment (see "Results"). Under these conditions, integrin
1 subunit, vinculin, talin, and FAK co-precipitated with
anti-dystrophin were decreased by 60-70%, although levels of these
proteins in the total cell lysate were not reduced greatly (Fig.
7B and "Results"). In contrast, antisense ODN directed
against
-sarcoglycan exerted minimal effects on cell adhesion, as
well as on the association of the dystrophin-DAP complex with focal
adhesion proteins. Thus,
- and
-sarcoglycans may be involved
directly in L6 cell adhesion by interacting with focal
adhesion-associated proteins. Of note, in skeletal muscle of patients
with various forms of limb-girdle muscular dystrophy, mutations in each
of the sarcoglycans result in the concomitant absence of the other
sarcoglycans from the sarcolemma (16-18, 21). Such findings are in
marked contrast to the observed absence of the effect of loss of the
sarcoglycan protein on the stability of the dystrophin-DAP complex in
L6 cells. This may reflect difference in molecules that contact or
surround sarcoglycans in the dystrophin-DAP complex in cultured L6
cells versus adult skeletal muscle.
Localization of dystrophin in integrin-rich cell areas has been
reported previously. By immunofluorescence staining, Kramarcy and
Sealock (38) observed that dystrophin and a 48-kDa protein, which is
recognized by anti-syntrophin, are colocalized to talin-positive focal
adhesion-like structures in cultured Xenopus muscle cells. In addition, Lakonishok et al. (39) observed transient
overlapping localization of 5
1 integrin
and dystrophin in a punctate lattice-like structure on the surface of
the cultured chick skeletal myotube during muscle development. We have
also reported that some focal adhesion-associated proteins are
co-precipitated with the dystrophin-DAP complex from
detergent-solubilized nonfusing muscle cell line BC3H1 cells that have
been cultured under differentiation conditions (22). On the other hand,
in platelets in which dystrophin is not expressed, a dystrophin-related
protein (utrophin) is a component of the membrane skeleton, and upon
binding of adhesive extracellular ligands to integrin
IIb
3, utrophin has been shown to
redistribute, along with other membrane skeleton proteins including
talin and vinculin, to the low-speed detergent-insoluble fraction from
the high-speed detergent-insoluble fraction (40).
The bidirectional interaction between the dystrophin-DAP complex and
the integrin-cytoskeletal system in cultured muscle cells suggests that
it could potentially play an important role in the regulation of the
function of each adhesion system. Integrins are the best studied
adhesion receptor, and much is known about the mechanism by which they
mediate the bidirectional transfer of information across the plasma
membrane (2, 7, 9). In contrast, little is known about the signaling
role of the dystrophin-DAP complex, for which dystroglycan is a
transmembrane adhesion receptor. As briefly discussed under
"Introduction," sarcoglycans seem to be particularly important for
the function of the dystrophin-DAP complex in striated muscle cells.
However, there has been no information available for the interaction of
sarcoglycans with other cellular proteins. It is intriguing to ask
whether there is a similar bidirectional interaction involving
sarcoglycans between the dystrophin-DAP complex and the
integrin-containing structures in the adult skeletal muscle. Previous
immunofluorescence studies of striated muscle have suggested that
dystroglycan, dystrophin, integrin 1 subunit, vinculin,
talin, and spectrin localize to the submembrane two-dimensional lattice
structures of costameres that mediate lateral attachment of the
contractile apparatus to the sarcolemma (6, 41-43).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. S. Froehner and K. Fujiwara for their generous gift of antibodies.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant-in-aid for Scientific Research on Priority Areas 343 from the Ministry of Education, Science and Culture, Research Grant for Cardiovascular Diseases 8A-1 from the Ministry of Health and Welfare of Japan, and a grant from Sankyo Life Science Foundation.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. Tel.: 81-6-833-5012;
Fax: 81-6-872-7485.
1 The abbreviations used are: FAK, focal adhesion kinase; DAP, dystrophin-associated protein; ODN, oligodeoxynucleotide; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin.
2 T. Yoshida, unpublished observation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|