Michael Lindenbaum

Dystroglycan-α, a dystrophin-associated glycoprotein, is a functional agrin receptor

Aggregation of acetylcholine receptors (AChRs) on skeletal muscle fibers is thought to be mediated by the basal lamina protein agrin. Structural similarities shared by agrin and laminin suggested that the laminin receptor dystroglycan-alpha, part of a dystrophin-receptor complex, might also bind agrin. We show here that dystroglycan-alpha and dystrophin-related protein (DRP/utrophin) are concentrated within AChR aggregates in cultures of C2 myotubes and that agrin binds specifically to dystroglycan-alpha in in vitro assays. This binding is calcium dependent and is inhibited by monoclonal antibody (MAb) IIH6 against dystroglycan-alpha, heparin, and laminin, but not by fibronectin. In S27 cells, which do not aggregate AChRs spontaneously, agrin and laminin binding to dystroglycan-alpha are dramatically decreased. Moreover, MAb IIH6 significantly inhibits agrin-induced AChR aggregation on C2 cells. We conclude that dystroglycan-alpha is an agrin-binding protein and part of a dystrophin-receptor complex involved in AChR aggregation.

Chimaeric mice deficient in dystroglycans develop muscular dystrophy and have disrupted myoneural synapses

Mutations in the dystrophin gene (DMD) and in genes encoding several dystrophin-associated proteins result in Duchenne and other forms of muscular dystrophy. α-Dystroglycan (Dg) binds to laminins in the basement membrane surrounding each myofibre and docks with β-Dg, a transmembrane protein, which in turn interacts with dystrophin or utrophin in the subplasmalemmal cytoskeleton. α- and β-Dgs are thought to form the functional core of a larger complex of proteins extending from the basement membrane to the intracellular cytoskeleton, which serves as a superstructure necessary for sarcolemmal integrity. Dgs have also been implicated in the formation of synaptic densities of acetylcholine receptors (AChRs) on skeletal muscle. Here we report that chimaeric mice generated with ES cells targeted for both Dg alleles have skeletal muscles essentially devoid of Dgs and develop a progressive muscle pathology with changes emblematic of muscular dystrophies in humans. In addition, many neuromuscular junctions are disrupted in these mice. The ultrastructure of basement membranes and the deposition of laminin within them, however, appears unaffected in Dg-deficient muscles. We conclude that Dgs are necessary for myofibre survival and synapse differentiation or stability, but not for the formation of the muscle basement membrane, and that Dgs may have more than a purely structural function in maintaining muscle integrity.

Neural regulation of α-dystroglycan biosynthesis and glycosylation in skeletal muscle

Abstract:α‐Dystroglycan (α‐DG) is part of a complex of cell surface proteins linked to dystrophin or utrophin, which is distributed over the myofiber surface and is concentrated at neuromuscular junctions. In laminin overlays of muscle extracts from developing chick hindlimb muscle, α‐DG first appears at embryonic day (E) 10 with an apparent molecular mass of 120 kDa. By E15 it is replaced by smaller (∼100 kDa) and larger (∼150 kDa) isoforms. The larger form increases in amount and in molecular mass (>200 kDa) as the muscle is innervated and the postsynaptic membrane differentiates (E10‐E20), and then decreases dramatically in amount after hatching. In myoblasts differentiating in culture the molecular mass of α‐DG is not significantly increased by their replication, fusion, or differentiation into myotubes. Monoclonal antibody IIH6, which recognizes a carbohydrate epitope on α‐DG, preferentially binds to the larger forms, suggesting that the core protein is differentially glycosylated beginning at E16. Consistent with prior observations implicating the IIH6 epitope in laminin binding, the smaller forms of α‐DG bind more weakly to laminin affinity columns than the larger ones. In blots of adult rat skeletal muscle probed with radiolabeled laminin or monoclonal antibody IIH6, α‐DG appears as a >200‐kDa band that decreases in molecular mass but increases in intensity following denervation. Northern blots reveal a single mRNA transcript, indicating that the reduction in molecular mass of α‐DG after denervation is not obviously a result of alternative splicing but is likely due to posttranslational modification of newly synthesized molecules. The regulation of α‐DG by the nerve and its increased affinity for laminin suggest that glycosylation of this protein may be important in myofiber‐basement membrane interactions during development and after denervation.

The basement membrane at the neuromuscular junction: a synaptic mediatrix

The basement membrane at the neuromuscular junction directs formation of pre- and postsynaptic elements at this synapse. Efforts to understand the molecular basis for development of the postsynaptic specialization have brought new insights into extracellular matrix proteins and their cell-surface receptors. Recent evidence for an agrin receptor and mice null for the s-laminin gene have reinforced the function of the basement membrane in both orthograde and retrograde signalling across the synapse.

Neurofilament phosphorylation in cultured bovine adrenal chromaffin cells is stimulated by phorbol ester.

Abstract Primary cultures of bovine adrenal chromaffin cells contain neurofilament proteins that are hypophosphorylated. When the cells were grown in medium containing 32Pi and 0.1 μM 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA), 32P‐labelling of the three neurofilament subunits was increased 6‐ to 20‐fold relative to controls, the highest level of stimulation occurring for the mid‐sized subunit. Addition of the protease inhibitor leupeptin to the growth medium had no effect on TPA‐stimulated phosphorylation. The increased 32P incorporation was accompanied by a marked reduction in the gel electrophoretic mobilities of the two largest subunits. The augmented phosphorylation was observed 10 min after addition of TPA to a concentration of 0.1 μM or after 1 h of incubation in the presence of 0.01 μM TPA. One‐dimensional peptide mapping and phosphoamino acid analysis indicated that TPA stimulated the phosphorylation of seryl residues at new sites in the mid‐sized subunit. All of the latter subunit contained in the cytoskeletal fraction of chromaffin cells was converted to a more highly phosphorylated state after the cells were grown in the presence of TPA for 1 h.