Although new genes and proteins are continuously identified that are involved in the dystrophic phenotype, the one question that remains unanswered is the mechanism by which this phenotype occurs. Several animal model systems are being developed to explore this question and, through these models, it has become clear that the sarcoglycans help in the maintenance of skeletal muscle, although how this is accomplished remains elusive. Interestingly, although mutations in dystrophin and the sarcoglycans produce a similar phenotype, dystrophin staining in the sarcoglycanopathies is either normal or slightly reduced, indicating that the selective loss of the sarcoglycan complex is sufficient to cause muscular dystrophy.
Here, we describe the novel interaction between members of the sarcoglycan complex and FLN2, a muscle-specific relative of filamin 1 (FLN1). We demonstrate that both γ- and δ-sarcoglycan interact with FLN2 specifically through in vivo and in vitro studies. In addition, we show that FLN2 is located in two intracellular pools, with ~3% of the cellular content at the sarcolemmal membrane and the remaining ~97% contained within the cytoplasm, presumably at the Z-line of the contractile apparatus as seen by EM. Strikingly, in LGMD patients, DMD patients, and knockout mice lacking either γ- or δ-sarcoglycan, the membrane component of FLN2 increases to ~30%. In contrast, in a patient with an undetermined myopathy, FLN2 expression at the membrane appeared normal.
Filamin (ABP-280) was first identified in chicken gizzard and rabbit peripheral blood as a protein capable of inducing actin polymerization (Hartwig and Stossel 1975
; Wang et al. 1975
). Subsequent biochemical and molecular studies have shown that three different types of filamin proteins exist in mammals (FLN1
, and a third muscle-specific form, here named FLN2
). Virtually all cells express at least one filamin form at some point in development, often changing forms as the cell undergoes terminal differentiation (Stossel and Hartwig 1975
; Shizuta et al. 1976
; Maestrini et al. 1993
; Takafuta et al. 1998
). All three filamins are encoded by separate genes: FLN1
is located on chromosome Xq28; β-filamin is located on chromosome 3p14.3-p21.1; and an EST, shown here to be part of the FLN2
transcript, is located on chromosome 7q32-q35 (Gorlin et al. 1993
; Maestrini et al. 1993
Most reports of filamin function include a role in actin polymerization, a process that is critical for the regulation of the contractile apparatus in skeletal muscle as well as cell structure, organization of membrane receptors with signaling molecules, and mechanoprotection in other tissues. These processes can regulate cell behavior by providing the cell with the information necessary for making decisions regarding cell shape, adhesion and migration, growth and differentiation, and apoptosis and survival. An example of one of these processes is found in platelets, where the transient binding of FLN1 to the GP Ib-IX receptor complex has been shown to be important in platelet activation by inducing changes in cellular shape, adhesion, and membrane organization. Interestingly, the binding of both of these proteins to the GP Ib-IX complex is dependent on the stress-induced conformational change of the receptor complex (Andrews and Fox 1991
; Andrews and Berndt 1998
In addition to establishing cell structure and reorganizing receptor/signaling molecules, FLN1 also has been shown to be crucial in protecting cells from external stress, a process commonly referred to as mechanoprotection. This phenomenon has been chiefly studied in fibroblasts where FLN1 is recruited to integrin receptor complexes in response to the stress-induced binding of ECM proteins. This signal triggers the rearrangement of the actin cytoskeleton to accommodate for the sensed tension. Remarkably, after force application, cells deficient for FLN1 show membrane disruption and a >90% increase in cell death compared with FLN1 containing cells (Glogauer et al. 1998
). Therefore, in the absence of FLN1, these cells are unable to maintain membrane integrity when exposed to external forces, ultimately leading to cell death.
With the identification of FLN2 as a sarcoglycan interacting protein, the sarcoglycans join the list of proteins that are both involved in signal transduction and the dystrophic phenotype. Moreover, this interaction connects other proteins involved in dystrophy with the sarcoglycans in a signaling process that has been extensively studied in other systems, and is now unfolding in skeletal muscle. Mutations in the integrin α7 subunit, a muscle-specific integrin, result in congenital myopathy (Vachon et al. 1997
; Hayashi et al. 1998
), whereas mutations in calpain-3, caveolin-3, and the sarcoglycans result in an LGMD phenotype. Family members of all three of these proteins have been found to contribute in various signaling cascades. Moreover, the integrins are also involved in mechanoprotection pathways in fibroblasts where they bind to FLN1 (Glogauer et al. 1998
). Although these studies focus on the α2 subunit, it has been established that both FLN1 and β-FLN bind β1 integrin in other cells.
Given the data presented here and the known functions of FLN1, a mechanistic model for some forms of muscular dystrophy can be explored. The FLN2 membrane increase in LGMD and DMD patients suggests that FLN2 is binding other membrane bound proteins other than the sarcoglycans. A logical candidate for this second interacting protein would be β1 integrin given that both of the other filamin family members bind to this subunit in other cells. If this is true, it is possible that the sarcoglycans and the integrins both bind FLN2 at the membrane (). FLN1 has been shown to bind a variety of receptors, validating this possibility. Signals received by the integrins might recruit FLN2 to the membrane until a signal via the sarcoglycans is received, thereby allowing FLN2 to translocate back to the actin cytoskeleton. If selected components of the DGC are missing from the membrane, integrin bound FLN2 remains at the membrane failing to translocate back to bind F-actin. Data from patient and mouse studies support this scenario in which membrane bound FLN2 goes from 3% (normal) to 20–40% in individuals lacking either γ-sarcoglycan, δ-sarcoglycan, or dystrophin. Another important connection between filamin proteins and muscular dystrophy are the calpains. FLN1 is cleaved by nonmuscle calpain to regulate actin-myosin filament formation (Kwak et al. 1993a
). Thus, the overall regulation of FLN2 levels could be accomplished by the muscle-specific calpain, calpain-3. To date, no substrate has been identified for calpain-3, leaving FLN2 as an attractive candidate.
In summary, the identification of the interaction between FLN2 and the sarcoglycans should help in establishing a more definitive function for the sarcoglycan complex in skeletal muscle. It was originally believed that the DGC had simply a structural role in the maintenance of skeletal muscle by anchoring muscle cells to the extracellular matrix (Campbell 1995
). Examination of FLN2 and several proteins involved in the dystrophic phenotype reveals that it is more likely a dynamic process such as signal transduction. Furthermore, as a new member of the dystrophin–glycoprotein complex, FLN2
becomes a candidate disease gene in LGMD patients who do not have mutations in any of the known LGMD genes. Recently, two LGMD families were reported to be genetically linked to chromosome 7q to a region that may include FLN2
(Speer et al. 1999
). Thus, it will be interesting to determine if these patients have disease causing mutations in FLN2