(Received for publication, December 16, 1994)
From the
Syntrophin represents three cytoplasmic components of the
dystrophin-glycoprotein complex that links the cytoskeleton to the
extracellular matrix in skeletal muscle. -Syntrophin has now been
translated in vitro and shown to associate directly with all
three components of the syntrophin triplet and with dystrophin. The in vitro translated 71-kDa non-muscle dystrophin isoform,
containing the cysteine-rich/C-terminal domain, can also interact with
the syntrophin triplet. The syntrophin binding motif in dystrophin was
localized to exons 73 and 74 including amino acids 3447-3481 by
comparing the interactions of
-syntrophin and seven overlapping
human dystrophin fusion proteins. More than one syntrophin interaction
site in this binding motif was suggested.
-Syntrophin also
interacts directly with a C-terminal utrophin fusion protein.
-Syntrophin is localized to the muscle sarcolemma as well as to
the neuromuscular junction in control mouse muscle. However, similar to
utrophin,
-syntrophin is only present at the neuromuscular
junction in mdx mouse muscle in which dystrophin is absent.
Our data suggest that
-syntrophin binds all syntrophin isoforms,
and syntrophin directly interacts with dystrophin through more than one
binding site in dystrophin exons 73 and 74 including amino acids
3447-3481.
The dystrophin-glycoprotein complex (DGC) ()is
composed of five sarcolemmal glycoproteins (
-dystroglycan, 50-DAG,
-dystroglycan, 43-DAG and 35-DAG), a 25-kDa transmembrane protein
(25-DAP), and two intracellular proteins (59-DAP and
dystrophin)(1, 2, 3, 4, 5, 6) .
The
-dystroglycan (156-DAG) binds laminin and dystrophin binds to
actin filaments, indicating that one function of the
dystrophin-glycoprotein complex is to provide a link between the
extracellular matrix and the actin
cytoskeleton(5, 6) . 59-DAP (now named syntrophin) is
a 59-kDa cytoplasmic protein triplet in the purified
dystrophin-glycoprotein
complex(1, 2, 3, 4, 5, 6) .
Together with dystrophin and the 87-kDa protein homologous to
dystrophin, syntrophin is also highly enriched in AChR-rich Torpedo postsynaptic membrane(7, 8, 9) . cDNA
cloning and peptide sequencing further demonstrate that the syntrophin
triplet contains a family of homologous but distinct syntrophin
proteins encoded by several separate
genes(10, 11, 12) . The greatly reduced
expression of dystrophin-glycoprotein complex in mdx mice and
Duchenne muscular dystrophy (DMD) patients suggests that
syntrophin-containing dystrophin-glycoprotein complex is important in
maintaining the normal structure and function of muscle
sarcolemma(8, 11, 13, 14) .
Several lines of biochemical evidence suggest that dystrophin and
syntrophin may directly interact with each other. Torpedo dystrophin can bind either Torpedo 52/58-kDa protein or
the corresponding mouse protein in protein overlay assays(15) .
The 59-kDa protein band corresponding to -syntrophin in the
purified DGC separated on SDS-PAGE can be overlaid by dystrophin fusion
protein (16) . Dystrophin, utrophin, 87-kDa postsynaptic
dystrophin homologous protein, and DP71 were able to be
immunoprecipitated by an anti-syntrophin antibody(17) .
Transgenic mdx mice, which express a 71-kDa non-muscle
dystrophin isoform in skeletal muscle, were able to rescue all
components of the dystrophin-glycoprotein complex including
syntrophin(18, 19) . The biochemical evidence above
suggests that some syntrophins, if not all, directly interact with
dystrophin. However, the protein interactions among dystrophin and all
protein components in the syntrophin triplet are still elusive.
Here, -syntrophin has been translated in vitro and
shown to directly bind dystrophin as well as all the components of the
syntrophin triplet. In vitro translated 71-kDa non-muscle
dystrophin isoform, which contains the cysteine-rich/C-terminal domain,
can also associate with the syntrophin triplet. More than one
syntrophin binding site in dystrophin has been identified in dystrophin
exons 73 and 74 including amino acids 3447-3481. The data suggest
that syntrophin isoforms may associate directly with dystrophin in
vivo as a complex through multiple interaction sites.
To test the hypothesis that syntrophin may directly interact
with dystrophin, the full-length open reading frame of -syntrophin
was amplified by PCR and subcloned into pGEM3 vector containing the
50-nucleotide alfalfa mosaic virus consensus initiation site as
described(20) . This construct was used to synthesize a
[
S]methionine-labeled probe by coupled in
vitro transcription and translation in the TNT system (Promega).
An overlay assay was carried out to test for syntrophin-binding
proteins in skeletal muscle surface membrane and purified DGC. Fig. 1shows that
-syntrophin binds to all three components
of the syntrophin triplet as well as dystrophin. An 87-kDa protein,
which is not enriched in purified DGC, also bound
-syntrophin.
This protein could be related to the 87-kDa postsynaptic membrane
protein, which has been demonstrated to colocalize with the AChR and
associate with Torpedo dystrophin and
syntrophin(8, 9) . Our data demonstrate that
-syntrophin binds directly to dystrophin in skeletal muscle. It
also provides the first evidence that syntrophins may form a syntrophin
complex in vivo.
Figure 1:
Identification of
syntrophin-binding proteins in skeletal muscle cell membrane and
purified dystrophin-glycoprotein complex. a, autoradiogram of
SDS-polyacrylamide gel of in vitro translated S-labeled
-syntrophin ([
S]
-syn). b,
Coomassie Blue (CB)-stained SDS-polyacrylamide gel of rabbit
skeletal muscle crude membranes (Mic), surface membrane (SM), and purified dystrophin-glycoprotein complex (DGC); IB, corresponding immunoblot stained with a
mix of affinity-purified anti-dystrophin and anti-syntrophin triplet
antibodies; Overlay, autoradiogram of an identical
nitrocellulose transfer overlaid by in vitro translated
[
S]
-syntrophin
probe.
To further define syntrophin-dystrophin
interactions, the murine non-muscle 71-kDa dystrophin isoform
corresponding to the sequence of DP-71 was in vitro translated
with [S]methionine as described
above(20, 21) . This probe was used to overlay
skeletal muscle surface membranes and purified DGC separated by
SDS-PAGE. This dystrophin isoform bound the syntrophin triplet
suggesting that all the syntrophins including
- as well as
-type may directly bind to dystrophin (Fig. 2). Previous
work by Ozawa's group indicated that one of 43-DAGs was overlaid
by dystrophin fusion protein containing cysteine-rich and C-terminal
domains(16) . This interaction was not detected in our
experiments, probably because of the lower concentration of in
vitro translated dystrophin in the overlay assay. Interaction of
dystrophin with
-dystroglycan (one of the 43-DAGs) can be seen
using another protein binding assay. (
)
Figure 2:
Identification of dystrophin-binding
proteins in skeletal muscle cell membrane and purified
dystrophin-glycoprotein complex. a, autoradiogram of
SDS-polyacrylamide gel of in vitro translated S-labeled dystrophin C terminus ([
S]Dct). b, IB, corresponding immunoblot stained with affinity-purified
anti-syntrophin triplet antibodies; Overlay, Autoradiogram of
the same nitrocellulose transfer overlaid by in vitro translated
S-labeled dystrophin C terminus
probe.
To determine
regions important for syntrophin binding in dystrophin, seven
overlapping human dystrophin GST fusion proteins were synthesized.
Protein overlay assay on these fusion proteins was performed with in vitro translated S-labeled
-syntrophin (Fig. 3a). The relative positions of these fusion
proteins in human dystrophin molecule are indicated in Fig. 3b. The association between these fusion proteins
and syntrophin is also described in Fig. 3b. The
largest dystrophin fusion protein, which contains both the
cysteine-rich and the C-terminal domains, bound
-syntrophin. In
this region, only the C-terminal domain (amino acids 3435-3685),
covering the last 251 amino acids, is responsible for this binding.
Comparison of the association of syntrophin and the dystrophin fusion
proteins, which contain amino acids 3435-3482 or 3481-3685,
confines the syntrophin binding motif to amino acids 3447-3481.
Interestingly, two other fusion proteins (3054-3463 and
3464-3685), which divide this motif in half, were able to
independently bind syntrophin, which suggests that there are more than
one binding site in this motif. If all syntrophins directly bind
dystrophin as a complex in vivo, the presence of more than one
syntrophin interaction sites may be required. Fusion proteins
containing the dystrophin N terminus or rod domain were also
synthesized, but showed no binding activity to syntrophin (data not
shown). This is consistent with the observation that all syntrophin
isoforms bind to the dystrophin C terminus (Fig. 2).
Figure 3:
Characterization of the syntrophin binding
motif in dystrophin. a, Coomassie Blue-stained
SDS-polyacrylamide gel of GST control and seven different human
dystrophin C-terminal domains expressed as GST fusion proteins in total
bacterial lysates (top), and autoradiogram of corresponding
overlay with [S]
-syntrophin on these
immobilized fusion proteins (bottom). The relative locations
of these fusion proteins in human dystrophin are indicated below. b, alignment of these fusion proteins against the dystrophin
C-terminal region identifying a dystrophin-syntrophin interaction motif
containing more than one independent syntrophin binding site. The first
and last amino acids of each fusion protein and the interaction motif
were numbered based on their locations in the primary structure. The blackbox represents the cysteine-rich domain. The
interactions between these fusion proteins and
-syntrophin are
indicated on the right.
Previously, we reported that utrophin associates with the same
complex of dystrophin-associated proteins(23) . In order to
test the direct interaction of syntrophin and utrophin, mouse utrophin
C terminus was expressed as a MBP fusion protein. Fig. 4a shows that this fusion protein was also bound by in vitro translated [S]
-syntrophin in an
overlay assay. As the negative control, mouse dystrophin N-terminal MBP
fusion protein containing amino acids 1-246 shows no binding on
syntrophin (Fig. 4a). This suggests that syntrophin may
interact with utrophin at sites homologous to the corresponding
sequence in dystrophin. Comparing the deduced protein sequences of
dystrophin and utrophin, many amino acid residues are conserved in this
syntrophin binding motif(22, 24) . These amino acid
residues are probably critical for syntrophin interactions in both
dystrophin and utrophin.
Figure 4:
Direct interaction of syntrophin and
utrophin. a, Coomassie Blue (CB)-stained
SDS-polyacrylamide gel of a control MBP fusion protein containing
dystrophin N-terminal domain (Dnt) and utrophin C-terminal
domain expressed as MBP fusion protein (DRP); Overlay, Corresponding overlay with S-labeled
-syntrophin. b, immunofluorescence on control (cont; top) and mdx (middle) mouse
quadriceps muscle stained with anti-
-syntrophin antibody (anti-
-syn). Neuromuscular junctions were
identified by double staining with rhodamine-labeled
-bungarotoxin
(
-BGT) in mdx mouse muscle (bottom).
-Syntrophin is present throughout the
sarcolemma in control mouse skeletal muscle (Fig. 4b).
However, the staining of
-syntrophin is greatly reduced in the
sarcolemma of mdx mice which lack dystrophin. Notably,
-syntrophin remains enriched at the neuromuscular junction (Fig. 4b) where utrophin is located(25) . This
further suggests that
-syntrophin associates with both dystrophin
and utrophin in vivo.
Thus far, our results indicate that
syntrophin isoforms bind to each other, perhaps forming a syntrophin
complex. This complex may directly associate with dystrophin or
utrophin, which may be involved in anchoring dystrophin and utrophin to
muscle sarcolemma. Another member of the syntrophin triplet, -A1,
contains an extended N-terminal hydrophobic sequence(12) . It
is possible that the syntrophin complex associates with the cell
membrane by the
-A1 N-terminal hydrophobic domain, thereby
anchoring dystrophin or utrophin to the cytoskeleton. Syntrophin also
contains two pleckstrin homology domains(26) . Pleckstrin
homology domains are found in many intracellular signaling or
cytoskeletal proteins and have been shown to bind the
subunits of G proteins and phosphatidylinositol 4,5-bisphosphate (27, 28, 29) . Thus, the pleckstrin homology
domain of syntrophin may also be involved in membrane association of
syntrophin.
The correct localization of the dystrophin-glycoprotein
complex in the sarcolemma is crucial for its function, based on
Duchenne muscular dystrophy
studies(30, 31, 32) . Identification of
mutations in the DMD gene demonstrated the importance of the unique
C-terminal domain in dystrophin(33, 34, 35) .
Most mutations in the C-terminal domain cause severe Duchenne-type
dystrophy, probably due to deficient interactions between dystrophin
and its associated proteins. However, the exact function of this domain
remains elusive. Previously, two patients with small C-terminal domain
truncations were analyzed(36) . One patient with truncated
C-terminal domain from amino acids 3444 to 3685 had a severe DMD
phenotype, whereas another patient with a truncation from amino acids
3485 to 3685 had a milder phenotype. By comparing these two
truncations, it was suggested that the domain that contains amino acids
3444-3485 in dystrophin molecule is of functional importance and
may be linked to certain severe DMD phenotypes(36) .
Interestingly, this sequence exactly matches the syntrophin-binding
domain reported here, which covers amino acids 3447-3481. This
may indicate that the inability of dystrophin to associate with
syntrophin may be partially responsible for the severe DMD phenotypes.
However, transgenic mice expressing a murine dystrophin mini-gene
lacking exons 71-74 display normal muscle function and express
all the dystrophin-associated proteins, including -syntrophin, in
the muscle sarcolemma(37) . This suggests that there may be
more syntrophin binding sites beyond exons 71-74 in dystrophin
that were not detected by overlay experiments. Alternatively,
syntrophin may have been recruited by other unknown syntrophin-binding
proteins other than dystrophin.