Comparison of innervation and agrin-induced tyrosine phosphorylation of the nicotinic acetylcholine receptor

Abstract

Studies of the regulation of tyrosine phosphorylation at the neuromuscular junction during development and following denervation suggest that tyrosine phosphorylation of the nicotinic acetylcholine receptor is regulated by neuronal innervation of muscle. The finding that agrin, a neuronally derived extracellular matrix protein also induces tyrosine phosphorylation of the nicotinic receptor, suggests that nerve-induced tyrosine phosphorylation may be mediated by agrin. To study this further, we have examined the regulation of tyrosine phosphorylation of the nicotinic receptor by innervation in vitro using muscle-neuron cocultures. Innervation of chick myotubes by chick ciliary ganglia neurons induced tyrosine phosphorylation of the nicotinic receptor with the same subunit specificity seen with bath applied purified agrin. Both innervation and agrin-induced phosphorylation of the nicotinic receptor resulted in an increase in tyrosine and serine phosphorylation. In addition, thermolysin phosphopeptide maps of the subunits after innervation or agrin- treatment were identical. The similarity in the agrin- and nerve- induced phosphorylation of the acetylcholine receptor suggests that agrin mediates the nerve-induced phosphorylation during development in vivo and that phosphorylation of the acetylcholine receptor may play an important role in the development of the neuromuscular junction.

Functional Interaction of Src Family Kinases with the Acetylcholine Receptor in C2 Myotubes

“…6B) was not consistently observed. Tyrosine phosphorylation of certain proteins, such as the ␦ subunit of the AChR in chick, is reported to be sensitive to degradation during boiling in SDS loading buffer (15). However, we did not see additional phosphotyrosine bands specifically precipitated by ␣-bungarotoxin-Sepharose beads, even when unboiled samples were used and when immunoblots were analyzed with an alternative phosphotyrosine antiserum (data not shown).…”

“…Tyrosine phosphorylation of the AChR may also be related to one of the earliest steps in synapse formation, the clustering of AChRs in the postsynaptic membrane underlying the nerve terminal (5). Studies on the development of the chick neuromuscular synapse have shown that the earliest detectable AChR clusters in vivo contain phosphotyrosine and that in vitro AChR clustering and phosphotyrosine co-staining in chick myotubes depend on innervation by co-cultured neurons (15). Both in nerve-muscle cultures and in vivo, the aggregation of AChRs at the nascent neuromuscular junction is caused by agrin released from motor nerves (16,17).…”

“…Both in nerve-muscle cultures and in vivo, the aggregation of AChRs at the nascent neuromuscular junction is caused by agrin released from motor nerves (16,17). The addition of agrin to cultured myotubes induces both widespread AChR cluster formation and tyrosine phosphorylation of the AChR (15,18,19). In mammalian muscle, this phosphorylation, which is specific for the ␤ subunit of the receptor, reaches a peak in 1 h and precedes AChR clustering (20).…”

Tyrosine Phosphorylation of Nicotinic Acetylcholine Receptor Mediates Grb2 Binding

“…This nerve-derived protein originally was isolated for its striking ability to cause AChR aggregation in chick myotubes (Godfrey et al, 1984). The observation that agrin induces tyrosine phosphorylation of the ␤ (Wallace et al, 1991;Qu and Huganir, 1994;Wallace, 1994;Ferns et al, 1996), and perhaps the ␦ (Qu and Huganir, 1994), subunit of the AChR has led to the hypothesis that this is a critical, and perhaps a prerequisite, step in the AChR clustering pathway. A recently identified musclespecific receptor tyrosine kinase, MuSK, has been shown to be essential for NMJ formation (Valenzuela et al, 1995;.…”

“…Phosphorylation of the AChR by both serine and tyrosine kinases is associated with an increased rate of receptor desensitization (Huganir et al, 1986;Hopfield et al, 1988). In addition, agrin-induced tyrosine phosphorylation of the AChR may play a role in its clustering to high densities in the postsynaptic membrane of the neuromuscular junction (Qu et al, 1990;Wallace et al, 1991;Qu and Huganir, 1994;Wallace, 1994;Ferns et al, 1996).…”