Abstract
The rat L6 skeletal muscle cell line was used to study expression of the dystrophin-containing glycoprotein complex and its interaction with the integrin system involved in the cell-matrix adhesion reaction. A complex of dystrophin and its associated proteins was fully expressed in L6 myotubes, from which anti-dystrophin or anti-alpha-sarcoglycan co-precipitated integrin alpha 5 beta 1 and other focal adhesion-associated proteins vinculin, talin, paxillin, and focal adhesion kinase. Immunostaining and confocal microscopy revealed that dystrophin, alpha-sarcoglycan, integrin alpha 5 beta 1, and vinculin exhibited overlapping distribution in the sarcolemma, especially at focal adhesion-like, spotty structures. Adhesion of cells to fibronectin- or collagen type I-coated dishes resulted in induction of tyrosine phosphorylation of alpha- and gamma-sarcoglycans but not beta-sarcoglycan. The same proteins were also tyrosine-phosphorylated when L6 cells in suspension were exposed to Arg-Gly-Asp-Ser peptide. All of these tyrosine phosphorylations were inhibited by herbimycin A. On the other hand, treatment of L6 myotubes with alpha- and gamma-sarcoglycan antisense oligodeoxynucleotides resulted in complete disappearance of alpha- and gamma-sarcoglycans and in significant reduction of levels of the associated focal adhesion proteins, which caused about 50% reduction of cell adhesion. These results indicate the existence of bidirectional communication between the dystrophin-containing complex and the integrin adhesion system in cultured L6 myocytes.
Highlights
Several classes of cell adhesion receptors, including integrins, dystroglycan, cadherins, and members of the immunoglobulin family, are coexpressed by skeletal muscle and presumably play critical roles during skeletal muscle differentiation and development
We have provided several lines of evidence suggesting that the dystrophin-dystrophin-associated proteins (DAPs) complex in cultured rat L6 myotubes has specific bidirectional interactions with the ␣51 integrin adhesion system: 1) anti-dystrophin or anti-␣-sarcoglycan co-precipitated integrin ␣51 and other focal adhesionassociated proteins together with the components of the dystrophin-DAP complex (Figs. 1A and 5B)
Anti-1 integrin subunit or anti-FAK co-precipitated the components of the dystrophinDAP complex, antibodies against ␣V integrin subunit, ezrin, and cortactin did not precipitate them (Figs. 1B and 5B and “Results”). 2) Immunofluorescence study using confocal microscopy revealed that ␣-sarcoglycan and vinculin exhibited overlapping distribution in the sarcolemma, especially at the spotty structures in the basal cell area, when L6 cells were plated on fibronectin-coated dishes (Fig. 2). 3) Adhesion of L6 cells to fibronectin or collagen type I resulted in induction of tyrosine phosphorylation of ␣- and ␥-sarcoglycans (Figs. 3–5)
Summary
In our recent preliminary report [22], we have presented evidence that the dystrophin-DAP complex is associated with the focal adhesion assembly proteins, such as the integrin 1 subunit, vinculin, and FAK in serum-deprived, differentiated. We have shown that loss of ␣-sarcoglycan induced by the antisense ODN treatment results in a significant inhibition of adhesion to the substrate by these cells. In this communication, we report that muscle-specific ␣- or ␥-sarcoglycans were tyrosine-phosphorylated when differentiated rat skeletal L6 cells were adhered to the extracellular matrix. The results indicate the existence of bidirectional communication between the dystrophin-DAP complex and the integrin adhesion system in these cultured cells
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