Neural agrin activates a high-affinity receptor in C2 muscle cells that is unresponsive to muscle agrin

Abstract

During synaptogenesis, agrin, released by motor nerves, causes the clustering of acetylcholine receptors (AChRs) in the skeletal muscle membrane. Although muscle alpha-dystroglycan has been postulated to be the receptor for the activity of agrin, previous experiments have revealed a discrepancy between the biological activity of soluble fragments of two isoforms of agrin produced by nerves and muscles, respectively, and their ability to bind alpha-dystroglycan. We have determined the specificity of the signaling receptor by investigating whether muscle agrin can block the activity of neural agrin on intact C2 myotubes. We find that a large excess of muscle agrin failed to inhibit either the number of AChR clusters or the phosphorylation of the AChR induced by picomolar concentrations of neural agrin. These results indicate that neural, but not muscle, agrin interacts with the signaling receptor. Muscle agrin did block the binding of neural agrin to isolated alpha-dystroglycan, however, suggesting either that alpha-dystroglycan is not the signaling receptor or that its properties in the membrane are altered. Direct assay of the binding of muscle or neural agrin to intact myotubes revealed only low-affinity binding. We conclude that the signaling receptor for agrin is a high-affinity receptor that is highly specific for the neural form.

Fig. 1.

Excess muscle agrin does not inhibit AChR clustering induced by neural agrin. Soluble agrin was added to C2 myotubes for 18 hr, and the cultures were stained with rhodamine-conjugated α-bugarotoxin to assay AChR clustering.Top left, Concentration curves for neural and muscle agrin. The results of two independent experiments are shown. Top right, The concentration curve for neural agrin with (▪) and without (○) 1000-fold excess muscle agrin. As the neural agrin concentration was increased, the muscle agrin concentration was increased proportionally. The data for neural agrin alone are similar to that shown in top left panel. Both curves (top left, top right) were plotted on a log scale.Bottom left, Two concentrations of neural agrin (10 and 100 pm) plus a 10-, 100-, or 1000-fold excess of muscle agrin. In both experiments, an excess of muscle agrin failed to decrease the number of AChR clusters induced by neural agrin.

Fig. 2.

Muscle agrin does not competitively inhibit phosphorylation of the AChR β subunit induced by neural agrin. C2 myotubes were treated with neural agrin (C-Ag_4,8) for 1 hr in the presence or absence of a 100-fold excess of muscle agrin (C-Ag_0,0). AChRs were isolated from cell extracts using α-bugarotoxin-conjugated Sepharose beads and immuno- blotted with a phosphotyrosine antibody as described in Materials and Methods. Lane 1, Blank beads⁺; lane 2, no agrin;lane 3, 10 nm muscle agrin;lane 4, 100 pm neural agrin;lane 5, 100 pm neural agrin + 10 nm muscle agrin. A 100-fold excess of muscle agrin did not alter the level of neural agrin-induced AChR β phosphorylation. The two faint tyrosine-phosphorylated bands of ∼106 kDa in the muscle agrin-treated extracts appeared inconsistently and were not further characterized.

Fig. 3.

Muscle agrin competitively inhibits neural agrin binding to α-dystroglycan on nitrocellulose blots. Membrane extracts from (A) Torpedo electric organ (T) and (B) C2 myotubes (C) were isolated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with¹²⁵I-labeled neural agrin (0.5 and 0.8 nm, respectively) without and with 50 nm neural (C-Ag_4,8) and muscle (C-Ag_0,0) agrin. Radiolabeled agrin bound to a protein of 200 kDa (A) and 150 kDa (B) was identified as α-dystroglycan. This binding was inhibited by an excess of either unlabeled neural or muscle agrin.

Fig. 4.

Binding characteristics of neural and muscle agrin to cultured C2 myotubes. Agrin binding to intact C2 myotubes was measured by RIA as described in Materials and Methods. Top left, C2 myotubes were incubated with increasing concentrations of muscle agrin in the presence (○) or absence (▪) of calcium (2 mm EDTA). Top right, Neural (○) and muscle (▪) agrin-binding curves were constructed with C2 myotubes. Both curves (top left, top right) were plotted on a log scale. Bottom left, C2 myotubes were incubated with 500 μm muscle agrin and increasing concentrations of heparin. The low-affinity agrin binding to intact myotubes is dependent on the presence of calcium and is relatively insensitive to heparin.