is a component of the dystrophin associated glycoprotein complex (DGC) which, in muscle, is thought to serve as a transmembrane link between the submembraneous cytoskeleton and the basal lamina (Campbell and Kahl, 1989
; Yoshida and Ozawa, 1990
; Ervasti and Campbell, 1991
). Other components of the complex include at least seven transmembrane glycoproteins, β-DG, sarcospan (Crosbie et al., 1997
), α-, β-, γ-, δ-, and ε-sarcoglycan (SG; McNally et al., 1998
) as well as the syntrophins, a family of three intracellular PDZ domain containing proteins (for review see Carbonetto and Lindenbaum, 1995
). α- and β-DG are derived from cleavage of a common polypeptide precursor (Ibraghimov-Beskrovnaya et al., 1992
) and remain associated with one another on the cell surface (Bowe et al., 1994
). β-DG associates with either dystrophin or its autosomal homologue utrophin via an SH3 domain-binding region in its COOH terminus (Jung et al., 1995
). This region has also been shown to interact with the adapter protein Grb 2 (Yang et al., 1995
) raising the possibility that modulation of DG-cytoskeletal interactions may be mediated by a signaling pathway involving small GTP-binding proteins.
In contrast to the transmembrane protein β-DG, α-DG is a heavily glycosylated, mucin-like protein (Smalheiser and Kim, 1995
) anchored on the extracellular surface of the myotube. In vitro studies have demonstrated that α-DG can bind the extracellular matrix component laminin (LN) with high affinity (Smalheiser and Schwartz, 1987
; Douville et al., 1988
; Ibraghimov-Beskrovnaya et al., 1992
; Gee et al., 1993
). LN is a heterotrimer of α, β, and γ chains, each of which is a member of multigene families. α-DG binds to the last two globular domains in the COOH-terminal extension of the LN α1 and α2 chains (Gee et al., 1993
). α-DG has been shown to colocalize with LN in skeletal and cardiac muscle (Klietsch et al., 1993
) and a number of other cells including peripheral nerve (Yamada et al., 1994
), astrocytes, Purkinje neurons (Tian et al., 1996
), and kidney epithelium (Durbeej et al., 1995
). During muscle development, α-DG upregulation coincides temporally with the onset of innervation and ECM deposition (Leschziner, A., and S. Carbonetto, unpublished observations). These findings are consistent with the hypothesis that α-DG is a receptor in situ linking LN in the ECM to the subsarcolemmal cytoskeleton and thus may be important in the organization of these extracellular and subplasmalemmal networks. The existence of this putative transmembrane linkage is further supported by genetic evidence. For example, naturally occurring mutations in mice and humans that alter the expression of dystrophin and secondarily the DGC give rise to Duchenne and Becker muscular dystrophies, which are characterized by severe progressive damage to the sarcolemma (reviewed in Campbell, 1995
; Worton, 1995
). The phenotypes of these lesions bear considerable resemblance to those resulting from mutations affecting the gene encoding the α2 chain of LN 2 (α2β1γ1; merosin), which is enriched in the ECM of skeletal muscle (Xu et al., 1994
; Sunada et al., 1995
), or mutations affecting expression of the SG complex (Roberds et al., 1994
; Bönnemann et al., 1995
; Jung et al., 1996a
,b; Nigro et al., 1996
; Carrie et al., 1997
). This convergence of biochemical and genetic evidence underscores the importance of interactions between the ECM and the DGC in the maintenance of sarcolemmal integrity.
To date, no naturally occurring mutations in α-DG have been identified which would substantiate and elucidate its role as an ECM receptor. Recently, targeted mutations abolishing DG expression in mice have been shown to result in early embryonic lethality due to disruption of the Reichert's membrane (Williamson et al., 1997
; Côté, P., M. Lindenbaum, and S. Carbonetto. 1997. Mol. Biol. Cell.
8:222a). This indicates that DG expression is absolutely required for embryonic survival and strongly implicates DG in the maintenance of Reichert's basement membrane but sheds little light on its function(s) in more differentiated tissues such as skeletal muscle.
To test the assertion that α-DG is a LN receptor required for the elaboration of the ECM of muscle, we have perturbed its expression with stable transfection of C2 myoblasts with an antisense DG expression construct. In the process, we have generated two clonal cell lines 11F and 11E that express 40–50% and 10–20%, respectively, of the levels of α-DG protein in parental C2 cells after differentiation. These cells maintain the ability to fuse and form multinucleate myotubes and express normal or near normal levels of other DGC components including α-SG, but have greatly reduced levels of LN expression on their surfaces relative to C2 cells. This reduction correlates well with the level of α-DG in these clones. We also show that the residual, unbound α-DG on the surface of 11F myotubes is redistributed upon addition of exogenous LN. However, little or no binding by exogenous LN is seen in 11E myotubes, which express the lowest levels of α-DG. After transfer to fusion medium there is an increase in cell death and increased numbers of apoptotic nuclei in 11F and 11E cultures, which again correlates with levels of α-DG expressed in these clones. In 11E cells the integrity of the plasma membrane is not obviously compromised, as revealed by exclusion of the vital dye Trypan blue, and is consistent with apoptotic, not necrotic, cell death. We conclude that α-DG serves as a LN receptor in muscle and that interactions between the ECM and the DGC are required for maintenance of muscle viability.