Activation of muscle-specific receptor tyrosine kinase and binding to dystroglycan are regulated by alternative mRNA splicing of agrin

Agrin induces the aggregation of postsynaptic proteins at
the neuromuscular junction (NMJ). This activity requires the
receptor-tyrosine kinase MuSK. Agrin isoforms differ in
short amino acid stretches at two sites, called A and B, that
are localized in the two most C-terminal laminin G (LG)
domains. Importantly, agrin isoforms greatly differ in their
activities of inducing MuSK phosphorylation and of binding
to alpha-dystroglycan. By using site-directed mutagenesis, we
characterized the amino acids important for these activities
of agrin. We find that the conserved tripeptide asparagineglutamate-
isoleucine in the eight-amino acid long insert at
the B-site is necessary and sufficient for full MuSK phosphorylation
activity. However, even if all eight amino acids were
replaced by alanines, this agrin mutant still has significantly
higher MuSK phosphorylation activity than the splice version
lacking any insert. We also show that binding to alpha-dystroglycan
requires at least two LG domains and that amino acid inserts at
the A and the B splice sites negatively affect binding.