Isolation and characterization of a-dystroglycan, a receptor for laminin and agrin

When brain proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose are probed with $ sp{125}$I-labeled laminin, a single broad band of $ sim$120 kDa binds laminin specifically. This laminin binding protein, LBP120, is closely to dystroglycan-$ alpha$, part of a complex of proteins associated with dystrophin, the product of the gene that is absent in Duchenne muscular dystrophy. Five peptides from purified bovine brain LBP120, ranging in size from 7 to 19 residues, are up to 100% identical to the predicted amino acid sequence of muscle dystroglycan-$ alpha$ (Ibraghimov-Beskrovnaya, O., Ervasti J. M., Leveille, C. J., Slaughter, C. A., Sernett, S. W., and Campbell, K. P., (1992) Nature 355, 696-702). The glycosylation and extracellular localization of LBP120/dystroglycan-$ alpha$ are consistent with it being a cell surface laminin receptor. It binds, either as a native protein, or following SDS-PAGE and transfer to nitrocellulose, with high affinity (K$ sb{ rm d}$ = 90 nM) to native laminin and more specifically, to a proteolytic fragment of laminin (E3) composed of two globular (G)-type repeats and containing the major heparin binding domain. This binding is Ca$ sp{++}$ dependent and inhibited by low concentrations of heparin. The presence of G-type repeats in the acetylcholine receptor (AChR) aggregating protein agrin suggested that it might also be a ligand for dystroglycan-$ alpha$. Agrin, like laminin, binds specifically to dystroglycan-$ alpha$ in in vitro assays. This binding is calcium dependent and is inhibited by MAb IIH6 against dystroglycan-$ alpha$, by heparin and laminin, but not by fibronectin. Dystroglycan-$ alpha$ and utrophin (dystrophin-related protein (DRP)) are concentrated within AChR aggregates in cultures of C2 myotubes. In S27 cells, which do not aggregate AChRs spontaneously, agrin and laminin binding to dystroglycan-$ alpha$ are dramatically decreased. Moreover, MAb IIH6 significantly inhibit