(Received for publication, June 23, 1995)
From the
Dystrophin, the product of the Duchenne muscular dystrophy gene,
is tightly associated with the sarcolemmal membrane to a large
glycoprotein complex. One function of the dystrophin-glycoprotein
complex is to link the cytoskeleton to the extracellular matrix in
skeletal muscle. However, the molecular interactions of dystrophin with
the membrane components of the dystrophin-glycoprotein complex are
still elusive. Here, we demonstrate and characterize a specific
interaction between -dystroglycan and dystrophin. We show that
skeletal muscle and brain dystrophin as well as brain dystrophin
isoforms specifically bind to
-dystroglycan. To localize and
characterize the dystrophin and
-dystroglycan interaction domains,
we reconstituted the interaction in vitro using dystrophin
fusion proteins and in vitro translated
-dystroglycan. We
demonstrated that the 15 C-terminal amino acids of
-dystroglycan
constituted a unique binding site for the second half of the hinge 4
and the cysteine-rich domain of dystrophin (amino acids
3054-3271). This dystrophin binding site is located in a
proline-rich environment of
-dystroglycan within amino acids
880-895. The identification of the interaction sites in
dystrophin and
-dystroglycan provides further insight into the
structure and the molecular organization of the dystrophin-glycoprotein
complex at the sarcolemma membrane and will be helpful for studying the
pathogenesis of Duchenne muscular dystrophy.
Dystrophin is a large protein with a molecular mass of 427 kDa, which is absent in muscle from patients with Duchenne muscular dystrophy(1) . Based on its primary structure, dystrophin can be divided into four domains: the N-terminal actin binding domain, the large triple helical spectrin-like domain, the cysteine-rich domain, and the C-terminal domain(2) . Deletion of the cysteine-rich and C-terminal domains is associated with severe muscular dystrophy, which indicates that these domains play important roles in the stability of dystrophin(3, 4) .
Immunofluorescence
microscopy has established that dystrophin is located at the plasma
membrane of skeletal muscle(5) . Biochemical studies have
demonstrated that dystrophin is tightly associated through its
cysteine-rich and C-terminal domains with the sarcolemmal membrane to a
large glycoprotein
complex(6, 7, 8, 9, 10) .
Furthermore, cell membrane fractionation has shown that the
dystrophin-glycoprotein complex can only be dissociated by alkaline
treatment(6) . Dystrophin-glycoprotein complex (DGC) ()is composed of at least five transmembrane proteins
(50-kDa adhalin, 43-kDa
-dystroglycan, 43-kDa
dystrophin-associated glycoprotein A3b, 35-kDa dystrophin-associated
glycoprotein, and 25-kDa dystrophin-associated protein), one
extracellular protein (156 kDa
-dystroglycan), and four
cytoplasmic proteins (syntrophin triplet and
dystrophin)(6, 7, 8, 9, 11, 12, 13, 14, 15) .
In skeletal muscle, interactions between
-dystroglycan and laminin
2 (11, 12, 16) as well as between
dystrophin and cytoskeletal actin filaments (17) have been
identified, indicating that one function of the DGC is to provide a
link between the extracellular matrix and the cytoskeleton.
Furthermore, the loss of dystrophin leads to a great reduction of all
the dystrophin-associated proteins in Duchenne muscular dystrophy or mdx mouse skeletal muscle (18, 19) . Taken
together, these results suggest that the marked reduction of the DGC in
muscle from Duchenne muscular dystrophy patients and mdx mice
disrupts the linkage between the extracellular matrix and the
cytoskeleton, thereby rendering dystrophic muscle fibers more
susceptible to necrosis.
Protein overlay assays have previously
indicated that dystrophin cysteine-rich domain interacts with a 43-kDa
dystrophin-associated protein (-dystroglycan or A3b), although the
exact identity of this protein has yet to be conclusively
determined(20) . Recently, interactions between syntrophin and
the C-terminal domain (residues 3447-3481) of dystrophin have
also been
demonstrated(20, 21, 22, 23) .
However, the molecular organization of dystrophin with the membrane
components of the DGC is still elusive. Here, we report several
experiments aimed at determining whether
-dystroglycan directly
binds to dystrophin. We show that dystrophin from solubilized skeletal
muscle and brain binds to the cytoplasmic domain of
-dystroglycan
expressed as a GST fusion protein and coupled to glutathione-agarose
beads. The
-dystroglycan binding site on dystrophin was further
localized to the second half of hinge 4 and the cysteine-rich domain,
including amino acids 3054-3271, by using in vitro translated
-dystroglycan cytoplasmic domain and dystrophin
fusion proteins in an in vitro binding assay. Finally, we have
localized the dystrophin binding site to the extreme C terminus of
-dystroglycan, including amino acids 880-895.
Figure 1:
Skeletal muscle dystrophin binds to
-dystroglycan. A, rabbit skeletal muscle dystrophin
solubilized by pH 11 treatment was subjected to affinity chromatography
on various columns including GST-agarose (GST) and
GST-
-dystroglycan cytoplasmic domain (GST-
-DGct). The
starting material, solubilized membrane (pH 11), flow-through (Void),
agarose beads before chromatography (Beads(-)) and after
chromatography (Beads(+)) were resolved on SDS-PAGE. The gels were
electrotransferred to nitrocellulose membrane and immunoreacted with
anti-dystrophin DYS2 monoclonal antibody and affinity-purified
anti-syntrophin. Molecular weight standards
(
10
) are indicated on the left. B, rabbit skeletal muscle dystrophin solubilized by pH 11
treatment was immunoprecipitated with anti-dystrophin (Mab XIXC2)
protein G beads. The starting material, solubilized membrane (pH 11),
flow-through (Void), and protein G beads (Beads) were resolved on
SDS-PAGE. The gels were electrotransferred to nitrocellulose membrane
and immunoreacted with anti-dystrophin DYS2 monoclonal antibody and
affinity-purified anti-syntrophin polyclonal antibodies. Molecular mass
standards (
10
) are indicated on the left.
Figure 2:
Brain dystrophin and dystrophin isoform
bind to -dystroglycan. Rabbit brain dystrophin solubilized by pH
11 treatment was subjected to affinity chromatography on various
columns including GST-agarose (GST) and GST-
-dystroglycan
cytoplasmic domain (GST-
-DGct). The starting material, solubilized
membrane (pH 11), and agarose beads after chromatography (GST or
-DGct) were resolved by SDS-PAGE. The gels were electrotransferred
to nitrocellulose membrane and immunoreacted with affinity-purified
anti-dystrophin C-terminal domains. Molecular mass standard (
10
) are indicated on the left.
Figure 3:
Characterization of -dystroglycan
binding motif in dystrophin. A, in vitro translated
-dystroglycan cytoplasmic domain ([
S]
-DGct) was incubated with GST or GST-dystrophin fusion proteins
(containing amino acids 3054-3685, 3054-3446,
3435-3685, 3054-3271, or 3189-3448) coupled to
glutathione-agarose. The beads were subjected to SDS-PAGE, stained with
Coomassie Blue (CB), dried, and subjected to autoradiography
(Autorad). Autoradiograph of in vitro translated
-dystroglycan cytoplasmic domain resolved on SDS-PAGE is shown on
the right ([
S]
-Dgct). Molecular
mass standards (
10
) are indicated on the left. B, alignment of the dystrophin fusion proteins
in the C-terminal portion of human dystrophin. The white, black, and gray box represents the hinge 4, the
cysteine-rich domain, and the C-terminal domain, respectively. The
amino acids of the fusion proteins were numbered based on their
locations in the primary structure of human dystrophin. Exons numbers
are indicated. The interactions between these fusion proteins and
-dystroglycan cytoplasmic domain are indicated on the left by (+) for interaction or(-) for no
interaction.
Figure 4:
Binding of -dystroglycan to
dystrophin fusion protein can be blocked by peptides of the cytoplasmic
domain of
-dystroglycan. Upper panel, peptide locations
in the cytoplasmic domain of
-dystroglycan; the peptide sequences
are underlined. Lower panel, In vitro translated
-dystroglycan cytoplasmic domain
([
S]
-DGct) was incubated in the presence
of 1 mM peptides (+PEP) or the absence of peptides, with
GST-dystrophin (amino acids 3054-3446) coupled to
glutathione-agarose. Control experiments were performed with GST
glutathione-agarose or GST-dystrophin (amino acids 3054-3446)
glutathione-agarose in the presence of 1 mM control peptide
(+PEPc). The proteins attached to beads were resolved on SDS-PAGE,
dried, and subjected to autoradiography. Autoradiograph of in vitro translated
-dystroglycan cytoplasmic domain resolved on
SDS-PAGE is shown on the right ([
S]
-Dgct).
To
confirm the localization of a unique dystrophin binding site at the
C-terminal domain of -dystroglycan, we determined whether or not
-dystroglycan lacking the 15 C-terminal amino acids is still able
to bind dystrophin. For this purpose, the cytoplasmic domain of
-dystroglycan lacking the 15 C-terminal amino acids was translated in vitro (
S-labeled
-DG775-880). In vitro binding assays were performed with this
S-labeled probe and dystrophin fusion protein. As shown in Fig. 5, the binding of
-dystroglycan to the cysteine-rich
domain of dystrophin was completely abolished by the truncation of the
C-terminal 15 amino acids of
-dystroglycan. This result
demonstrated that the C-terminal 15 amino acids of
-dystroglycan
are part of the dystrophin binding motif and that no other binding
motif is present on
-dystroglycan.
Figure 5:
Truncation of the 15 C-terminal amino
acids of -dystroglycan abolish the binding to dystrophin. A, autoradiograph of in vitro translated
-dystroglycan containing the full-length cytoplasmic domain
([
S]
-DGct) and the cytoplasmic domain
lacking the 15 C-terminal amino acids ([
S]
-DG775-880) resolved on SDS-PAGE. B, in vitro translated
-dystroglycan ([
S]
-DGct) or [
S]
-DG775-880 were
incubated with GST-dystrophin fusion protein, containing amino acids
3054-3446, coupled to glutathione-agarose. The beads were washed
and subjected to SDS-PAGE, dried, and subjected to autoradiography.
Molecular mass standards (
10
) are indicated
on the left.
However, despite the fact
that the truncation of the C-terminal 15 amino acids of
-dystroglycan abolished the binding to dystrophin, it does not
rule out the possibility that flanking N-terminal sequences are
contributing to the interaction. To test this possibility, we performed
a dystrophin affinity purification assay with a
-dystroglycan
C-terminal synthetic peptide (amino acids 880-895) immobilized on
Sepharose beads. As shown in Fig. 6A, dystrophin from
pH 11 solubilized skeletal muscle membranes was retained by
-dystroglycan C terminus peptide-Sepharose beads but not by beads
conjugated to an unrelated peptide. In addition,
-dystroglycan
C-terminal peptide-Sepharose beads successfully bound human dystrophin
fusion proteins containing amino acids 3054-3685 or amino acids
3054-3446 but did not bind to fusion proteins containing amino
acids 3435-3685 (Fig. 6B). Taken together, these
experiments demonstrate 1) the existence of a unique dystrophin binding
domain on
-dystroglycan C terminus, which contains amino acids
880-895 and 2) that this 15-amino acid sequence is sufficient by
itself for the association to dystrophin.
Figure 6:
Dystrophin binds to the 15 last amino
acids of -dystroglycan. A, rabbit skeletal muscle
dystrophin solubilized by pH 11 treatment was subjected to affinity
chromatography on Sepharose beads coupled to a synthetic peptide
covering the sequence of the 15 last amino acids of
-dystroglycan
(PEP-
-DGct) or a control peptide (PEPcont). The starting material,
solubilized membrane (pH 11), and the Sepharose beads after
chromatography were resolved on SDS-PAGE. The gels were
electrotransferred to nitrocellulose membrane and immunoreacted with
anti-dystrophin DYS2 monoclonal antibody. B, Upper panel shows a Coomassie Blue-stained polyacrylamide gel of 15 µg of
purified human dystrophin fusion protein (FP) containing amino
acids 3054-3685, amino acids 3054-3446, and amino acids
3435-3685. Lower panel, purified human dystrophin fusion
protein described above were incubated with Sepharose beads coupled to
a synthetic peptide covering the sequence of the 15 last amino acids of
-dystroglycan (PEP-
-DGct). After an extensive wash, the beads
were resolved on SDS-PAGE, and the gel was stained with Coomassie Blue.
Molecular mass standards (
10
) are indicated
on the left.
In conclusion, our results demonstrate that
-dystroglycan directly binds to dystrophin. Furthermore, we
identified the interacting motif on both proteins. One function of
dystrophin is to link, by way of the associated glycoprotein complex,
the actin cytoskeleton to the extracellular
matrix(11, 12, 13, 14, 15, 16, 17) .
Two domains of dystrophin responsible for its interaction with the
associated glycoprotein complex have now been characterized. The
C-terminal domain binds
syntrophin(20, 21, 22, 23) , and, as
we demonstrated here, the second half of hinge 4 and cysteine-rich
domain binds the C terminus of
-dystroglycan. Recently, Rafael and
colleagues (31) have generated a transgenic mdx mouse
expressing a dystrophin gene missing exons 71-74. Dystrophin
expression in this mdx transgenic mouse restored all the
dystrophin-associated proteins in muscle sarcolemma and prevented
dystrophic pathology, even in the absence of the syntrophin binding
motif in dystrophin. Consequently, the integrity of the
-dystroglycan motif in dystrophin, which appears to lie outside
exons 71-74, is sufficient to maintain the link between the
subsarcolemmal cytoskeleton and the extracellular matrix and maintain
the structural integrity of the muscle cells. Furthermore, syntrophin
is a dystrophin binding protein but does not exhibit any
dystrophin-associated glycoprotein binding activity(23) .
Therefore, it is unlikely that syntrophin is a direct molecular linker
that connects dystrophin to the associated glycoprotein complex. This
function may be fulfilled by
-dystroglycan.
- and
-dystroglycan are tightly associated and therefore likely exist as
a unique complex in various
tissues(32, 33, 34) . It is reasonable to
assume that this dystroglycan complex could link the underlying plasma
membrane cytoskeleton to the extracellular matrix by itself especially
in non-muscle tissues. So far, no pathologies resulting from the
absence of dystroglycan have been reported. However, this may be
because dystroglycan, which is ubiquitously expressed, plays an
essential role in cell function and/or development, and cells harboring
such mutations may be inviable.
On the basis of these results and
previous data(11, 35) , a schematic representation of
-dystroglycan structure can be drawn (Fig. 7). This model
of
-dystroglycan includes the putative N-terminal amino acid, the
potential N-linked glycosylation sites, the transmembrane
domain, and the dystrophin anchoring site. It is interesting to notice
that the dystrophin binding is located in a proline-rich environment
within the cytoplasmic domain of
-dystroglycan. We recently
demonstrated that the cytoplasmic domain of
-dystroglycan binds to
the SH3 domains of Grb2(36) . Therefore, It is reasonable to
assume that the interaction between dystrophin and
-dystroglycan
could be structurally related to a SH3-proline-rich sequence
interaction.
Figure 7:
Schematic structure of -dystroglycan.
Each amino acid is represented by a circle. The putative
N-terminal amino acid, corresponding to the cleavage site of the 97-kDa
dystroglycan precursor, is based on the results of Gee and colleagues (35) . Potential N-linked glycosylation sites (branched chains), transmembrane domain (gray
circles), and proline residues from the cytoplasmic domain (X) are indicated. The dystrophin anchoring site is indicated
by the light gray circles.
The identification of the interaction sites in
dystrophin and -dystroglycan provides further insight into the
structure and the molecular organization of the dystrophin-glycoprotein
complex at the sarcolemma membrane and will be helpful for studying the
pathogenesis of Duchenne muscular dystrophy.