J. Neurobiol. 44: 436-445 (2000)
Agrin is an extracellular synaptic protein that organizes the
postsynaptic apparatus, including acetylcholine receptors
(AChRs), of the neuromuscular junction. The COOH-terminal portion
of agrin has full AChR-aggregating activity in culture, and
includes three globular domains, G1, G2, and G3. Portions of the
agrin protein containing these domains bind to different cell
surface proteins of muscle cells, including a-dystroglycan
(G1-G2) and heparan sulfate proteoglycans (G2), whereas the G3
domain is sufficient to aggregate AChRs. We sought to determine
whether the G1 and G2 domains of agrin potentiate agrin activity in
vivo, as they do in culture. Fragments from the COOH-terminal
of a neuronal agrin isoform (4,8) containing G3, both G2 and G3,
or all three G domains were overexpressed in Xenopus
embryos during neuromuscular synapse formation in myotomal
muscles. RNA encoding these fragments of rat agrin was injected
into one-cell embryos. All three fragments increased the ectopic
aggregation of AChRs in noninnervated regions near the center of
myotomes. Surprisingly, ectopic aggregation was more pronounced
after overexpression of the smallest fragment, which lacks the
heparin- and a-dystroglycan-binding
domains. Synaptic AChR aggregation was decreased in embryos
overexpressing the fragments, suggesting a competition between
endogenous agrin secreted by nerve terminals and exogenous agrin
fragments secreted by muscle cells. These results suggest that
binding of the larger agrin fragments to a-dystroglycan
and/or heparan sulfate proteoglycans may sequester the fragments
and inhibit their activity in embryonic muscle. These
intermolecular interactions may regulate agrin activity and
differentiation of the neuromuscular junction in vivo.