* Howard Hughes Medical Institute, Department of Physiology and Biophysics, Department of Neurology, University of Iowa
College of Medicine, Iowa City, Iowa 52242; Department of Pediatrics and Department of Anatomy and Neurobiology,
Washington University School of Medicine, St. Louis, Missouri 63110; and § Department of Human Genetics, University of
Michigan, Ann Arbor, Michigan 48109
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The dystrophin-glycoprotein complex (DGC)
is a multisubunit complex that spans the muscle plasma
membrane and forms a link between the F-actin cytoskeleton and the extracellular matrix. The proteins of
the DGC are structurally organized into distinct subcomplexes, and genetic mutations in many individual
components are manifested as muscular dystrophy. We
recently identified a unique tetraspan-like dystrophin-associated protein, which we have named sarcospan
(SPN) for its multiple sarcolemma spanning domains
(Crosbie, R.H., J. Heighway, D.P. Venzke, J.C. Lee, and K.P. Campbell. 1997. J. Biol. Chem. 272:31221-31224).
To probe molecular associations of SPN within the
DGC, we investigated SPN expression in normal muscle as a baseline for comparison to SPN's expression in
animal models of muscular dystrophy. We show that, in
addition to its sarcolemma localization, SPN is enriched at the myotendinous junction (MTJ) and neuromuscular junction (NMJ), where it is a component of both the
dystrophin- and utrophin-glycoprotein complexes. We
demonstrate that SPN is preferentially associated with
the sarcoglycan (SG) subcomplex, and this interaction
is critical for stable localization of SPN to the sarcolemma, NMJ, and MTJ. Our experiments indicate that
assembly of the SG subcomplex is a prerequisite for
targeting SPN to the sarcolemma. In addition, the SG-
SPN subcomplex functions to stabilize -dystroglycan
to the muscle plasma membrane. Taken together, our
data provide important information about assembly
and function of the SG-SPN subcomplex.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE dystrophin-glycoprotein complex (DGC1; Campbell and Kahl, 1989; Ervasti et al., 1990
, 1991
;
Yoshida and Ozawa, 1990
; Ervasti and Campbell,
1991
) is comprised of peripheral and integral membrane proteins and provides a structural linkage between the extracellular matrix and the intracellular cytoskeleton of
muscle cells. Several forms of muscular dystrophy arise
from primary mutations in genes encoding dystrophin-associated proteins (for review see Campbell, 1995
; Straub
and Campbell, 1997
). Patients with mutations in the dystrophin gene develop either Duchenne or Becker muscular dystrophy, which is characterized by progressive wasting of skeletal muscles. Likewise, a nonsense mutation in
the murine dystrophin gene (mdx) eliminates expression
of dystrophin and, consequently, the DGC proteins are reduced at the sarcolemma. While the function of the DGC
is obviously essential for normal muscle physiology, its precise role in muscle function is unclear. It has been hypothesized that this transmembrane protein complex provides mechanical support to the plasma membrane during
myofiber contraction (Weller et al., 1990
; Petrof et al.,
1993
). More recently, data from several laboratories have
suggested that the DGC may also play a role in cellular
communication, as highlighted by the association of this
complex with known signaling molecules (Brenman et al.,
1995
; Yang et al., 1995
; Chang et al., 1996
).
The proteins that comprise the DGC are structurally organized into three distinct subcomplexes. These are the cytoskeletal proteins, dystrophin and syntrophins; the dystroglycans (DGs; and
subunits); and the sarcoglycans
(SGs;
,
,
, and
subunits). Exactly how these proteins
are arranged with respect to one another is uncertain, but
interactions between subcomplexes are clearly important
for targeting to the sarcolemma, as well as for membrane stabilization. Recent reports have demonstrated that the
NH2 terminus of dystrophin interacts directly with F-actin
in an extended, lateral fashion, similar to many actin side-binding proteins (Rybakova et al., 1996
; Rybakova and
Ervasti, 1997
; Amann et al., 1998
). Dystrophin connects
with the other DGC subcomplexes through its COOH-terminal domain, which binds directly to the COOH terminus of
-DG, an integral membrane protein with a single
transmembrane helix (Jung et al., 1995
).
-DG, in turn,
binds
-DG, anchoring it to the extracellular surface of the
sarcolemma.
-DG serves as a receptor for laminin 2, thereby completing the physical connection between the
actin cytoskeleton and the extracellular matrix (Ervasti and Campbell, 1993
).
The SG subcomplex is composed of four distinct single-pass transmembrane glycoproteins, referred to as -,
-,
-,
and
-SG (for review see Lim and Campbell, 1998
). The
SGs, in conjunction with
-DG, mediate attachment of
-DG to the muscle plasma membrane. A defect in any
one of the SGs results in specific loss of the SG subcomplex, destabilization of
-DG, and sarcolemma damage (Holt et al., 1998
). Autosomal recessive limb-girdle muscular dystrophy (LGMD) types 2D, 2E, 2C, and 2F are
caused by mutations in
-,
-,
-, and
-SG, respectively
(Roberds et al., 1994
; Bönnemann et al., 1995
; Lim et al.,
1995
; Noguchi et al., 1995
; Piccolo et al., 1995
; Jung et al.,
1996
; Nigro et al., 1996a
,b; Passos-Bueno et al., 1996
).
Likewise, the BIO 14.6 hamster (Iwata et al., 1993
; Roberds et al., 1993
), which serves as an animal model for
LGMD2F, has a large deletion in the
-SG gene (Nigro et
al., 1997
). BIO 14.6 hamsters display both cardiomyopathic and myopathic features. Successful intervention of
disease progression has been achieved by introduction of a
recombinant
-SG adenovirus into skeletal muscle of the
BIO 14.6 hamster (Holt et al., 1998
). Targeted deletions of
the
- (Duclos et al., 1998b
) and
-SG (Hack et al., 1998
) genes in mice result in dystrophic muscle phenotypes and
have provided additional animal models for LGMD.
We have recently characterized a novel 25-kD dystrophin-associated protein and have shown that it is an integral member of the DGC (Crosbie et al., 1997, 1998
). We
have named this protein sarcospan (SPN) for its multiple
sarcolemma spanning helices, which are predicted based
on hydropathy analysis (Crosbie et al., 1997
). Dendrogram
analysis shows that SPN is a member of the transmembrane four or tetraspan superfamily of proteins (Crosbie
et al., 1997
). Each possess four transmembrane domains, a
large extracellular loop, and are thought to play important
roles in mediating transmembrane protein interactions
(Wright and Tomlinson, 1994
; Maecker et al., 1997
). These
characteristics make SPN unique among other dystrophin-associated proteins. Furthermore, given the propriety of
tetraspan proteins for mediating protein-protein interactions, SPN is structurally poised to be an important player
in facilitating interactions between subcomplexes of the
DGC. In the present study, we examine SPN expression in
several animal models of muscular dystrophy as a means
of assessing the molecular associations of SPN with subcomplexes of the DGC. We find that SPN interacts with
the SGs, forming an SG-SPN protein complex.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cDNA Isolation and Sequencing
SPN cDNA clones were isolated by hybridization screening of a CLONTECH rabbit skeletal muscle cDNA library with a PCR-derived SPN
cDNA probe encoding exons 1-3. Sequence analysis of the clones was
performed using dye terminator cycling and analyzed on a 373 stretch fluorescent automated sequencer (PE Applied Biosystems). The nucleotide
and deduced amino acid sequences of rabbit SPN have been deposited in
the GenBank/EMBL/DDBJ data bank with the accession number
AF120276. Multiple sequence alignment was performed using the DNAsis
sequence analysis software (Hitachi Software Engineering, Inc.). Also, we
have isolated SPN cDNA clones independently from a mouse skeletal
muscle library and found clones identical to those found by Scott et al.
(1994).
Northern Blotting
Adult mouse multiple tissue Northern blots (CLONTECH Laboratories, Inc.) containing 2 µg of poly (A)+ RNA per lane were probed with an expressed sequence tag corresponding to the 3' untranslated region of mouse SPN (GenBank accession number W83284). Identical results were obtained when blots were hybridized with PCR-amplified probes representing the entire coding region (GenBank accession number U02487) of mouse SPN.
Animal Models
Wild-type (wt; C57BL/10) and mdx (C57BL/10ScSn) mice, obtained from
Jackson ImmunoResearch Laboratories, Inc. were maintained at the University of Iowa Animal Care Unit in accordance with animal usage guidelines. The dystrophin transgenic mice have been described previously
(Cox et al., 1994; Rafael et al., 1994
, 1996
; Phelps et al., 1995
). Male F1B
and BIO 14.6 cardiomyopathic hamsters were obtained from BioBreeders. We have previously reported the generation and initial characterization of the
-SG deficient (Sgca-null) mice (Duclos et al., 1998b
). The targeted disruption of the
-SG gene was accomplished by replacement of
exons 2 and 3, and flanking intronic sequences with the neomycin resistance gene through homologous recombination (Duclos et al., 1998b
). Utrophin deficient (utrn
/
) and utrophin-dystrophin deficient mice
(mdx:utrn
/
) have been described previously (Grady et al., 1997a
,b).
Utrn
/
and mdx:utrn
/
mice were maintained at Washington University
(St. Louis, MO).
Antibodies
mAbs against - (20A6),
- (5B1), and
-SG (21B5), as well as mAbs
against
-DG (8D5) were generated in collaboration with Dr. Louise V.B.
Anderson (Newcastle General Hospital, Newcastle upon Tyne, UK).
mAb against
-DG (IIH6) have been described by Ervasti and Campbell
(1991)
. Antibodies against the laminin
2 chain (Allamand et al., 1997
)
and the NH2 terminus of rabbit SPN (Rabbit 216; Crosbie et al., 1997
)
have been described previously. For generating antibodies against mouse
SPN, two New Zealand White rabbits (rabbits 235 and 236; Knapp Creek
Farms) were injected at intramuscular and subcutaneous sites with a
COOH-terminal SPN-glutathione S transferase fusion protein (amino acids 186-216 of mouse SPN; CFVMWKHRYQVFYVGVGLRSLMASDGQLPKA). Affinity purification of SPN antibodies was accomplished
using Immobilon-P (Millipore Corp.) strips containing the COOH-terminal SPN-maltose-binding fusion protein. Antibody specificity was verified
for both immunofluorescence and immunoblotting by competition experiments using the COOH-terminal SPN fusion protein and peptides synthesized to the COOH-terminal region of mouse SPN (data not shown).
Immunofluorescence
Transverse muscle cryosections (7 µm) were analyzed by immunofluorescence as described in Crosbie et al. (1997). For extraocular muscle (EOM)
studies, rectus muscles (global layer) were examined. Affinity purified
rabbit 235 SPN antibody was incubated at a dilution of 1:50 and 1:10 with
mouse and hamster sections, respectively. After washing with TBS (10 mM
Tris-HCl, 150 mM NaCl, pH 7.4), the sections were incubated with Cy3-conjugated secondary antibodies at a dilution of 1:250 (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. For staining
of neuromuscular junctions (NMJs), samples were simultaneously incubated with fluorescein-conjugated
-bungarotoxin (1:1,000; Molecular
Probes, Inc.). After washing with TBS, the slides were mounted with
Vectashield mounting medium (Vector Labs Inc.) and observed under a
BioRad MRC-600 laser scanning confocal microscope. Digitized images
were captured under identical conditions.
Recombinant Adenovirus Injections
The human -SG cDNA sequence was subcloned into the pAdRSVpA adenovirus vector through standard methods of homologous recombination with Ad5 backbone dl309 by the University of Iowa Gene Transfer Vector
Core. Preparation of the recombinant adenovirus and the intramuscular
injections were performed as previously described (Holt et al., 1998
). In
brief, 109 viral particles in 100 µl of normal saline were injected into the
quadriceps femoris of 3-wk-old BIO 14.6 hamsters after the animals were
anesthetized by intraperitoneal injection of sodium pentobartital (Nembutal; Abbott Laboratories) at a calculated dose of 75 mg/kg. Quadriceps
muscle was collected 2 wk after the injection.
Preparation of Skeletal Muscle Membranes
KCl washed membranes from wt, mdx, and Sgca-null mice were prepared
from skeletal muscle as described previously (Duclos et al., 1998b).
Isolation of the SG-SPN Subcomplex by pH 11 Treatment
Purified DGC (Campbell and Kahl, 1989; Ervasti et al., 1990
, 1991
) from
rabbit skeletal muscle membranes was titrated to pH 11 using 1 M NaOH
and incubated for 1 h at room temperature with gentle mixing (Ervasti et al.,
1991
) in a buffer consisting of 50 mM Tris, 0.1% digitonin, 175 mM NaCl,
0.1 mM PMSF, 0.75 mM benzamidine. The alkaline treated DGC was concentrated fourfold using Centricon-10 filters (Amicon Corp.). The samples were loaded onto 5-30% linear sucrose gradients in a buffer of 50 mM
Tris-HCL, 500 mM NaCl, 0.1% digitonin, 0.1 mM PMSF, 0.75 mM benzamidine, pH 11. The gradients were centrifuged at 4°C in a Beckman Vti 65.1 vertical rotor for 2.5 h at 200,000 g. 16 0.8-ml fractions were collected
from the top of the gradient using an Isco model 640 density gradient fractionator. The protein samples (60 µl) were separated by 3-15% SDS-PAGE and immunoblotted, as described (vide infra).
Sucrose Gradient Separation of WGA Enriched Proteins from mdx Muscle
Quadriceps femoris muscle was dissected from mdx mice and snap frozen
in liquid nitrogen. Frozen tissue (1 g) was pulverized into small pieces with
a pestal and mortar filled with liquid nitrogen. The tissue was solubilized
by dounce homogenization in 10 ml of cold buffer A (50 mM Tris-HCl,
pH 7.8, 500 mM NaCl, 1.0% digitonin) with a cocktail of protease inhibitors (0.6 µg/ml pepstatin A, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.1 mM PMSF, 0.75 mM benzamidine, 5 µm calpain I inhibitor, and 5 µM calpeptin). The samples were spun at 142,400 g for 37 min at 4°C. The pellets were resolublized with 5 ml of buffer A, rotated at 4°C for 1 h, and
centrifuged as before. The two supernatants were combined and incubated overnight at 4°C with 1 ml of WGA-Sepharose (Vector Labs, Inc.).
The WGA-Sepharose was washed extensively (50 mM Tris-HCl, pH 7.8, 0.1% digitonin, 500 mM NaCl) and proteins were eluted with 0.3 M
N-acetyl glucosamine (Sigma Chemical Co.). Samples were concentrated
to 500 µl using a Centricon-30 filter and applied to a 5-30% sucrose gradient at pH 7.8, as described previously (Ervasti et al., 1991).
Immunoblotting
Protein samples were resolved under reducing conditions by 3-15% SDS-PAGE and transferred to PVDF (Immobilon-P) membranes (Millipore
Corp.). PVDF membranes were probed with anti-SG mAbs, as described
previously (Holt et al., 1998). For mouse SPN immunoblotting, the membranes were probed with affinity purified rabbit 235 antibody at a dilution
of 1:50. Note that for mouse SPN immunoblotting, proteins were resolved
on 3-15% SDS-PAGE under nonreducing conditions and transferred to
PVDF (Immobilon-P). For rabbit SPN staining, nitrocellulose blots were
probed with affinity purified rabbit 216 antibody as described (Crosbie et
al., 1997
). For
- and
-DG staining, SDS-polyacrylamide gels were transferred to nitrocellulose (Immobilon-NC) and probed with IIH6 (1:3 dilution) and 20A6 (1:100 dilution). Following incubation with primary antibodies, blots were probed with the appropriate HRP-conjugated
secondary antibodies (1:5,000; Boehringer Mannheim Corp.) and developed using enhanced chemiluminescence (SuperSignal; Pierce Chemical Co.).
In Vivo Reconstitution Experiments
A human SPN expression construct was prepared by PCR amplification of
cDNA using primers containing appropriate restriction sites for subcloning into pcDNA3 (Pharmacia Biotech, Inc.). The SPN construct was engineered to encode a myc-tag at the COOH terminus. All constructs were
verified by direct DNA sequence analysis performed by the DNA Core
Facility at the University of Iowa (Iowa City, IA). Full-length myc-tagged
-,
-,
-, and
-SG pcDNA3 (Pharmacia Biotech Inc.) expression constructs have been previously described (Holt and Campbell, 1998
). Construction and design of the Grb2 cDNA expression vector has been described (Holt et al., 1996
). CHO cells were electroporated with SG and
SPN expression constructs (~5 µg of each plasmid DNA) at 340 V at 950 µF using a BioRad electroporator, as previously described (Holt and
Campbell, 1998
). 30 h after transfection, cells were analyzed for protein
expression by SDS-PAGE and immunoblotting. Membrane surface proteins were biotinylated using membrane impermeant sulfo-NHS-biotin
(Pierce Chemical Co.) as described previously for the SGs expressed in CHO cells (Holt and Campbell, 1998
). Immunoprecipitation using a
-SG
mAb (5B1) and analysis of protein samples by SDS-PAGE and immunoblotting with an anti-myc mAb (9E10) were performed as documented in
Holt and Campbell (1998)
.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SPN is the most recently identified dystrophin-associated
protein, and therefore the least characterized. Hydropathy
analysis of the primary amino acid sequences of human
(Heighway et al., 1996; Crosbie et al., 1997
) and murine
(Scott et al., 1994
) SPN predicts a protein with intracellular NH2 and COOH termini, and four transmembrane domains. We report determination of the primary structure
of rabbit SPN, as deduced from a rabbit skeletal muscle cDNA (Fig. 1 a). Multiple sequence alignment demonstrates that amino acid sequences derived from rabbit,
mouse, and human SPN are
75% identical (Fig. 1 a). Human and rabbit SPN contain a short insertion at the NH2
terminus, which is absent in mouse SPN. The four predicted transmembrane domains are extremely well conserved. SPN's membrane topology is strikingly different
from other dystrophin-associated proteins, which only
have a single pass transmembrane domain, and is reminiscent of the tetraspan superfamily of proteins (Wright and
Tomlinson, 1994
; Maecker et al., 1997
). Using phylogenetic analysis, we previously demonstrated that SPN is
closely related to the divergent family members Rom-1, peripherin, and uroplakin (Crosbie et al., 1997
). The tetraspans are thought to play important roles in mediating
interactions between transmembrane proteins as mechanisms to control cell growth and adhesion. We speculate
that SPN, a novel dystrophin-associated tetraspan, may be
facilitating interactions among proteins of the DGC and
perhaps mediating interactions of the DGC components with other sarcolemma proteins.
|
SPN Is a Unique Tetraspan Predominantly Expressed in Muscle
To examine the distribution of SPN in mouse tissue, we
performed RNA hybridization analysis. Hybridization of
mouse multiple tissue Northern blots with probes representing either the coding region or the 3' untranslated region of SPN gave identical results (Fig. 1 b). As shown in
Fig. 1, a 4.4-kb transcript is predominant in skeletal and
cardiac tissues, with minor transcript levels present in
lung, brain, and testis. Human skeletal and cardiac muscles
express two SPN transcripts (4.4 and 6.5 kb; Crosbie et al.,
1997). The additional 6.5-kb transcript in humans is likely the result of alternate splicing, since the 6.5-kb transcript does not hybridize to SPN exons 2 and 3 (Heighway et al.,
1996
).
SPN Localization to the Sarcolemma Is Dependent on the DGC
We have previously shown that SPN is expressed throughout the sarcolemma of normal human skeletal muscle
(Crosbie et al., 1997, 1998
). The limitations of patient biopsies with known mutations prompted our use of murine
models as a method to investigate the interactions of SPN
with the DGC. First, we examined expression of SPN in
quadriceps femoris (thigh), diaphragm, and cardiac muscles from mdx mice, a model for Duchenne muscular dystrophy. The mdx phenotype is inherited as an X-linked recessive trait, which stems from a premature stop codon in
exon 23 of the dystrophin gene, leading to absence of dystrophin protein (Bulfield et al., 1984
; Hoffman et al., 1987
;
Chamberlain et al., 1988
). As a consequence, the dystrophin-associated proteins are nearly ablated from the sarcolemma (Ohlendieck and Campbell, 1991
). Using indirect immunofluorescence on muscle cross sections, we
show that SPN is dramatically reduced in skeletal muscle
of mdx mice (Fig. 2). Our data also demonstrate that SPN
is expressed in normal cardiac tissue, which is consistent with the presence of SPN transcript in Northern blots (Fig.
1 b). As shown in Fig. 2, SPN is significantly reduced in
mdx cardiac tissue. The diaphragm muscle, which is the
most severely affected muscle in mdx mice, also lacks normal sarcolemma expression of SPN. In addition to its expression in skeletal muscle, recent experiments from our
group demonstrate that SPN is also present in smooth
muscle (Straub, V., and K.P. Campbell, personal communication). Lastly, we observed positive SPN staining in
muscle from the laminin
2 deficient dy (Arahata et al.,
1993
; Sunada et al., 1994
; Xu et al., 1994a
) and dy2J (Xu et
al., 1994b
; Sunada et al., 1995
) mice (data not shown), which are naturally occurring animal models of congenital
muscular dystrophy.
|
Association of SPN with Dystrophin Isoforms
To further explore the association of SPN with the DGC,
we examined SPN expression in a collection of dystrophin
transgenic mice. The transgenes, which were individually
expressed on an mdx background, encode truncated dystrophin products. Muscle from the transgenic mice was
previously evaluated for its morphology and for the ability
of the transgene to restore sarcolemma localization of the
DGC (Cox et al., 1994; Greenberg et al., 1994
; Rafael et al.,
1994
, 1996
; Phelps et al., 1995
). We detected SPN expression in skeletal muscle cross sections by indirect immunofluorescence staining with SPN antibodies, and found normal levels of SPN expression in muscle from
17-48
(Phelps et al., 1995
),
1-62 (Cox et al., 1994
; Greenberg et al.,
1994
),
71-74 (Rafael et al., 1994
, 1996
), and
75-78
(Rafael et al., 1996
) transgenic mice (Fig. 3). Despite large
deletions, these transgenes are able to restore SPN to the sarcolemma and, with the exception of the
1-62 transgene, alleviate muscular dystrophy. The
71-74 transgene
represents an alternately spliced dystrophin isoform that is
predominantly expressed in brain, and the
1-62 (Dp71)
transgene mimics a form of dystrophin present in lung,
spleen, testis, and retina.
|
SPN Enrichment at the NMJ Is Mediated by Dystrophin and Utrophin
To determine if SPN is associated with the utrophin-glycoprotein complex, and if replacement of dystrophin with
utrophin would affect SPN's localization to the sarcolemma, we examined NMJs from dystrophin, utrophin
(Grady et al., 1997a), and dystrophin-utrophin (Deconinck et al., 1997
; Grady et al., 1997b
) deficient muscle. The
NMJs were identified by staining cross sections of quadriceps femoris with fluorescein
-bungarotoxin, which selectively binds to acetylcholine receptors. By indirect immunofluorescence, we show that SPN is enriched at the NMJ
of innervated muscle (Fig. 4). This enrichment is maintained even after denervation, demonstrating that SPN is
associated with the postsynaptic membrane (data not shown). At the NMJ, dystrophin is replaced by the structurally and functionally similar protein, utrophin (Khurana
et al., 1991
; Nguyen et al., 1991
; Ohlendieck et al., 1991
;
Pons et al., 1991
; Matsumura et al., 1992
; Karpati et al.,
1993
). Enrichment of SPN at the NMJ is not altered by the
absence of dystrophin, as seen by positive NMJ staining in
the mdx muscle (Fig. 4). In this case, SPN's localization to
the NMJ is mediated by utrophin. Conversely, NMJ localization of SPN is preserved by dystrophin in utrn
/
muscle, as demonstrated by SPN NMJ staining in these mice.
Loss of SPN staining from the NMJ occurs only in the absence of both utrophin and dystrophin, as in the mdx:
utrn
/
double mutant mice.
|
In mdx mice, muscles with the greatest upregulation of
utrophin exhibit the least pathological changes (Porter et al.,
1998). For instance, the EOM are spared the pathological
effects in mdx mice and Duchenne muscular dystrophy patients, likely from the upregulation of utrophin (Matsumura et al., 1992
; Porter et al., 1998
). In support of this,
Tinsley et al. (1996
, 1998
) demonstrate that expression of
utrophin attenuates the dystrophic pathology in mdx mice,
suggesting that utrophin can functionally replace dystrophin within the complex. We examined the EOMs from
wt, mdx, and mdx:utrn
/
(Deconinck et al., 1997
; Grady
et al., 1997b
) mice for SPN expression as another method
to demonstrate that SPN is part of the utrophin-glycoprotein complex. We show that SPN is located at the sarcolemma of wt EOM and is maintained in the EOM of mdx
mice, despite absence of dystrophin (Fig. 5). The continued expression of SPN in the mdx EOM likely is mediated
through SPN's association with the utrophin-glycoprotein complex. Consistent with this idea, SPN expression is lost
in the EOM of mice lacking both dystrophin and utrophin
(mdx:utrn
/
; Fig. 5). These data are important as they
demonstrate that upregulation of utrophin retains SPN to
the sarcolemma and validates this as a reasonable therapy
for Duchenne muscular dystrophy.
|
SPN's Localization to the Sarcolemma Is Dependent on the SGs
The SGs (consisting of ,
,
, and
subunits) form a tight
subcomplex of four transmembrane glycoproteins within
the DGC (Ervasti et al., 1991
; Yoshida et al., 1994
; Jung
et al., 1996
). The integrity of this complex is maintained
despite harsh treatments with SDS (Jung et al., 1996
) and
n-octyl
-D-glucoside (Yoshida et al., 1994
). Absence of
any one of the SGs results in absence of the entire SG subcomplex and destabilization of
-DG from the sarcolemma
(Roberds et al., 1993
; Duclos et al., 1998a
; Holt et al.,
1998
). Furthermore, this subcomplex is critical for protecting the sarcolemma from contraction induced damage.
We wanted to determine if SPN depends on the SG subcomplex for proper membrane targeting by examining
-SG-deficient BIO 14.6 hamsters (Homburger et al., 1962
;
Okazaki et al., 1996
) for SPN expression. A large deletion
in the
-SG gene (Nigro et al., 1997
; Sakamoto et al., 1997
)
causes selective loss of the entire SG subcomplex from
BIO 14.6 skeletal muscle without affecting
-DG (Roberds
et al., 1993
; Mizuno et al., 1995
; Duclos et al., 1998b
). We
now demonstrate that SPN expression is absent from the
sarcolemma (Fig. 6), as well as the NMJ (data not shown)
of the BIO 14.6 hamster. Furthermore, we show that SPN
expression is restored to normal levels after delivery of an
adenovirus encoding
-SG into BIO 14.6 muscle (Fig. 6).
Control injections of
-SG did not restore proper localization of SPN or the SGs (Fig. 6). Recent experiments from
our laboratory have shown that injection of
-SG into
muscle of the BIO 14.6 hamster rescues expression of the entire SG subcomplex (Holt et al., 1998
). Muscle fibers expressing the restored SG-SPN subcomplex are spared the
pathological features of muscular dystrophy (i.e., sarcolemma damage and central nucleation) and have stable expression of
-DG at the plasma membrane (Holt et al.,
1998
). Thus, SPN and the SGs are required for normal
muscle physiology and prevention of dystrophic features.
|
In addition to this naturally occurring hamster model for
LGMD, our laboratory has created -SG null mice by a
targeted disruption of the murine
-SG gene (Duclos et al.,
1998b
). Like the BIO 14.6 hamsters, Sgca-null mice specifically lack the SG subcomplex (Duclos et al., 1998b
). We
now demonstrate that Sgca-null muscle is completely devoid of SPN (Fig. 7 a). The NMJ and myotendinous junction (MTJ), which we show are normally enriched for SPN
expression, also lack SPN in the Sgca-null mice (Fig. 7 a).
As further demonstration of the tight association of SPN
with the SGs, we immunoblotted KCl washed membranes
prepared from skeletal muscle of wt, mdx, and Sgca-null
mice. SPN is dramatically reduced in mdx membranes (~90% compared with wt), but SPN was not detected in
the Sgca-null membranes (Fig. 7 b).
|
Isolation of the SG-SPN Subcomplex
To demonstrate that SPN is tightly associated with the SGs, we isolated the SG-SPN subcomplex from skeletal muscle. We prepared purified DGC from rabbit skeletal muscle microsomes and titrated the complex to pH 11 to dissociate pH-sensitive protein-protein interactions. Alkaline-treated DGC was centrifuged through a 5-30% linear sucrose gradient. Proteins from the sucrose gradient fractions were separated by SDS-PAGE and immunoblotted with anti-DGC antibodies. As shown in Fig. 8 a, sucrose gradient sedimentation of alkaline-treated DGC separates the DG (fractions 6-9) and SG (fractions 9-12) subcomplexes from one another. SPN displays a sedimentation pattern similar to that of the SG subcomplex, indicating a preferential association of SPN with the SGs.
|
In addition to chemically disrupting the DGC, we analyzed the dissociation of SG and DG subcomplexes resulting from the absence of dystrophin. The dystrophin-associated proteins are present in the extrajunctional sarcolemma of mdx muscle, although at significantly reduced levels. Figs. 2 and 7 illustrate that ~10% of SPN expression is maintained at the mdx sarcolemma. We prepared glycoproteins by WGA-Sepharose chromatography of digitonin-solubilized mdx skeletal muscle. Without dystrophin, the SG and DG subcomplexes are no longer associated and can be separated by sucrose gradient centrifugation. The subcomplexes peak in separate fractions and the relative separations between the SG and DG containing fractions are similar for both mdx and pH 11 treated samples. As shown in Fig. 8 b, SPN migrates exclusively with the SG containing fractions.
Reconstitution of the SG-SPN Subcomplex
Using an in vivo cell expression system, we demonstrate
that SPN and the SGs are associated in a complex at the
plasma membrane. Myc-tagged human cDNA constructs
of the SGs (,
,
, and
) and SPN were transiently introduced into CHO cells by electroporation. Immunoblots of
cellular protein lysates with anti-myc antibodies demonstrate that each of the SGs and SPN, as well as the Grb2 negative control, are expressed at relatively equal quantities (Fig. 9). We confirm that these proteins are targeted to
the plasma membrane by treatment of cells with sulfo-NHS-biotin, which forms a covalent bond with free amines
of proteins at the cell surface. Clarified lysates from transfected CHO cells were incubated with avidin-Sepharose
to precipitate plasma membrane-associated proteins. As
shown in Fig. 9, SPN and the SGs are properly localized to the plasma membrane.
|
To demonstrate that the SGs and SPN are assembled
into a stable molecular complex, we performed immunoprecipitation experiments. CHO cells transfected with the
SGs plus SPN were immunoprecipitated using mAbs to
-SG. SPN coimmunoprecipitates along with the SGs from
CHO cells. To demonstrate the specificity of this association, control immunoprecipitation experiments from cells
expressing the SGs and myc-tagged Grb2 were performed.
Grb2 serves as a negative control since it is a soluble protein that is not expected to associate with the SGs at the
plasma membrane. The SGs and Grb2 were cotransfected
into CHO cells and cellular lysates were immunoprecipitated with the
-SG mAb. Grb2 is not found in the immune complex with the SGs (Fig. 9). These data provide
strong evidence that the simultaneous expression of the
SGs and SPN in CHO cells results in the formation of a
tight molecular complex.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The DGC spans the sarcolemma and links the intracellular
actin cytoskeleton of muscle cells to the extracellular matrix. Current evidence indicates that the DGC confers
structural stability to the muscle plasma membrane, thus
protecting it from stresses that develop during muscle
fiber contraction. In support of this theory, perturbations
in the dystrophin-associated components lead to loss of
membrane integrity. This is evidenced by increased permeability of muscle fibers to intravenously administered
Evans blue dye (Straub et al., 1997, 1998
; Holt et al., 1998
)
as well as leakage of muscle-specific enzymes into the serum. This loss of membrane integrity eventually manifests
itself as fiber degeneration. Thus, understanding the structural organization of the DGC is critical for understanding
the function of this complex.
Our findings represent the first account of SPN's localization in normal muscle, the expression of SPN in mutant
mice, and the molecular associations of SPN within the
DGC. We report that SPN, found at the sarcolemma of
skeletal, cardiac, and diaphragm muscles, is also expressed
at many specialized muscle membrane interfaces, including the NMJ (Fig. 4) and MTJ (Fig. 7), as well as at muscle
spindles (data not shown). SPN is also expressed in
smooth muscle, where it is part of a unique smooth muscle
SG-SPN complex (Straub, V., and K.P. Campbell, personal communication). Although SPN seems to be predominantly expressed in muscle, we detect SPN transcripts
in many nonmuscle tissues (Fig. 1 b; Crosbie et al., 1997).
Consistent with this, our examination of dystrophin transgenic mice indicates that SPN may be associated with nonmuscle isoforms of dystrophin, such as Dp71 (Fig. 3). Further experimentation is necessary to determine whether
SPN protein is present in these nonmuscle tissues. The discovery that a subset of dystrophin-associated proteins (i.e.,
dystrophin, DG, and
-SG) is present in a broad array of
cell types is a provocative finding since all tissues are not
subjected to the same shear stresses as muscle. This suggests that the DGC may serve a more fundamental role in
the cell, in addition to the structural one ascribed to the
DGC in muscle.
We now show that SPN's localization to the sarcolemma
is compromised in dystrophin and utrophin double null mice.
SPN's enrichment at the NMJ is achieved by its association with utrophin. It has been suggested that upregulation
of utrophin compensates for loss of dystrophin (Matsumura et al., 1992). Indeed, we have now demonstrated that the EOMs of mdx mice, which are spared from the
pathological features of muscular dystrophy, express utrophin (Porter et al., 1998
) and SPN throughout the sarcolemma. If utrophin can functionally replace dystrophin,
then it may be possible to upregulate utrophin expression
in Duchenne muscular dystrophy patients (Matsumura et al.,
1992
; Tinsley et al., 1996
, 1998
). Our current data lend credence to the proposed theory that sarcolemma expression
of utrophin would completely restore the dystrophin-associated proteins to the muscle plasma membrane.
The DGC can be broken down into at least three interconnected subcomplexes: dystrophin, the DGs, and the SGs. Using several independent criteria, we demonstrate that SPN's localization to the sarcolemma is dependent on an intact SG subcomplex. SPN is completely absent from the sarcolemma, NMJ, and MTJ of the SG-deficient BIO 14.6 hamster and Sgca-null mouse. The preferential association of SPN with the SGs is demonstrated by biochemical isolation of the SG-SPN subcomplex. Alkaline treatment of purified DGC causes dissociation of the complex into distinct subcomplexes, where SPN preferentially associates with the SG containing fractions (Fig. 8 a). Likewise, in the absence of dystrophin, the remaining extrajunctional dystrophin-associated proteins dissociate into distinct protein complexes, where SPN's specific interactions with the SGs are maintained (Fig. 8 b).
Furthermore, we reconstitute the SG-SPN complex in a
recently developed heterologous cell system, which lacks
muscle specific proteins (Fig. 9; Holt and Campbell, 1998).
Previous work from our group has shown that mutations
in an individual SG result in intracellular accumulation of
the SG subcomplex (Holt and Campbell, 1998
). These experiments suggest that obligatory steps in the biosynthetic
pathway for SG subcomplex assembly cannot occur if individual SG proteins are aberrant or missing (Holt et al.,
1998
; Holt and Campbell, 1998
).
Taken together, our in vivo experiments now indicate that assembly of the SG subcomplex is a prerequisite for targeting and stabilization of SPN to the sarcolemma, as illustrated in Fig. 10. We currently do not know the molecular basis of the interaction between the SG-SPN and DG subcomplexes. It is clear, however, that proper structural alignment of these two subcomplexes, along with dystrophin, is required for DGC function and prevention of muscular dystrophy. The data presented in the current study are also consistent with our finding that SG-deficient LGMD patients also lack SPN (Crosbie, R.H., and K.P. Campbell, personal communication).
|
Although SPN is tightly associated with the SGs, SPN
bears no structural homology to the SGs. To date, there
are five known SGs, including the ubiquitously expressed
-SG, which exhibits >40% amino acid identity to
-SG
(Ettinger et al., 1997
; McNally et al., 1998
).
-SG shares all
the structural features of the skeletal muscle SGs, but is
also expressed in many nonmuscle tissues.
-,
-, and
-SG
are type II transmembrane proteins, while
- and
-SG are
type I membrane proteins with an NH2-terminal signal sequence.
-SG expression is not perturbed by targeted
deletion of the
-SG gene, suggesting that
-SG is not an
additional member of the
-,
-,
-,
-tetrameric SG subcomplex in skeletal muscle (Duclos et al., 1998b
). Each of
the SGs have a five cysteine residue motif in its extracellular domain, which is unique to this group of proteins. The
SGs also possess one or more consensus sites for glycosylation and treatment with PNGase F has been shown to shift the molecular weight of these proteins. SPN, on the
other hand, has many characteristics that distinguish it
from the SGs. Most obviously, SPN is predicted to have
multiple transmembrane domains and has no consensus
sites for N-linked glycosylation. Consistent with this, treatment of purified DGC with PNGase F does not alter
SPN's molecular weight (data not shown). Thus, SPN represents the first non-SG protein to be associated with the
SG subcomplex of the DGC.
The tight association of SPN with the SGs is consistent with SPN's homology to the tetraspan superfamily of proteins. The tetraspans are thought to function as facilitators of transmembrane protein interactions, and we suspect SPN serves to coordinate protein-protein interactions within the DGC. The results of our study provide support for this notion, since we find that SPN is intimately associated with at least one subcomplex of the DGC. Further examination of SPN's interaction with other DGC subcomplexes should provide significant insight into how the DGC is structurally organized, which is critical for understanding the function of this complex.
![]() |
Footnotes |
---|
Address correspondence to Kevin P. Campbell, Howard Hughes Medical Institute, University of Iowa College of Medicine, 400 Eckstein Medical Research Building, Iowa City, IA 52242. Tel.: (319) 335-7867. Fax: (319) 335-6957. E-mail: kevin-campbell{at}uiowa.edu WWW site: http: //
Received for publication 23 December 1998 and in revised form 2 March 1999.
R.H. Crosbie is supported by the Robert G. Sampson postdoctoral research fellowship from the Muscular Dystrophy Association. C.S. Lebakken is supported by the Iowa Cardiovascular Interdisciplinary Research Fellowship (HL07121). V. Straub was supported by the Deutsche
Forschungsgemeinschaft (Str 498/1-1). R.M. Grady was supported by a
National Research Service Award. J.R. Sanes was supported by the National Institutes of Health (NIH R01NS1915). This research was also supported by a grant from the Muscular Dystrophy Association to K.P.
Campbell and J.R. Sanes. K.P. Campbell is an investigator of the Howard
Hughes Medical Institute.
We thank the University of Iowa Diabetes and Endocrinology Research Center (NIH DK25295) and the University of Iowa DNA Sequencing Core Facility. We are indebted to Beverly L. Davidson (University of Iowa) and the University of Iowa Gene Transfer Vector Core (supported in part by the Carver Foundation). We also thank L.E. Lim (University of Iowa) for adenoviral injected BIO 14.6 muscle samples and F. Duclos for Sgca-null muscle samples. We are greatly indebted to Louise V.B. Anderson for mAbs. We also thank J. Heighway for helpful discussions of the manuscript.
![]() |
Abbreviations used in this paper |
---|
DG, dystroglycan;
DGC, dystrophin-
glycoprotein complex;
EOM, extraocular muscle;
LGMD, limb-girdle
muscular dystrophy;
mdx, murine dystrophin gene;
MTJ, myotendinous
junction;
NMJ, neuromuscular junction;
SG, sarcoglycan;
Sgca-null, -SG
deficient mice;
SPN, sarcospan;
utrn
/
, utrophin deficient;
mdx:utrn
/
, utrophin-dystrophin deficient;
wt, wild-type.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Allamand, V.,
Y. Sunada,
M.A. Salih,
V. Straub,
C.O. Ozo,
M.H. Al-Turaiki,
M. Akbar,
T. Kolo,
H. Colognato,
X. Zhang, et al
.
1997.
Mild congenital
muscular dystrophy in two patients with an internally deleted laminin alpha-2-chain.
Hum. Mol. Genet
6:
747-752
|
2. |
Amann, K.J.,
B.A. Renley, and
J.M. Ervasti.
1998.
A cluster of basic repeats in
the dystrophin rod domain binds F-actin through an electrostatic interaction.
J. Biol. Chem
273:
28419-28423
|
3. | Arahata, K., Y.K. Hayashi, R. Koga, K. Goto, J.H. Lee, Y. Miyagoe, H. Ishii, T. Tsukahara, S. Takeda, M. Woo, et al. 1993. Laminin in animal models for muscular dystrophy: defect of laminin M in skeletal and cardiac muscles and peripheral nerve of the homozygous dystrophic dy/dy mice. Proc. Jpn. Acad. 69B:259-264. |
4. | Bönnemann, C.G., R. Modi, S. Noguchi, Y. Mizuno, M. Yoshida, E. Gussoni, E.M. McNally, D.J. Duggan, C. Angelini, and E.P. Hoffman. 1995. Beta-sarcoglycan (A3b) mutations cause autosomal recessive muscular dystrophy with loss of the sarcoglycan complex. Nat. Genet 11: 266-273 |
5. | Brenman, J.E., D.S. Chao, H. Xia, K. Aldape, and D.S. Bredt. 1995. Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell 82: 743-752 |
6. | Bulfield, G., W.G. Siller, P.A. Wright, and K.J. Moore. 1984. X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc. Natl. Acad. Sci. USA 81: 1189-1192 [Abstract]. |
7. | Campbell, K.P.. 1995. Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage. Cell 80: 675-679 |
8. | Campbell, K.P., and S.D. Kahl. 1989. Association of dystrophin and an integral membrane glycoprotein. Nature 338: 259-262 |
9. | Chamberlain, J.S., J.A. Pearlman, D.M. Muzny, R.A. Gibbs, J.E. Ranier, C.T. Caskey, and A.A. Reeves. 1988. Expression of the murine Duchenne muscular dystrophy gene in muscle and brain. Science 239: 1416-1418 |
10. |
Chang, W.J.,
S.T. Iannaccone,
K.S. Lau,
B.S.S. Masters,
T.J. McCabe,
K. McMillan,
R.C. Padre,
M.J. Spencer,
J.G. Tidball, and
J.T. Stull.
1996.
Neuronal
nitric oxide synthase and dystrophin-deficient muscular dystrophy.
Proc.
Natl. Acad. Sci. USA
93:
9142-9147
|
11. | Cox, G.A., Y. Sunada, K.P. Campbell, and J.S. Chamberlain. 1994. Dp71 can restore the dystrophin-associated glycoprotein complex in muscle but fails to prevent dystrophy. Nat. Genet 8: 333-339 |
12. |
Crosbie, R.H.,
J. Heighway,
D.P. Venzke,
J.C. Lee, and
K.P. Campbell.
1997.
Sarcospan: the 25 kDa transmembrane component of the dystrophin-glycoprotein complex.
J. Biol. Chem.
272:
31221-31224
|
13. | Crosbie, R.H., H. Yamada, D.P. Venzke, M.P. Lisanti, and K.P. Campbell. 1998. Caveolin-3 is not an integral component of the dystrophin-glycoprotein complex. FEBS Lett 427: 279-282 |
14. | Deconinck, A.E., J.A. Rafael, J.A. Skinner, S.C. Brown, A.C. Potter, L. Metzinger, D.J. Watt, J.G. Dickson, J.M. Tinsley, and K.E. Davies. 1997. Utrophin-dystrophin deficient mice as a model for Duchenne muscular dystrophy. Cell 90: 717-727 |
15. |
Duclos, F.,
O. Broux,
N. Bourg,
V. Straub,
G.L. Feldman,
Y. Sunada,
L.E. Lim,
F. Piccolo,
S. Cutshall,
F. Gary, et al
.
1998a.
![]() |
16. |
Duclos, F.,
V. Straub,
S.A. Moore,
D.P. Venzke,
R.F. Hrstka,
R.H. Crosbie,
M. Durbeej,
C.S. Lebakken,
A.J. Ettinger,
J. van der Meulen, et al
.
1998b.
Progressive muscular dystrophy in ![]() |
17. | Ervasti, J.M., and K.P. Campbell. 1991. Membrane organization of the dystrophin-glycoprotein complex. Cell 66: 1121-1131 |
18. | Ervasti, J.M., and K.P. Campbell. 1993. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J. Cell Biol. 122: 809-823 [Abstract]. |
19. | Ervasti, J.M., K. Ohlendieck, S.D. Kahl, M.G. Gaver, and K.P. Campbell. 1990. Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle. Nature 345: 315-319 |
20. |
Ervasti, J.M.,
S.D. Kahl, and
K.P. Campbell.
1991.
Purification of dystrophin
from skeletal muscle.
J. Biol. Chem.
266:
9161-9165
|
21. |
Ettinger, A.J.,
G. Feng, and
J.R. Sanes.
1997.
![]() |
22. |
Grady, R.M.,
J.P. Merlie, and
J.R. Sanes.
1997a.
Subtle neuromuscular defects
in utrophin-deficient mice.
J. Cell Biol.
136:
871-881
|
23. | Grady, R.M., H. Teng, M.C. Nichol, J.C. Cuttingham, R.S. Wilkinson, and J.R. Sanes. 1997b. Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy. Cell 90: 729-738 |
24. | Greenberg, D.S., Y. Sunada, K.P. Campbell, D. Yaffe, and U. Nudel. 1994. Exogenous Dp71 restores the levels of dystrophin associated proteins but does not alleviate muscle damage in mdx mice. Nat. Genet 8: 340-344 |
25. |
Hack, A.A.,
C.T. Ly,
F. Jiang,
C.J. Clendenin,
K.S. Sigrist,
R.L. Wollmann, and
E.M. McNally.
1998.
![]() |
26. | Heighway, J., D.C. Betticher, P.R. Hoban, H.J. Altermatt, and R. Cowen. 1996. Coamplification in tumors of KRAS2, type 2 inositol 1,4,5 triphosphate receptor gene, and a novel human gene, KRAG. Genomics 35: 207-214 |
27. | Hoffman, E.P., R.H. Brown Jr., and L.M. Kunkel. 1987. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51: 919-928 |
28. |
Holt, K.H., and
K.P. Campbell.
1998.
Assembly of the sarcoglycan complex: insights for LGMD.
J. Biol. Chem
273:
34667-34670
|
29. |
Holt, K.H.,
S.B. Waters,
S. Okada,
K. Yamauchi,
S.J. Decker,
A.R. Saltiel,
D.G. Motto,
G.A. Koretzky, and
J.E. Pessin.
1996.
Epidermal growth factor
receptor targeting prevents uncoupling of the Grb2-SOS complex.
J. Biol.
Chem
271:
8300-8306
|
30. |
Holt, K.H.,
L.E. Lim,
V. Straub,
D.P. Venzke,
F. Duclos,
R.D. Anderson,
B.L. Davidson, and
K.P. Campbell.
1998.
Functional rescue of the sarcoglycan
complex in the BIO 14.6 hamster using ![]() |
31. | Homburger, F., J.R. Baker, C.W. Nixon, and R. Whitney. 1962. Primary, generalized polymyopathy and cardiac necrosis in an inbred line of Syrian hamsters. Med. Exp. 6: 339-345 . |
32. | Iwata, Y., H. Nakamura, Y. Mizuno, M. Yoshida, E. Ozawa, and M. Shigekawa. 1993. Defective association of dystrophin with sarcolemmal glycoproteins in the cardiomyopathic hamster heart. FEBS Lett 329: 227-231 |
33. |
Jung, D.,
B. Yang,
J. Meyer,
J.S. Chamberlain, and
K.P. Campbell.
1995.
Identification and characterization of the dystrophin anchoring site on ![]() |
34. | Jung, D., F. Leturcq, Y. Sunada, F. Duclos, F.M. Tome, C. Moomaw, L. Merlini, K. Azibi, M. Chaouch, C. Slaughter, et al . 1996. Absence of gamma-sarcoglycan (35 DAG) in autosomal recessive muscular dystrophy linked to chromosome 13q12. FEBS Lett 381: 15-20 |
35. | Karpati, G., S. Carpenter, G.E. Morris, K.E. Davies, C. Guerin, and P. Holland. 1993. Localization and quantitation of the chromosome 6-encoded dystrophin-related protein in normal and pathological human muscle. J. Neuropathol. Exp. Neurol. 52: 119-128 |
36. | Khurana, T.S., S.C. Watkins, P. Chafey, J. Chelly, F.M. Tome, M. Fardeau, J.C. Kaplan, and L.M. Kunkel. 1991. Immunolocalization and developmental expression of dystrophin related protein in skeletal muscle. Neuromuscul. Disord 1: 185-194 |
37. | Lim, L.E., and K.P. Campbell. 1998. The sarcoglycan complex in limb-girdle muscular dystrophy. Curr. Opin. Neurol. 11: 443-452 |
38. | Lim, L.E., F. Duclos, O. Broux, N. Bourg, Y. Sunada, V. Allamand, J. Meyer, I. Richard, C. Moomaw, C. Slaughter, et al . 1995. Beta-sarcoglycan: characterization and role in limb-girdle muscular dystrophy linked to 4q12. Nat. Genet 11: 257-265 |
39. |
Maecker, H.T.,
S.C. Todd, and
S. Levy.
1997.
The tetraspanin superfamily: molecular facilitators.
FASEB J
11:
428-442
|
40. | Matsumura, K., J.M. Ervasti, K. Ohlendieck, S.D. Kahl, and K.P. Campbell. 1992. Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle. Nature 360: 588-591 |
41. | McNally, E.M., C.T. Ly, and L.M. Kunkel. 1998. Human epsilon-sarcoglycan is highly related to alpha-sarcoglycan (adhalin), the limb-girdle muscular dystrophy 2D gene. FEBS Lett. 422: 27-32 |
42. | Mizuno, Y., S. Noguchi, H. Yamamoto, M. Yoshida, I. Nonaka, S. Hirai, and E. Ozawa. 1995. Sarcoglycan complex is selectively lost in dystrophic hamster muscle. Am. J. Pathol. 146: 530-536 [Abstract]. |
43. | Nguyen, T.M., J.M. Ellis, D.R. Love, K.E. Davies, K.C. Gatter, G. Dickson, and G.E. Morris. 1991. Localization of the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating brain cell lines. J. Cell Biol. 115: 1695-1700 [Abstract]. |
44. | Nigro, V., E. de Sa, Moreira, G. Piluso, M. Vainzof, A. Belsito, L. Politano, A.A. Puca, M.R. Passos-Bueno, and M. Zatz. 1996a. Autosomal recessive limb-girdle muscular dystrophy, LGMD2F, is caused by a mutation in the delta-sarcoglycan gene. Nat. Genet 14: 195-198 |
45. |
Nigro, V.,
G. Piluso,
A. Belsito,
L. Politano,
A.A. Puca,
S. Papparella,
E. Rossi,
G. Viglietto,
M.G. Esposito,
C. Abbondanza, et al
.
1996b.
Identification of a
novel sarcoglycan gene at 5q33 encoding a sarcolemmal 35 kDa glycoprotein.
Hum. Mol. Genet
5:
1179-1186
|
46. |
Nigro, V.,
Y. Okazaki,
A. Belsito,
G. Piluso,
Y. Matsuda,
L. Politano,
G. Nigro,
C. Ventura,
C. Abbondanza,
A.M. Molinari, et al
.
1997.
Identification of the
Syrian hamster cardiomyopathy gene.
Hum. Mol. Genet
6:
601-607
|
47. | Noguchi, S., E.M. McNally, K. Ben, Othmane, Y. Hagiwara, Y. Mizuno, M. Yoshida, H. Yamamoto, C.G. Bönnemann, E. Gussoni, P.H. Denton, et al . 1995. Mutations in the dystrophin-associated protein gamma-sarcoglycan in chromosome 13 muscular dystrophy. Science 270: 819-822 [Abstract]. |
48. | Ohlendieck, K., and K.P. Campbell. 1991. Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice. J. Cell Biol. 115: 1685-1694 [Abstract]. |
49. | Ohlendieck, K., J.M. Ervasti, K. Matsumura, S.D. Kahl, C.J. Leveille, and K.P. Campbell. 1991. Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle. Neuron 7: 499-508 |
50. | Okazaki, Y., H. Okuizumi, T. Ohsumi, O. Nomura, S. Takada, M. Kamiya, N. Sasaki, Y. Matsuda, M. Nishimura, O. Tagaya, et al . 1996. A genetic linkage map of the Syrian hamster and localization of cardiomyopathy locus on chromosome 9qa2.1-b1 using RLGS spot-mapping. Nat. Genet. 13: 87-90 |
51. |
Passos-Bueno, M.R.,
E.S. Moreira,
M. Vainzof,
S.K. Marie, and
M. Zatz.
1996.
Linkage analysis in autosomal recessive limb-girdle muscular dystrophy
(AR LGMD) maps a sixth form to 5q33-34 (LGMD2F) and indicates that
there is at least one more subtype of AR LGMD.
Hum. Mol. Genet
5:
815-820
|
52. | Petrof, B.J., J.B. Shrager, H.H. Stedman, A.M. Kelly, and H.L. Sweeney. 1993. Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc. Natl. Acad. Sci. USA. 90: 3710-3714 [Abstract]. |
53. | Phelps, S.F., M.A. Hauser, N.M. Cole, J.A. Rafael, R.T. Hinkle, J.A. Faulkner, and J.S. Chamberlain. 1995. Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice. Hum. Mol. Genet. 4: 1251-1258 [Abstract]. |
54. | Piccolo, F., S.L. Roberds, M. Jeanpierre, F. Leturcq, K. Azibi, C. Belford, A. Carrie, and D. Recan. 1995. Primary adhalinopathy: a common cause of autosomal recessive muscular dystrophy of variable severity. Nat. Genet 5: 1963-1969 . |
55. | Pons, F., N. Augier, J.O. Leger, A. Robert, F.M. Tome, M. Fardeau, T. Voit, L.V. Nicholson, D. Mornet, and J.J. Leger. 1991. A homologue of dystrophin is expressed at the neuromuscular junctions of normal individuals and DMD patients, and of normal and mdx mice. Immunological evidence. FEBS Lett 282: 161-165 |
56. |
Porter, J.D.,
J.A. Rafael,
R.J. Ragusa,
J.K. Brueckner,
J.I. Trickett, and
K.E. Davies.
1998.
The sparing of extraocular muscle in dystrophinopathy is lost
in mice lacking utrophin and dystrophin.
J. Cell Sci.
111:
1801-1811
|
57. | Rafael, J.A., Y. Sunada, N.M. Cole, K.P. Campbell, J.A. Faulkner, and J.S. Chamberlain. 1994. Prevention of dystrophic pathology in mdx mice by a truncated dystrophin isoform. Hum. Mol. Genet. 3: 1725-1733 [Abstract]. |
58. | Rafael, J.A., G.A. Cox, K. Corrado, D. Jung, K.P. Campbell, and J.S. Chamberlain. 1996. Forced expression of dystrophin deletion constructs reveals structure-function correlations. J. Cell Biol 134: 93-102 [Abstract]. |
59. |
Roberds, S.L.,
J.M. Ervasti,
R.D. Anderson,
K. Ohlendieck,
S.D. Kahl,
D. Zoloto, and
K.P. Campbell.
1993.
Disruption of the dystrophin-glycoprotein
complex in the cardiomyopathic hamster.
J. Biol. Chem.
268:
11496-11499
|
60. | Roberds, S.L., F. Leturcq, V. Allamand, F. Piccolo, M. Jeanpierre, R.D. Anderson, L.E. Lim, J.C. Lee, F.M.S. Tome, N.B. Romero, et al . 1994. Missense mutations in the adhalin gene linked to autosomal recessive muscular dystrophy. Cell 78: 625-633 |
61. |
Rybakova, I.N., and
J.M. Ervasti.
1997.
Dystrophin-glycoprotein complex is
monomeric and stabilizes actin filaments in vitro through a lateral association.
J. Biol. Chem
272:
28771-28778
|
62. | Rybakova, I.N., K.J. Amann, and J.M. Ervasti. 1996. A new model for the interaction of dystrophin with F-actin. J. Biol. Chem. 135: 661-672 . |
63. |
Sakamoto, A.,
K. Ono,
M. Abe,
G. Jasmin,
T. Eki,
Y. Murakami,
T. Masaki,
T. Toyo, and
-oka, and F. Hanaoka.
1997.
Both hypertrophic and dilated cardiomyopathies are caused by mutation of the same gene, delta-sarcoglycan, in
hamster: an animal model of disrupted dystrophin-associated glycoprotein
complex.
Proc. Natl. Acad. Sci. USA
94:
13873-13878
|
64. | Scott, A.F., A. Elizaga, J. Morrell, A. Bergen, and M.B. Penno. 1994. Characterization of a gene coamplified with Ki-ras in Y1 murine adrenal carcinoma cells that codes for a putative membrane protein. Genomics 20: 227-230 |
65. | Straub, V., and K.P. Campbell. 1997. Muscular dystrophies and the dystrophin- glycoprotein complex. Curr. Opin. Neurol 10: 168-175 |
66. |
Straub, V.,
J.A. Rafael,
J.S. Chamberlain, and
K.P. Campbell.
1997.
Animal
models for muscular dystrophy show different patterns of sarcolemmal disruption.
J. Cell Biol
139:
375-385
|
67. |
Straub, V.,
F. Duclos,
D.P. Venzke,
J.C. Lee,
S. Cutshall,
C.J. Leveille, and
K.P. Campbell.
1998.
Molecular pathogenesis of muscle degeneration in the
![]() |
68. |
Sunada, Y.,
S.M. Bernier,
C.A. Kozak,
Y. Yamada, and
K.P. Campbell.
1994.
Deficiency of merosin in dystrophic dy mice and genetic linkage of laminin
M chain gene to dy locus.
J. Biol. Chem
269:
13729-13732
|
69. | Sunada, Y., S.M. Bernier, A. Utani, Y. Yamada, and K.P. Campbell. 1995. Identification of a novel mutant transcript of laminin alpha 2 chain gene responsible for muscular dystrophy and dysmyelination in dy2J mice. Hum. Mol. Genet 4: 1055-1061 [Abstract]. |
70. | Tinsley, J., N. Deconinck, R. Fisher, D. Kahn, S. Phelps, J.M. Gillis, and K. Davies. 1998. Expression of full-length utrophin prevents muscular dystrophy in mdx mice. Nat. Med 4: 1441-1444 |
71. | Tinsley, J.M., A.C. Potter, S.R. Phelps, R. Fisher, J.I. Trickett, and K.E. Davies. 1996. Amelioration of the dystrophic phenotype of mdx mice using a truncated utrophin transgene. Nature 384: 349-353 |
72. | Weller, B., G. Karpati, and S. Carpenter. 1990. Dystrophin-deficient mdx muscle fibers are preferentially vulnerable to necrosis induced by experimental lengthening contractions. J. Neurol. Sci. 100: 9-13 |
73. | Wright, M.D., and M.G. Tomlinson. 1994. The ins and outs of the transmembrane 4 superfamily. Immunol. Today 15: 588-594 |
74. | Xu, H., P. Christmas, X.-R. Wu, U.M. Wewer, and E. Engvall. 1994a. Defective muscle basement membrane and lack of M-laminin in the dystrophic dy/dy mouse. Proc. Natl. Acad. Sci. USA 91: 5572-5576 [Abstract]. |
75. |
Xu, H.,
X.-R. Wu,
U.M. Wewer, and
E. Engvall.
1994b.
Murine muscular dystrophy caused by a mutation in the laminin ![]() ![]() |
76. |
Yang, B.,
D. Jung,
D. Motto,
J. Meyer,
G. Koretzky, and
K.P. Campbell.
1995.
SH3 domain-mediated interaction of dystroglycan and Grb2.
J. Biol. Chem
270:
11711-11714
|
77. | Yoshida, M., and E. Ozawa. 1990. Glycoprotein complex anchoring dystrophin to sarcolemma. J. Biochem. (Tokyo) 108: 748-752 [Abstract]. |
78. | Yoshida, M., A. Suzuki, H. Yamamoto, S. Noguchi, Y. Mizuno, and E. Ozawa. 1994. Dissociation of the complex of dystrophin and its associated proteins into several unique groups by n-octyl beta-D-glucoside. Eur. J. Biochem. 222: 1055-1061 [Abstract]. |