Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin–glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin–glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a γ- and δ-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.

Dystrobrevin is a component of the dystrophin-associated protein complex and has been shown to interact directly with dystrophin, ␣1-syntrophin, and the sarcoglycan complex. The precise role of ␣-dystrobrevin in skeletal muscle has not yet been determined. To study ␣-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting proteins. Three overlapping clones were identified that encoded an intermediate filament protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 intermediate filament proteins. A positive interaction between DMN and ␣-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.T he severe muscle wasting disorder, Duchenne muscular dystrophy, is caused by abnormalities in the dystrophin gene (1). The dystrophin protein is expressed in heart and skeletal muscle, where it is part of the dystrophin-associated protein complex. Dystrophin's N-terminal domain binds to actin, whereas the WW domain and the total cysteine-rich domain bind to ␤-dystroglycan (2), a component of the dystroglycan subcomplex. This subcomplex links to laminin, a major component of the basal membrane, thereby forming the linkage between an intracellular protein, actin, and the extracellular matrix.A second subcomplex of the dystrophin-associated protein complex includes four transmembrane proteins (␣-, ␤-, ␥-, and ␦-sarcoglycan) (3). Each has been shown to be involved in different forms of limb-girdle muscular dystrophy (LGMD 2D, 2E, 2C, and 2F) (4-8). ␣-Sarcoglycan is a type 1 transmembrane protein and is expressed in heart and skeletal muscle. ␤-, ␥-, and ␦-sarcoglycans are type 2 transmembrane proteins containing a cluster of cysteine residues in their extracellular domains. These four proteins form the sarcoglycan complex, which is thought to be involved in some type of signaling pathway (9).A third subcomplex of the dystrophin-associated protein complex involves ␣-dystrobrevin (10-12) and the syntrophins (␣1, ␤1, and ␤2) (13-15). These intracellular proteins directly bind to dystrophin (16,17). In addition, the N-terminal region of ␣-dystrobrevin associates with the sarcoglycan complex (18). There are at least ...

Calpain 3 (C3) is the only muscle-specific member of the calcium-dependent protease family. Although neither its physiological function nor its in vivo substrates are known, C3 must be an important protein for normal muscle function as mutations in the C3 gene result in limb-girdle muscular dystrophy type 2A. Previous reports have shown that the ubiquitous calpains (mu and m) proteolyze filamins in nonmuscle cells. This observation suggests that the muscle-specific filamin C (FLNC) is a good candidate substrate for C3. Binding studies using recombinant proteins establish that recombinant C3 and native FLNC can interact. When these two proteins are translated in vitro and incubated together, C3 cleaves the C-terminal portion of FLNC. Cleavage is specific as C3 fails to cleave FLNC lacking its C-terminal hinge and putative dimerization domains. Cotransfection experiments in COS-7 cells confirm that C3 can cleave the C-terminus of FLNC in live cells. The C-terminus of FLNC has been shown to bind the cytoplasmic domains of both delta- and gamma-sarcoglycan. Removal of the last 127 amino acids from FLNC, a protein that mimics FLNC after C3 cleavage, abolishes this interaction with the sarcoglycans. These studies confirm that C3 can cleave FLNC in vitro and suggest that FLNC may be an in vivo substrate for C3, functioning to regulate protein-protein interactions with the sarcoglycans. Thus, calpain-mediated remodeling of cytoskeletal-membrane interactions, such as those that occur during myoblast fusion and muscle repair, may involve regulation of FLNC-sarcoglycan interactions.