Dystroglycan Is Not Required for Localization of Dystrophin, Syntrophin, and Neuronal Nitric-oxide Synthase at the Sarcolemma but Regulates Integrin α7B Expression and Caveolin-3 Distribution*

Dystroglycan is part of the dystrophin-associated protein complex, which joins laminin in the extracellular matrix to dystrophin within the subsarcolemmal cytoskeleton. We have investigated how mutations in the components of the laminin-dystroglycan-dystrophin axis affect the organization and expression of dystrophin-associated proteins by comparing mice mutant for merosin (α2-laminin, dy), dystrophin (mdx), and dystroglycan (Dag1) using immunohistochemistry and immunoblots. We report that syntrophin and neuronal nitric-oxide synthase are depleted in muscle fibers lacking both dystrophin and dystroglycan. Some fibers deficient in dystroglycan, however, localize dystrophin at the cell surface at levels similar to that in wild-type muscle. Nevertheless, these fibers have signs of degeneration/regeneration including increased cell surface permeability and central nuclei. In these fibers, syntrophin and nitric-oxide synthase are also localized to the plasma membrane, whereas the sarcoglycan complex is disrupted. These results suggest a mechanism of membrane attachment for dystrophin independent of dystroglycan and that the interaction of sarcoglycans with dystrophin requires dystroglycan. The distribution of caveolin-3, a muscle-specific component of caveolae recently found to bind dystroglycan, was affected in dystroglycan- and dystrophin-deficient mice. We also examined alternative mechanisms of cell-extracellular matrix attachment to elucidate how the muscle basement membrane may subsist in the absence of dystroglycan, and we found the α7B splice variant of the α7 integrin receptor subunit to be up-regulated. These results support the possibility that α7B integrin compensates in mediating cell-extracellular matrix attachment but cannot rescue the dystrophic phenotype.

Striated muscle fibers interact with a specialized extracellular matrix, the basement membrane, upon which they depend for survival and function. This interaction is mediated primarily by a large oligomeric dystrophin-associated protein (DAP) 1 complex (1, 2) as well as by integrin receptors (3). Based on their biochemical properties, the DAP complex can be subdivided into three subcomplexes as follows: the dystroglycan complex, the sarcoglycan-sarcospan complex, and the cytoplasmic complex (dystrophin, syntrophin, and dystrobrevin) (4). The ␣ and ␤ subunits of dystroglycan constitute the core of the DAP complex because they establish a transmembrane link between laminins and dystrophin (5). Integrins, heterodimeric (␣/␤) transmembrane receptors, are widely recognized as mediators of cell-extracellular matrix interaction involved in the regulation of such fundamental processes as proliferation, migration, differentiation, adhesion, and survival. The ␣ 7 ␤ 1 integrin, a highly expressed receptor on the surface of myoblasts and myotubes, also binds laminins and is critical for muscle fiber survival and differentiation (6).
A number of studies have demonstrated that myopathies of variable severity arise when genetic lesions are introduced in molecules involved in linking the basement membrane to the cytoskeleton (for recent reviews see Refs. 7 and 8). In fact, the most prevalent form of muscle disorder, Duchenne muscular dystrophy, is caused by a mutation in the dystrophin gene (DMD) that breaks this interaction at the level of the peripheral cytoskeleton (9,10). The concomitant reduction of the DAP complex with dystrophin in striated muscles of Duchenne muscular dystrophy patients and in the mdx mouse (11)(12)(13) suggests a critical role for the DAP complex in the maintenance of membrane integrity. The integrins have also been implicated in muscle disease (14 -17). Most relevant to human diseases are observations that mutations in the ␣ 7 integrin subunit gene produce a congenital myopathy that appears to result from disruptions in the myotendonous junctions (15)(16)(17). Furthermore, an increase in ␣ 7B integrin transcript and protein levels is seen in cases of Duchenne and Becker muscular dystrophy and in the murine mdx model (18,19). This up-regulation suggests that integrins may attempt to compensate for the loss of dystrophin/DAP-mediated linkage. Interestingly, sarcolemmal disruption is not an obligatory step in the pathogenesis of muscular dystrophies as, for example, in the "classical" form of congenital muscular dystrophy and in the dy mouse. The severe dystrophy observed both in congenital muscular dystrophy patients and in the dy mouse is caused by mutations in the laminin-␣ 2 gene and does not appear to involve damage to the sarcolemma.
The muscle-specific form of caveolin, caveolin-3, is a major structural component of caveolae that has recently been implicated in the etiology of muscular dystrophy (20 -22). Although it does not associate tightly with the DAP complex (23), caveo-lin-3 was recently found to bind directly to the same domain as dystrophin on the cytoplasmic tail of ␤-dystroglycan (24). Mutations in caveolin-3 lead to limb girdle muscular dystrophy 1C (21), and it was also found to be overexpressed in muscle from Duchenne muscular dystrophy patients and from mdx mice (25,26). The pathogenic mechanism of caveolin-3 remains elusive, but precise regulation of expression levels appears to be important for normal muscle function because a null mutation (27,28) and induced overexpression (29) both lead to muscle pathology in mice.
In a previous report (30), we demonstrated that chimeric mice with little or no dystroglycan had a severe histopathology closely resembling that of patients with Duchenne muscular dystrophy and that was more severe than that of the mdx mouse. In the present study, we have explored the composition of the DAP complex in dystroglycan-deficient muscle to determine whether interactions inferred from biochemical studies or from mutations of dystroglycan-interacting proteins are confirmed by depletion of dystroglycan in situ. We find that the depletion of dystroglycan-interacting proteins results not only from displacement from the sarcolemma toward the cytosol but also from a decrease in overall protein levels. Furthermore dystrophin was found not to be absolutely dependent on ␤-dystroglycan for membrane targeting. We have also examined the distribution of caveolin-3 in dystroglycan-deficient muscle and found its distribution identical to that of the mdx mouse. Finally, the ␣ 7B splice variant of the integrin ␣ 7 subunit was found to be selectively up-regulated. Because dystroglycandeficient muscles have basement membranes that are indistinguishable from controls (30), this receptor may compensate for the loss of dystroglycan in basement membrane assembly.

EXPERIMENTAL PROCEDURES
Animals-Embryonic stem (ES) cell lines rendered null for dystroglycan were described previously (30). Briefly, we generated two targeting vectors, one harboring a hygromycin resistance gene at the 5Ј end of the second exon of the dystroglycan gene (Dag1) and one with the neomycin resistance gene also at the 5Ј end of the second exon. R1 ES cells (31) were electroporated with the hygromycin resistance vector and submitted to drug selection, and homologous recombinants were identified by Southern blotting. The same procedure was repeated on single copy Dag1 mutants with the neomycin resistance targeting vector to target the other copy. Two dystroglycan null clones, 3C12 and 3H1, were chosen for blastocyst injection. Mice from the resulting litters were selected initially on the basis of their coat color for further analysis. Mice with mutations in the dystrophin (B6Ros.Cg-Dmd mdx-3Cv ) and ␣ 2 -laminin (129/ReJ-Lama2 dy ) gene were obtained from The Jackson Laboratory (Bar Harbor, ME). The mdx-3Cv mice are deficient in muscle and non-muscle dystrophin isoforms as a result of a mutation in a consensus splice acceptor site (32). The animals were maintained and humanely sacrificed in accordance to the guidelines of the Canadian Council on Animal Care.
Creatine Kinase Assay-Approximately 0.5 ml of blood was collected from mice that were anesthetized by using 13 mg/kg xylazine and 100 mg/kg ketamine. Total creatine kinase (CK) levels in serum was measured with an Hitachi 917 automated clinical chemistry analyzer (Hitachi, San Francisco, CA) using a CK assay kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. In cases where CK levels exceeded 2300 units/liter, samples were diluted to ensure that measurements taken were within the linear range of the assay.
Electron Microscopy-Blocks of muscle (2-3 mm 3 ) were postfixed for 3 h in 3.5% glutaraldehyde, rinsed, and fixed for 1 h in 1% osmium tetroxide. The samples were dehydrated and embedded in Durcupan (Sigma). Ultrathin sections were collected (60 -65 nm) on 100 or 200 square mesh grids, counterstained with lead citrate, and examined with a CM10 Philips transmission electron microscope.
Immunohistochemistry-Hindlimb muscles (gastrocnemius, soleus, and femoral biceps) were dissected and frozen in liquid nitrogen-cooled isopentane in Tissue-Tek OCT compound (Sakura, Torrance, CA). Remaining hindlimb muscles were isolated from the bone and frozen in liquid nitrogen for the preparation of protein extracts. In chimeric mice, the contribution from dystroglycan null ES cells to the hindlimb musculature was assessed by glucose-phosphate isomerase (GPI) assay (34) on cellulose acetate plates (Helena Laboratories, Beaumont, TX) as well as by immunoblotting and immunohistochemistry using an antiserum against ␤-dystroglycan. Fresh frozen muscle was sectioned at 10 m in a cryostat; sections were collected on gelatin-coated slides and stored at Ϫ80°C until required. Experimental and control tissues were placed on the same slide to ensure they were submitted to the same experimental conditions. Slides were thawed at 37°C, incubated in 10% normal goat serum in Dulbecco's PBS (blocking solution) for 30 min at room temperature, incubated overnight with the primary antibody in blocking solution at 4°C, washed with Dulbecco's PBS, incubated with Oregon Green 488 goat anti-rabbit IgG antiserum (Molecular Probes, Eugene, OR) in blocking solution for 1 h at room temperature, and washed with Dulbecco's PBS. As required, slides were then incubated with rhodamine isothiocyanate-labeled ␣-bungarotoxin and/or 4Ј,6-diamidino-2phenylindole (DAPI, Molecular Probes), before mounting with Immun-oFluor (ICN, Costa Mesa, CA). For the analysis of serial sections of transversally sectioned muscle, corresponding areas on adjacent sections were identified in phase contrast using a CCD camera (Hamamatsu Photonics, Bridgewater, NJ), and then photographs were taken on a Axioscope fluorescence microscope (Carl Zeiss, Thornwood, NY).
Extract Preparation and Immunoblotting-Hindlimb muscles from wild-type control, mdx, dy, and chimeric mice were minced in Dulbecco's PBS with 1ϫ protease inhibitor mixture (Roche Molecular Biochemicals) and spun gently at 150 ϫ g. The pellet was used for total protein extraction using a rotating Teflon Dounce homogenizer at 800 rpm in extraction buffer (25 mM Tris, pH 7.5, 25 mM glycine, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 1ϫ protease inhibitor mixture). A wild-type control sample was always processed alongside the experimental sample under identical conditions. The protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad). To confirm that there was a high contribution of dystroglycan-null ES cells to muscle, a sample of protein extract from chimeric mice was analyzed by GPI assay (Fig. 4A, results were replicated in two additional chimeras) (30). Each sample (40 g per lane) was electrophoresed on an SDS-polyacrylamide gel of appropriate percentage depending on the protein examined and electrotransferred for 1 h at 100 V onto nitrocellulose. Membranes were blocked using 5% nonfat dry milk, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween for 1 h and incubated with the primary antibody in blocking buffer for 2 h at room temperature, washed, and probed with horseradish peroxidase-conjugated goat antimouse or goat anti-rabbit antisera (Jackson ImmunoResearch, West Grove, PA) at a 1/2000 dilution. Blots were developed using the ECL chemiluminescence system (Amersham Biosciences). Densitometric analysis was performed using the NIH image analysis program.

RESULTS
Chimeric mice with reduced levels of dystroglycan develop histopathologic signs typical of dystrophic muscle (30). In addition, they have CK levels 20 -100 times above normal ( Fig.  1A) consistent with a breakdown in cell surface permeability. Electron microscopy analysis of the gastrocnemius muscle from chimeric mice shows major disruptions in the ultrastructure of some muscle fibers (Fig. 1, B and C). The nuclei often contained vacuoles and prominent nucleoli (Fig. 1B). The sarcoplasmic reticulum was dilated (Fig. 1B) similar to skeletal muscle in mdx mice and patients with Duchenne muscular dystrophy (35,36). We have also observed "cytomembrane swirls" (Fig. 1C) that appear as concentric layers of intracellular membranes with a morphology distinct from "fingerprint bodies" that are seen in several myopathies (37,38).
Dystroglycan Is Not Required for the Localization of Dystrophin and the Cytoplasmic DAP Complex to the Sarcolemma-In a previous study (30) we had observed several fibers that were positively labeled for dystrophin even within chimeric muscle that appeared to be null for dystroglycan when muscle extracts were assayed by immunoblotting or by GPI electrophoresis. At the time we were unable to monitor dystroglycan expression within single myofibers because the anti-mouse IgG secondary antiserum used to recognize the dystroglycan monoclonal antibody also reacted with endogenous immunoglobulins that had infiltrated the dystroglycan-depleted muscle (30). Thus, the localization of dystrophin to the sarcolemma of a subset of these fibers could be due to dystroglycan expression in chimeric myofibers with a small number of wild-type nuclei. Therefore, we generated a rabbit anti-␤-dystroglycan antiserum, which revealed that in muscle that was dystroglycan-deficient by immunoblotting and GPI assays, ϳ1-3% of the fibers were dystroglycan-positive ( Fig. 2G and not shown).
By staining serial sections of muscle and identifying the same region on all the sections, it was possible to determine the make-up of the whole DAP complex in a single fiber. In the vast majority of myofibers in dystroglycan-deficient chimeras dystroglycan was undetectable (Fig. 2G) as were dystrophin (Fig.  2J), the sarcoglycans (Fig. 2, H and I), and the cytoplasmic complex (␣ 1 -syntrophin and nNOS) (Fig. 2, K and L). These fibers had an immunohistochemical profile similar to dystrophin null muscle (Fig. 2, S-X) but distinct from merosin null muscle (M-R) where DAPs are largely unaffected (12). In a few instances where fibers in dystroglycan-deficient muscle had detectable levels of dystroglycan, dystrophin, the sarcoglycans (␣ and ␥), and the cytoplasmic DAP complex (syntrophin and nNOS) were localized at the plasmalemma (Fig. 2, G-L, arrowheads) at levels similar to that in wild-type mice (Fig. 2, A-F). However, even when dystroglycan was poorly expressed, it was often concentrated at neuromuscular junctions, identified by double labeling with ␣-bungarotoxin (39; not shown), and colocalized with molecules of the DAP complex. Presumably, recruitment of residual dystroglycan to junctions during development (40) can stabilize the complex at those sites (39) and may help render these regions resistant to disruption.
In some fibers where there was no trace of dystroglycan or ␣-sarcoglycan (Fig. 3, A and B), dystrophin was clearly detectable at the plasmalemma, and these fibers were typically found in small groups in the muscle (Fig. 3C). In these instances syntrophin and nNOS co-distributed with dystrophin at the cell surface (Fig. 3, D and E). Thus, dystrophin and a cytoplasmic complex of at least syntrophin and nNOS can be localized at the plasma membrane independently of dystroglycan or the sarcoglycans. The membrane anchor responsible for this interaction is unknown as are member(s) of the cytoplasmic complex that can interact with it (discussed below). It is important to note, however, that the complex lacking dystroglycan was insufficient to prevent muscle degeneration because these myofibers were atrophic and/or had central nuclei typical of myofiber regeneration (Fig. 3F) and were occasionally found to contain high levels of endogenous immunoglobulins (not shown) indicative of a breakdown in the sarcolemma.
Absence of Dystroglycan Leads to a Reduction in Expression of the DAP Complex-To assess whether the reduced immunohistochemical labeling at the sarcolemma of mutant muscle reflects a reduction in expression of the DAPs or simply a displacement from the sarcolemma to the cytosol, we performed immunoblots for dystrophin and members of the DAP complex. The expression level of the DAP complex in total protein extracts (1% Triton X-100) of dystroglycan-deficient chimeric mice was compared with that of mdx and dy mice. Replicate blots were made and probed with antibodies to ␣-dystroglycan, ␤-dystroglycan, ␣-sarcoglycan, ␤-sarcoglycan, ␥-sarcoglycan, dystrophin, ␣ 1 -syntrophin, ␤ 1 -syntrophin, and nNOS (Fig. 4B). As shown previously, the absence of dystrophin in the mdx mouse leads to a reduction in the expression of DAP components including dystroglycan, sarcoglycans, syntrophin, dystrobrevin, and nNOS (12,(41)(42)(43). Dystrophin levels were considerably reduced in dystroglycan-deficient chimeric muscle, and the effect of dystroglycan depletion mirrored that of dystrophin depletion in the mdx mouse resulting in a significant reduction of the sarcoglycan complex and ␣ 2 -dystrobrevin. nNOS, which binds to syntrophin, was also substantially decreased in dystroglycan-deficient mice but only moderately affected in mdx mice. The reduction in these DAPs did not appear merely to result from the degeneration of myofibers in these animals because dy mice, which have a severe muscular dystrophy, expressed these DAPs in amounts equal to those in wild-type skeletal muscle (Fig. 4B and Fig. 2, M-R).
In contrast, ␣ 1 -syntrophin showed little change in expression levels in dystroglycan-deficient muscle. Thus targeting to the membrane of ␣ 1 -syntrophin rather than expression, per se, seems disrupted in the absence of dystroglycan. Consistent with this an internal pool of ␣-syntrophin is visible immunohistochemically in some myofibers (Fig. 2K). This pattern is different from that in dystrophin null muscle where ␣ 1 -syntrophin is essentially undetectable (Fig. 2W). In mdx, dy, and dystroglycan-deficient muscle the anti-␤ 1 -syntrophin antibody revealed a protein of 56 kDa in addition to the 59-kDa protein. This protein is most likely a product of proteolysis that occurs in all types of dystrophy.
Finally, the predominantly synaptic isoform of ␣-dystrobrevin, ␣ 1 -dystrobrevin, was not affected in dy, mdx, or chimeric mice, which again appears to reflect the stability of the postsynaptic density at neuromuscular junctions. These differences in DAPS in dystroglycan-deficient, mdx, and dy muscles reveal distinct interactions for dystroglycan, dystrophin, and merosin in the DAP complex as well as in regulation of expression of other members of the DAP complex.
Redistribution of Caveolin-3 in Dystroglycan-deficient Chimeric Muscle-Caveolin-3 is a muscle-specific component of small membrane invaginations (caveolae) found in many cell types and has recently been implicated in human myopathies (21,22). Because caveolin-3 binds to dystroglycan (24), we investigated its distribution in dystroglycan-deficient mice. Immunostaining with antiserum specific to caveolin-3 stained the sarcolemma of chimeric muscle in a fashion closely resembling that of mdx mice (compare Fig. 5, H, I, K with F and G). Not only was the staining in chimeras and mdx mice more intense than in wild-type and dy mice (Fig. 5, A, B, D, and E), but the antigen was in a wider band at the cell surface. In immunoblots, caveolin-3 levels were increased by ϳ36%, suggesting that dystrophin and dystroglycan are in a genetic pathway that regulates the distribution of caveolin-3 at the cell surface (Fig. 5L).
The ␣ 7B Integrin Is a Candidate for Compensation in Dystroglycan-deficient Chimeric Muscle-Dystroglycan is thought to be a laminin receptor essential for basement membrane assembly (44,45). Yet the function of basement membranes in maintaining the integrity of the cell surface and in the etiology of muscular dystrophies is complex. For example, in its composition and ultrastructure, the muscle basement membrane from dystroglycan-depleted chimeric mice appears to be normal (30). Because the ␣ 7 ␤ 1 integrin is a major integrin receptor for laminins in muscle and the ␣ 7B integrin is up-regulated in cases of Duchenne and Becker muscular dystrophy (18,19), we examined the expression and the distribution of ␣ 7 integrin subunit splice variants in dystroglycan-deficient muscle. Immunoblot analysis of total protein extracts probed with anti-␣ 7A integrin and anti-␣ 7B integrin antisera revealed that levels of ␣ 7A integrin are decreased in dystroglycan-deficient chimeric muscle, whereas levels of ␣ 7B integrin are increased (Fig. 6, A  and B). Immunofluorescence with anti-␣ 7B antibody revealed areas of increased immunoreactivity, which in longitudinal sections appeared to follow the whole length of the fiber (Fig. 6,  C and D). These areas of increased ␣ 7B staining often contained atrophic/centrally nucleated fibers and were interspersed with degenerating fibers. Thus, in the absence of dystroglycan a splice variant of the ␣ 7 integrin subunit is selectively up-regulated and may help assemble and maintain the basement membrane, but this is insufficient to sustain muscle fiber integrity. DISCUSSION ␣and ␤-dystroglycan are thought to form the functional core of the DAP complex, but detailed study of these proteins has been hampered by the absence of natural mutations in the DAG1 gene as well as by the early embryonic lethality of targeted mutations of DAG1 in mice (46). In previous work we have generated chimeric mice with skeletal muscle deficient in dystroglycan, which have a muscular dystrophy and defects in nerve-muscle synapses (30,39). Here we present evidence on interactions in situ of members of the DAP complex in the absence of ␣and ␤dystroglycan.
Dystrophin interacts with ␤-dystroglycan primarily via amino acids 3054 -3271, although amino acids 3271-3446 are necessary for optimal binding (47,48). These regions are near the C terminus and encompass a WW domain, two putative calcium-binding EF hand motifs, and a putative zinc finger domain (49,50). Mutations within the C-terminal of dystrophin are rare and usually result in the absence of detectable dystrophin protein. However, at least five patients have been reported who completely lacked the C terminus, including the cysteine-rich region, but have dystrophin localized correctly to the sarcolemma (51)(52)(53)(54)(55). In three of these patients proteins of the DAP complex were greatly reduced (55).
Here we describe for the first time a situation where dystrophin is localized to the sarcolemma in myofibers that have undetectable dystroglycan. These data complement the data discussed above and reveal that the sarcolemma contains a non-dystroglycan anchor for dystrophin. The nature of this anchor is obscure. nNOS is known to bind caveolin-3 (56) and syntrophin (57, 58) and may provide a means for dystrophin,  4. Expression of dystrophin, the sarcoglycan complex, and nNOS, but not ␣ 1 -syntrophin, is affected in dystroglycandeficient muscle. Electrophoresis of GPI isozymes (A) demonstrated that the chimeric muscle (ch) used for subsequent immunoblotting analysis had a very high contribution from dystroglycan-null ES cells. Muscle extracts from 129 mice (129, strain from which the ES cells were derived) and C57Bl6/J (C57, strain used to produce host blastocysts) were used as controls. 1% Triton X-100 extracts (B) of chimeric mouse (ch), laminin-␣ 2 -deficient dy (dy), and dystrophin-deficient mdx (mdx) hindlimb muscles were separated by SDS-PAGE, blotted to nitrocellulose, and probed for ␣-dystroglycan (␣-DG), ␤-dystroglycan (␤-DG), ␣-sarcoglycan (␣-SG), ␤-sarcoglycan (␤-SG), ␥-sarcoglycan (␥-SG), dystrophin (dyst), ␣ 1 -syntrophin (␣1-SY), ␤ 1 -syntrophin (␤1-SY, arrowhead), ␣ 1 -dystrobrevin (␣1-DB), ␣ 2 -dystrobrevin (␣2-DB, arrowhead), and neuronal nitric-oxide synthase (nNOS). A wild-type control (wt) was processed alongside each experimental extract. The expression pattern of the DAP complex generally follows that of the mdx mouse in dystroglycan-deficient animals with the exception of ␣ 1 -syntrophin, which is present at normal levels in chimeric muscle but reduced in mdx. The open arrowhead points to a 56-kDa protein detected by the ␤ 1 -syntrophin antiserum present in extracts from all three dystrophic muscles (ch, dy, and mdx), which is likely to be a product of proteolysis. A strong ␣ 1 -dystrobrevin signal in all lanes confirmed that equivalent amounts of proteins were loaded. independent of dystroglycan, to associate with the sarcolemma. Alternatively, the syntrophins have pleckstrin homology domains that are known to direct membrane targeting of their host proteins by binding to polyphosphoinositides (59,60). Indeed, we have found that nNOS and ␣ 1 -syntrophin were localized at the sarcolemma in dystroglycan-negative/dystrophinpositive fibers (Fig. 3), consistent with the possibility that either or both of these proteins anchor dystrophin to the sarcolemma in the absence of dystroglycan. Other possibilities include direct binding of dystrophin to the lipid bilayer through its spectrin-like domains (61) or maintenance of dystrophin at the periphery via its interaction with actin or via an unidentified membrane protein.
It is important to note that this non-dystroglycan anchor is insufficient to maintain muscle integrity. Dystroglycan-negative/ dystrophin-positive myofibers have increased cell-surface permeability reflected by the infiltration of immunoglobulins into the sarcoplasm 2 despite localization of dystrophin, syntrophin, and nNOS at the sarcolemma. Consistent with this, forced expression of dystrophin constructs lacking the entire cysteine-rich domain in mdx mice did not alter dystrophin targeting to the sarcolemma (62). Also consistent with our observations in chimeric mice, the muscular dystrophy progressed unabated in mdx mice expressing these dystrophin constructs (62). FIG. 6. The ␣ 7B integrin is a candidate for molecular compensation in dystroglycan-null muscle. A, 1% Triton X-100 extracts of C57Bl/6 (c57), 129Re/J (129), and chimeric mouse (ch) hindlimb muscles were separated by SDS-PAGE, blotted to nitrocellulose, and probed for ␤-dystroglycan (␤-DG), ␣ 7A integrin (␣7A), and ␣ 7B integrin (␣7B). In dystroglycan-depleted muscle the ␣ 7A integrin expression is decreased, whereas the expression of the ␣ 7B integrin is substantially increased. B, histogram depicting the percentage of variation relative to the wild type of individual chimeric mice as quantified by densitometric analysis of immunoblots for ␣ 7A (open bars) and ␣ 7B (filled bars) integrins. The increase in the ␣ 7B integrin is also shown immunohistochemically in wild-type (C) and in chimeric gastrocnemius muscle (D). In the latter the staining is increased along the whole length of the fiber. The arrow indicates a degenerating fiber. Bar indicates 100 m.
ple, loss of any one of the sarcoglycans leads to abnormal expression of all others at the sarcolemma (63)(64)(65)(66). Also reduction of dystrophin levels in cases of ␥-sarcoglycan deficiency (67) together with chemical cross-linking studies (2) indicate that dystrophin and ␥-sarcoglycan interact directly. ␣-Sarcoglycan seems to be only loosely associated with the other sarcoglycans, whereas ␦-sarcoglycan appears to be most closely associated with ␤-dystroglycan (68). The disruption of ␥-sarcoglycan only results in a mild reduction of ␣and ⑀-sarcoglycans with no effect on the expression of ␣and ␤-dystroglycan (65). In chimeric muscle with reduced dystroglycan, ␣and ␥-sarcoglycan are found exclusively in myofibers that are dystroglycan-positive (Fig. 2). Thus, unlike syntrophin and nNOS, the presence of dystrophin at the sarcolemma in the absence of dystroglycan does not restore the sarcoglycan complex (Fig. 3). Matsumura et al. (69) have predicted a direct interaction of sarcoglycans with the extracellular matrix and proposed a model in which the sarcoglycans bind both the dystroglycans and the extracellular matrix to stabilize the DAP complex. If this is so, our results suggest that the absence of dystroglycan may destabilize the complex or, perhaps, that it is required for assembly or insertion of the sarcoglycan complex at the cell surface.
Caveolin oligomers are major structural components of caveolae, small (50 -100 nm) vesicle-like structures at the plasma membrane that have been implicated in endocytosis, signal transduction, and organization of lipids in the membrane (28,70). Mutations in caveolin-3 give rise to limb girdle muscular dystrophy 1C (21,22), and mice with a targeted mutation in the caveolin-3 gene or mice overexpressing caveolin-3 have dystrophic-like muscles. Biochemical studies indicate that in skeletal muscle caveolin-3 (71) can interact with nNOS (56) as well as with the C terminus of ␤-dystroglycan (24). By comparing different muscular dystrophies, one finds that in mice null for caveolin and chimeric mice deficient in dystroglycan there is a substantial increase in serum levels of creatine kinase indicative of muscle degeneration (Ref. 28, Fig.  1). Although in dystroglycan-deficient mice (30) muscle degeneration is considerably more severe (cf. Ref. 28). Moreover, mice (28) or human (72) mutants in caveolin-3 show no changes in the amount of ␤-dystroglycan, dystrophin, or ␣-sarcoglycan. ␣-Dystroglycan, however, may be absent at the sarcolemma (72). This suggests a common pathway for muscle pathology in these mutants and emphasizes the value of genetic studies in analyzing the interactions of DAPs in situ because biochemical studies have suggested that ␣and ␤-dystroglycan are obligate partners (4). Mice lacking caveolin also do not appear to target ␤-dystroglycan, dystrophin, or ␣-sarcoglycan to cholesterolsphingolipid rafts (28) which are normally rich in caveolin. In mice deficient in dystroglycan, caveolin expression is increased and appears microscopically as a broad band at the sarcolemma with occasional whispy extensions into the proximal sarcoplasm (Fig. 5). Immunohistochemically and in Western blots the increase in caveolin expression appears similar to that seen in Duchenne muscular dystrophy (26). This mislocalization of caveolin may reflect its interaction with the PPXY motif in ␤-dystroglycan (24). Also in dystroglycan-deficient muscle, nNOS is essentially absent from the sarcolemma, whereas caveolin is increased, again suggesting that the interaction of these molecules may be necessary for localization to caveolae rather than for targeting to the sarcolemma per se. We are currently exploring whether dystroglycan regulates the formation of caveolae and whether dystroglycan-deficient mice may have other features of mice null for caveolin (28).
The notion that DAPs are involved in the assembly of basement membranes is supported by observations in skeletal mus-cle (45,73) and other tissues (44,46). Consistent with this, merosin, perlecan, and acetylcholinesterase are depleted from the basement membrane at neuromuscular junctions of dystroglycan-deficient muscle (39). In contrast, basement membranes are not similarly affected in the extrajunctional regions of skeletal muscle deficient in dystroglycan (30). This may result from compensation in extrajunctional regions by other extracellular matrix receptors, most obviously the ␣ 7 ␤ 1 integrin that forms the dominant laminin-binding integrin in mature skeletal muscle. The ␣ 7 integrin subunit has also been reported to be up-regulated in cases of Duchenne muscular dystrophy and in the mdx mouse (18,19), both of which have apparently normal basement membranes. On the other hand, dy mice that have disrupted basement membranes (74,75) have decreased levels of ␣ 7 integrins (18,19,76) with no effect on dystroglycan expression. In our studies with dystroglycan-deficient muscle, the ␣ 7B splice variant was found to be up-regulated both in extracts of chimeric muscle, and the ␣ 7A variant was downregulated. The ␣ 7B subunit was also increased immunohistochemically at the surface of the gastrocnemius muscle (Fig. 6, C and D). This muscle was chosen for analysis because it is composed primarily of type IIb and type IId fibers (77) which normally express low or moderate levels, respectively, of ␣ 7B integrin at the sarcolemma. These results support others (18,19) demonstrating a regulatory pathway between the DAP complex and integrin receptors and are consistent with the idea that dystroglycans are involved in selective regulation of integrin transcription (18).
Mice and humans with mutations in the ␣ 7 integrin gene are myopathic (15,16). In a recent study (78), the ␣ 7BX2 isoform of the ␣ 7 integrin was overexpressed on a dystrophin (mdx)/utrophin double knockout genetic background, which resulted in a considerable amelioration of the dystrophy in the double knockout. One can speculate that in dystroglycan-deficient muscle where levels of ␣ 7B integrin are maintained, there may be compensatory effects in basement membrane assembly and muscle degeneration, which raises questions about whether these alterations affect the etiology of other muscular dystrophies where dystroglycan expression is compromised.