From the
Dystroglycan is a novel laminin receptor that links the
extracellular matrix and sarcolemma in skeletal muscle. The
dystroglycan complex containing
Dystroglycan was first identified as a component of the
dystrophin-glycoprotein
complex
(1, 2, 3, 4, 5) .
Grb2 is a
25-28-kDa protein containing SH2 and 3 domains
(17) and
has been found to be the mammalian homolog of sem-5 protein
product in Caenorhabditis elegans, which is involved in vulval
development and in sex myoblast migration
(18) . Grb2 functions
as an adapter protein through its SH2 or SH3 domains to interact with
proteins containing phosphotyrosine or proline-rich
domains
(17, 18, 19, 20, 21, 22) .
Grb2 is also an important protein linking receptor tyrosine kinases to
small GTP-binding protein signaling, such as growth factor-induced
cytoskeleton
organization
(17, 18, 19, 20, 21, 22, 23, 24, 25) .
Furthermore, integrin-mediated signal transduction is also coupled to
activation of the Grb2/Ras pathway
(26) .
Here, we demonstrate
that
Affinity chromatography with
It has been
demonstrated that the extracellular matrix affects many aspects of cell
behavior such as cell adhesion, migration, proliferation, and
differentiation
(29, 30) . Attachment to the
extracellular matrix is also required for maintaining cell viability
(31-33). It is possible that the extracellular
matrix-dystroglycan-Grb2 interaction is essential for muscle cell
viability. The disruption of this interaction may affect normal muscle
cell function and lead to muscle cell apoptosis or necrosis in several
types of muscular dystrophy where muscle cell attachment to the
extracellular matrix is
perturbed
(1, 7, 8, 9, 10) .
We thank Oxana Beskrovnaya, Wei Guo, and Yoshihide
Sunada for helpful discussion and technical assistance. We thank Dr.
Fiona J. McDonald for providing SH3 domain-containing fusion protein
constructs. We thank Leland Lim, Steven L. Roberds, and Victoria Scott
for the helpful comments.
- and
-dystroglycan also
serves as an agrin receptor in muscle, where it may regulate
agrin-induced acetylcholine receptor clustering at the neuromuscular
junction.
-Dystroglycan has now been expressed in vitro and shown to directly interact with Grb2, an adapter protein
involved in signal transduction and cytoskeletal organization. Protein
binding assays with two Grb2 mutants, Grb2/P49L and Grb2/G203R, which
correspond to the loss-of-function mutants in the Caenorhabditis
eleganssem-5, demonstrated that the
dystroglycan-Grb2 association is through
-dystroglycan C-terminal
proline-rich domains and Grb2 Src homology 3 domains. Affinity
chromatography has also shown endogenous skeletal muscle Grb2 interacts
with
-dystroglycan. Immunoprecipitation experiments have
demonstrated that Grb2 associates with
/
-dystroglycan in
vivo in both skeletal muscle and brain. The specific
dystroglycan-Grb2 interaction may play an important role in
extracellular matrix-mediated signal transduction and/or cytoskeleton
organization in skeletal muscle that may be essential for muscle cell
viability.
-Dystroglycan binds laminin and dystrophin binds actin filaments,
indicating that one function of the dystrophin-glycoprotein complex is
to provide a link between the extracellular matrix and the cell
cytoskeleton
(5, 6) . Disruption of the
dystrophin-glycoprotein complex in several forms of muscular dystrophy
suggests that this extracellular matrix-cytoskeleton linkage is
important in maintaining muscle cell
viability
(1, 7, 8, 9, 10) .
Recently, the dystroglycan complex was also shown to serve as an agrin
receptor in muscle, where it may regulate agrin-induced acetylcholine
receptor clustering at the neuromuscular
junction
(11, 12, 13) . The dystroglycan complex
has also been suggested to play a role in kidney epithelial
morphogenesis.
(
)
These functional features
indicate that dystroglycan may function to transduce extracellular
signals into cells or regulate cell cytoskeleton organization in a
manner similar to that of growth factor receptors or integrins. The
structural analysis of dystroglycan
(5, 14) indicates
that
-dystroglycan contains a phosphotyrosine consensus sequence
and several proline-rich regions that could associate with Src homology
2 and 3 (SH2
(
)
and 3) domains of cytoskeletal or
signaling proteins
(15, 16) .
-dystroglycan directly interacts with Grb2. Protein binding
assays with two Grb2 mutants, Grb2/P49L and Grb2/G203R, which
correspond to the loss-of-function mutants in the C. eleganssem-5, demonstrate that the specific association
is through
-dystroglycan C-terminal proline-rich domains and Grb2
SH3 domains. Protein affinity purification and immunoprecipitation
assays further indicate that Grb2 associates with
/
-dystroglycan in vivo in both skeletal muscle and
brain. The data suggest that the specific dystroglycan-Grb2 interaction
may play an important role in extracellular matrix-mediated signal
transduction and cytoskeleton organization in different tissues through
the dystroglycan complex.
Generation of Glutathione S-transferase Fusion
Proteins
The rabbit -dystroglycan (GenBank accession number
X64393) cytoplasmic domain was amplified from a dystroglycan cDNA clone
by polymerase chain reaction (PCR) with the following primers: sense
(5`-CCCGGGTTACCGCAAGAAGCGGAAGG-3`) and antisense
(3`-AAGGAATTCGTGGGCGATGGTCTGC-5`). The resulting product was subcloned
into pGEX-2TK vector cDNA encoding. The glutathione
S-transferase (GST) fusion protein (
-DGct) was introduced
into Escherichia coli DH5
cells. Overnight cultures were
diluted 1:10, incubated for 1 h, and induced for 3 h with 1 mM
isopropyl-
-D-thiogalactopyranoside. The GST fusion
protein was purified on a glutathione-Sepharose 4B affinity column
(Pharmacia Biotech Inc.). The expressed fusion protein was
immunoreactive to the antibody against the C-terminal 15 amino acid
residues of
-dystroglycan
(5) . The GST fusion proteins
containing different SH3 domains were purified in the same way. The
human cDNA-encoding Grb2 protein (GenBank accession number M96995) was
amplified using the sense (5`-GGCGGATCCGAAGCCATCGCCAAATATGACTTC-3`) and
antisense (3`-CAGTGGGGGCACTTGGCCTTGCAGACTTAAGGG-5`) primers and
subcloned into pGEX-2TK. The loss-of-binding Grb2 mutant cDNAs were
constructed in the pALTER-1 vector using the Altered Sites mutagenesis
kit (Promega) with the following primers: Grb2 P49L, antisense
(3`-CTATGTAGTTCTTGAGAATGAAGG-5`); Grb2 G203R, antisense
(3`-GCGGGGAAAATCCTGGTCTGCCCTGG-5`). The underlined codons
represent the nucleotide substitutions. These cDNAs were subcloned into
pGEX-2TK. These constructs encoding all of the fusion proteins were
then transformed into the DH5
competent cells. Fusion proteins
were purified as described above.
Protein Overlay Assay
The SH3 domain-containing
fusion proteins were electrophoretically separated on 3-12%
SDS-polyacrylamide gel and transferred onto nitrocellulose membrane.
The membrane was blocked for 2 h at 4 °C in blocking buffer (0.1%
gelatin, 5% bovine serum albumin, and 0.1% Tween 20 in
phosphate-buffered saline, pH 7.5) and was then incubated with purified
fusion protein -DGct (10 µg/ml) in overlay buffer (150
mM NaCl, 20 mM Hepes, 2 mM MgCl
,
1 mM dithiothreitol, and 5% bovine serum albumin, pH 7.5)
overnight at 4 °C. After washing twice with overlay buffer, the
membrane was processed for Western blot with antibody against 15
-dystroglycan C-terminal amino acid residues.
In Vitro Transcription and Translation of
The S-Labeled
-Dystroglycan Cytoplasmic
Domain
-dystroglycan cytoplasmic domain was
amplified by PCR with the following primers: sense
(5`-CCATGGTCTGCTACCGCAAG-3`) and antisense
(3`-TCTAGAGGGTTAAGGGGGAA-5`). The resulting product was subcloned into
pGEM3 containing the 50-nucleotide alfalfa mosaic virus consensus
initiation site
(27) . This construct was used to synthesize a
S-labeled probe by coupled in vitro transcription
and translation with the TNT system (Promega) as described. The DNA
template was incubated at 30 °C for 2 h with TNT rabbit
reticulocyte lysate, T7 RNA polymerase, and an amino acid mixture
containing [
S]methionine (Amersham Corp.). The
S-labeled
-dystroglycan cytoplasmic domain was
analyzed by SDS-PAGE and autoradiography.
Protein Binding Assay
GST fusion protein-coupled
glutathione-Sepharose beads were equilibrated in binding buffer (150
mM NaCl, 0.1% Tween 20, 10 mM Hepes, pH 7.4) and
incubated with in vitro translated
[S]
-DGct probe (10
µl
ml
of binding buffer) for 12 h at 4
°C. Due to degradation, more G203R fusion protein was used to
achieve an equal amount of intact fusion protein in each lane. The
beads were then washed three times with binding buffer and separated on
3-12% SDS-polyacrylamide gel, and the gel was dried and exposed
to film (X-Omat AR, Kodak).
Affinity Chromatography
Rabbit skeletal muscle was
homogenized in 20 mM Tris-HCl, 1 M NaCl, 1% CHAPS
with protease inhibitors: aprotinin (76.8 nM), benzamidine
(0.83 mM), leupeptin (1.1 µM), pepstatin A (0.7
µM), and phenylmethylsulfonyl fluoride (0.23 mM),
pH 7.5. The homogenate was incubated at 4 °C for 4 h with mixing
and then centrifuged at 100,000 rpm for 30 min at 4 °C. The
supernatant was diluted 1:10 with 20 mM Tris-HCl (pH 7.5), 1
mM CaCl and protease inhibitors as above. The
diluted homogenate was further centrifuged at 100,000 rpm for 30 min to
remove precipitated protein. After being precleared with
glutathione-Sepharose 4B for 3 h, the solubilized skeletal muscle
homogenate was incubated with the fusion protein bound to
glutathione-Sepharose overnight at 4 °C. After incubation, the
Sepharose was washed with 20 mM Tris-HCl, pH 7.5, 150
mM NaCl, 1 mM CaCl
, 0.5% CHAPS. The
proteins bound to Sepharose were separated by 3-12% SDS-PAGE and
analyzed by immunoblot assay with anti-Grb2 antibody.
Protein Immunoprecipitation
CHAPS-solubilized
skeletal muscle or brain homogenate was incubated with
anti--dystroglycan antibody IIH6-Sepharose overnight at 4 °C
after being precleared with Sepharose 6B. After extensive washing with
Tris-buffered saline, the proteins attached to beads were resolved by
SDS-PAGE with starting material and void in parallel. The gel was
transferred to nitrocellulose and subjected to immunoblotting with
anti-
/
-dystroglycan antibodies or anti-Grb2 antibody.
Immunoblotting
Proteins were separated on
3-12% gradient SDS-polyacrylamide gels and transferred onto
nitrocellulose. Immunoblot staining was performed either with anti-Grb2
antibody (Santa Cruz) or with affinity-purified
anti-/
-dystroglycan antibody from sheep
anti-dystrophin-glycoprotein complex polyclonal
antisera
(1, 2, 3) .
RESULTS AND DISCUSSION
cDNA containing the rabbit -dystroglycan cytoplasmic
domain was amplified by PCR and subcloned into the pGEX-2TK expression
vector to make the GST fusion protein,
-DGct. A protein overlay
assay was performed to test the association between
-dystroglycan
(
-DGct) and SH3 domain-containing proteins. Fig. 1shows the
interaction between
-dystroglycan and the four SH3
domain-containing proteins, Gap, Grb2, Lck, and Src. Notably, only Grb2
associates with
-dystroglycan in this assay. Four additional SH3
domain-containing proteins (Abl, Crk, phosphatidylinositol 3-kinase,
and spectrin) were also examined and showed no
-dystroglycan
binding activity (data not shown).
-Dystroglycan and its
cytoplasmic domain were also translated in vitro in the
presence of [
S]methionine. Using a protein
overlay assay, the
S-labeled
-dystroglycan only bound
Grb2 among the eight SH3 domain-containing proteins (data not shown).
Additionally, the
S-labeled
-dystroglycan cytoplasmic
domain ([
S]
-DGct) was also retained by Grb2
fusion protein Sepharose beads (Fig. 2, a-c). GST
or GST-conjugated Sepharose beads, the negative control, did not bind
-dystroglycan ( Fig. 1and Fig. 2, a-c)
.
Figure 1:
-dystroglycan binds Grb2
in vitro. a, Coomassie Blue (CB) stained
SDS-polyacrylamide gel of different GST fusion proteins of SH3
domain-containing proteins including human GAP (amino acids
275-351, GAP), human GRB2 (amino acids 1-217,
GRB2), murine LCK (amino acids 5-124, LCK), and
chicken Src SH3 (SRC). GST alone serves as negative control.
b, overlay of the corresponding GST fusion proteins
transferred on nitrocellulose with fusion protein
-DGct containing
-dystroglycan cytoplasmic domain and detected with the antibody
against the C-terminal 15 amino acid residues of
-dystroglycan
(5).
Figure 2:
Specific
association between -dystroglycan and Grb2 SH3 domains.
a, autoradiogram of SDS-polyacrylamide gel of in vitro translated
S-labeled
-dystroglycan cytoplasmic
domain, [
S]
-DGct. b, Coomassie
Blue-stained SDS-polyacrylamide gel of purified GST fusion protein with
wild type Grb2 (WT) and its mutants Grb2/P49L (P49L)
and Grb2/G203R (G203R). GST was used as the negative control.
c, autoradiogram of corresponding binding of
S-labeled
-dystroglycan cytoplasmic domain by these
wild type and mutant Grb2 GST fusion protein-conjugated glutathione
beads. d, overlay of the corresponding GST fusion proteins
transferred on nitrocellulose with
-dystroglycan cytoplasmic
domain fusion protein and detected with the antibody against the
C-terminal 15 amino acid residues of
-dystroglycan
(5).
The primary structure of -dystroglycan contains several
proline-rich regions at its C terminus
(5, 14) . To test
further whether the Grb2-
-dystroglycan interaction occurs via an
SH3-proline-rich domain association, two Grb2 mutants, Grb2/P49L and
Grb2/G203R, which correspond to the loss-of-function mutants in the
C. elegans Grb2 homolog,
sem-5(18, 21, 22) , were used in a
fusion protein binding assay and an overlay assay (Fig. 2). Each
of these Grb2 mutants possesses a point mutation in either the
N-terminal or the C-terminal SH3 domain, which abolishes or greatly
reduces association with Grb2-binding
proteins
(18, 21, 22) . The
S-labeled in vitro translated
-dystroglycan
cytoplasmic domain, [
S]
-DGct, was used in
the fusion protein binding assay (Fig. 2a). Due to
degradation, an increase in G203R fusion protein was used to achieve an
equal amount of intact fusion protein in each lane
(Fig. 2b). As shown in Fig. 2c, the
N-terminal SH3 domain loss-of-function mutation (P49L) results in loss
of
-dystroglycan binding. The C-terminal SH3 domain mutant (G203R)
results in reduced binding of
-dystroglycan. In parallel
experiments, protein overlay assay using
-dystroglycan as the
probe showed that the P49L and the G203R mutants display tremendously
decreased binding activity (Fig. 2d). There is almost no
detectable binding affinity between
-dystroglycan and Grb2/G203R
in the protein overlay assay, possibly because of the higher
sensitivity of the protein binding assay or overloading of G203R fusion
protein in the binding assay. The data above suggest that Grb2 binds to
the
-dystroglycan cytoplasmic proline-rich domains with both its
N- and C-terminal SH3 domains, but the N-terminal SH3 domain may be the
stronger binding site. Like the Grb2-SOS interaction, the association
between Grb2 and
-dystroglycan may be mediated primarily through
the N-terminal SH3 domain, although high affinity binding may require
the coordinate interaction of both Grb2 SH3
domains
(21, 22) .
-DGct was used to investigate whether
-dystroglycan can
associate with endogenous skeletal muscle Grb2. As shown in
Fig. 3
, Grb2 in CHAPS-solubilized skeletal muscle homogenate was
retained by
-DGct-Sepharose beads but not by the beads conjugated
with fusion protein of
-dystroglycan extracellular domain
(
-DGnt). In addition, Sepharose beads conjugated with Grb2/R86K,
which has the loss-of-function mutation in the SH2 domain, precipitated
-dystroglycan from CHAPS-solubilized muscle homogenate (data not
shown). This is consistent with the protein binding and overlay assays,
indicating that the association between endogenous Grb2 and
-dystroglycan is mediated through the SH3 domains. To test whether
/
-dystroglycan and Grb2 associate in vivo in
different tissues, anti-
-dystroglycan antibody IIH6
(6) was
used to immunoprecipitate
-dystroglycan and Grb2 from
CHAPS-solubilized skeletal muscle and brain homogenates. Both Grb2 and
-dystroglycan were co-immunoprecipitated by IIH6 but not by
several other unrelated antibodies ( Fig. 4and data not shown).
In Fig. 4a, most Grb2 protein in skeletal muscle was
immunoprecipitated by IIH6. This suggests that Grb2 mainly associates
in vivo in skeletal muscle with dystrophin-associated protein
dystroglycan, where dystrophin constitutes approximately 5% of the
membrane protein
(3, 28) . In brain, only a small part of
Grb2 associates with dystroglycan (Fig. 4b). Grb2 and
-dystroglycan were also co-immunoprecipitated by IIH6 from the
C2C12 muscle cell line (data not shown). Thus,
- and
-dystroglycan and Grb2 are associated in vivo in skeletal
muscle and brain.
Figure 3:
Skeletal
muscle Grb2 binds to -dystroglycan. CHAPS-solubilized skeletal
muscle homogenate was subjected to affinity chromatography on various
columns including GST-Sepharose (GST),
-dystroglycan
N-terminal domain Sepharose (
-DGnt), and
-dystroglycan C-terminal domain Sepharose (
-DGct).
The starting material, skeletal muscle homogenate (Homo.),
flow-through (Void), Sepharose beads before incubating
(Beads(-H)) and after incubating
(Beads(+H)) with muscle homogenate, and washing were
analyzed on SDS-PAGE. The gels were transferred to nitrocellulose and
subjected to immunoblotting with anti-Grb2
antibody.
Figure 4:
In
vivo association between dystroglycan and Grb2 in skeletal muscle and
brain. CHAPS-solubilized skeletal muscle (a) or brain
(b) homogenate was subjected to immunoprecipitation with
anti--dystroglycan monoclonal antibody IIH6. The starting
material, skeletal muscle homogenate (Homo.), flow-through
(Void), and IIH6 Sepharose beads after incubation with muscle
homogenate (Beads) were separated by SDS-PAGE and transferred
to nitrocellulose. The nitrocellulose blots were stained with
affinity-purified anti-
/
-dystroglycan antibody
(anti-
/
-DG) or anti-Grb2 antibody
(anti-Grb2).
Through protein overlay and protein binding
assays, we have demonstrated that Grb2 directly associates with
-dystroglycan. Experiments performed using mutated GST-Grb2 fusion
proteins demonstrated that this association is likely to be mediated
through both SH3 domains of Grb2. The fact that both dystroglycan and
Grb2 are expressed in many tissues indicates that they may have
ubiquitous cellular functions
(5, 14, 17) .
Protein immunoprecipitation assays suggest that
- and
-dystroglycan and Grb2 are associated in both skeletal muscle and
brain. This gives rise to the hypothesis that the dystroglycan complex
may regulate cellular functions, such as certain cytoskeletal
processes, through Grb2 in different tissues. Consistent with this
hypothesis is the finding that growth factor-induced stress fiber
formation at focal adhesions and ruffle induction involves small
GTP-binding protein (Ras, Rac, and Rho)
activation
(23, 24, 25) . Grb2 has been shown to
modulate the activities of these small GTP-binding proteins that
stimulate cytoskeleton organization in vivo (23-25).
Furthermore, integrin-mediated signal transduction in NIH3T3
fibroblasts is coupled to activation of the Grb2/Ras
pathway
(26) . As a laminin or agrin receptor, the dystroglycan
complex may function in an analogous manner and regulate cytoskeleton
organization through Grb2-involved signal transduction triggered by the
extracellular matrix or neurally released agrin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.