Acetylcholine receptors (AChRs) and other postsynaptic molecules cluster spontaneously on cultured C2 myotubes. The frequency of clustering is enhanced by neural agrin, neuraminidase, or calcium through a signaling pathway which includes tyrosine phosphorylation of a muscle-specific kinase (MuSK) and the AChR β-subunit. Vicia villosa agglutinin (VVA) lectin, previously shown to potentiate agrin-induced clustering on C2 myotubes, is shown here to also potentiate neuraminidase- and calcium-induced clustering of AChRs, while having no effect on the level of tyrosine phosphorylation of MuSK or the AChR β-subunit. We propose that VVA lectin increases the frequency of AChR clustering through a mechanism that is distinct from agrin signaling, and that may involve α-dystroglycan.

To investigate the role of acetylcholine receptors (AChRs) in the aggregation of postsynaptic molecules on muscle cells, we utilized the 1R- genetic variant of C2 muscle cells which has very little expression of AChRs in its cell membrane. On C2 myotubes, AChRs cluster spontaneously, with the frequency of clustering greatly enhanced by motor neuron-derived agrin. Signal transduction events driven by agrin, including the tyrosine phosphorylation of muscle-specific kinase (MuSK) and the AChR beta subunit, have been implicated as requirements of postsynaptic scaffold assembly. We show here that some molecules of the postsynaptic scaffold spontaneously aggregate and colocalize on 1R- myotubes at very low frequency, including an as yet unidentified agrin binding molecule, beta-dystroglycan and MuSK. Agrin is unable to increase the frequency of these aggregations, but does cause tyrosine phosphorylation of MuSK. We conclude that free molecules can associate into aggregates independently of AChRs, but AChRs are required for high-frequency molecular aggregation driven by the agrin signalling pathway. MuSK tyrosine phosphorylation appears to precede a requisite event involving AChRs that aggregates postsynaptic molecules.

The neural factor agrin induces the aggregation of acetylcholine receptors (AChRs) and other synaptic molecules on cultured myotubes. This aggregating activity can be mimicked by experimental manipulations that include treatment with neuraminidase or elevated calcium. We report evidence that neuraminidase and calcium act through the agrin signal transduction pathway. The effects of neuraminidase and calcium on AChR clustering are additive with that of agrin at low concentrations and cosaturating at high concentrations. In addition, like agrin, both neuraminidase and calcium cause rapid tyrosine phosphorylation of the muscle-specific kinase (MuSK) and the AChR-beta subunit. Our results argue that all three agents act directly on components of the same signal transduction complex. We suggest that sialic acids on components of the complex inhibit interactions necessary for signal transduction and that disinhibition can result in activation. In such a model, agrin could activate signal transduction by disinhibition or by circumventing the inhibition.

The neural factor agrin induces the aggregation of acetylcholine receptors (AChRs) and other synaptic molecules on cultured myotubes. This aggregating activity can be mimicked by experimental manipulations that include treatment with neuraminidase or elevated calcium. We report evidence that neuraminidase and calcium act through the agrin signal transduction pathway. The effects of neuraminidase and calcium on AChR clustering are additive with that of agrin at low concentrations and cosaturating at high concentrations. In addition, like agrin, both neuraminidase and calcium cause rapid tyrosine phosphorylation of the muscle‐specific kinase (MuSK) and the AChR‐β subunit. Our results argue that all three agents act directly on components of the same signal transduction complex. We suggest that sialic acids on components of the complex inhibit interactions necessary for signal transduction and that disinhibition can result in activation. In such a model, agrin could activate signal transduction by disinhibition or by circumventing the inhibition. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 356–365, 1999

Acetylcholine receptor (AChR) clustering is an early event in neuromuscular synapse formation that is commonly studied using muscle cell culture. Motor neuron-derived agrin induces the postsynaptic tyrosine phosphorylation of both a muscle-specific kinase (MuSK) and the AChR beta-subunit. These phosphorylation events are required for AChR clustering, suggesting an agrin-driven signaling pathway. Both the phosphorylation events and AChR clustering can also be induced by neuraminidase, an enzyme that cleaves sialic acid from glycoconjugates, suggesting that neuraminidase is able to activate the agrin signaling pathway. A postulated signal for postsynaptic differentiation at sites of nerve-muscle contact during vertebrate development is the enzymatic removal of basal lamina components. We show here that bath-applied sialic acid has an effect directly opposite that of agrin or neuraminidase. Sialic acid not only decreases AChR clustering but also diminishes the tyrosine phosphorylation of MuSK and the AChR beta-subunit signal-transduction events normally driven by agrin. However, sialic acid does not prevent agrin-binding molecules from colocalizing with the decreased number of AChR clusters that do form, suggesting that sialic acid is acting to inhibit the agrin signaling pathway downstream of agrin binding to the muscle cell membrane. We propose a regulatory role for sialic acid in the signal transduction events of neuromuscular synapse formation, in which agrin or neuraminidase can overcome this sialic acid repression, resulting in the clustering of AChRs and other postsynaptic molecules.