Full-length agrin isoform activities and binding site distributions on cultured Xenopus muscle cells

Agrin is a large multidomain protein involved in the induction of postsynaptic differentiation of the neuromuscular junction. As a step toward further understanding the mechanisms by which agrin induces the aggregation of acetylcholine receptors (AChRs), we have characterized the activity of purified, full-length chick agrin isoforms on Xenopus muscle cells. Incubation with agrin isoforms led to the formation of numerous small AChR clusters primarily on the ventral surface of cells with differing efficacies: Y(4)Z(8) > Y(4)Z(11) = Y(4)Z(19), with Y4B0 being ineffective. Agrin activity appeared to be tyrosine phosphorylation-dependent as the kinase inhibitor tyrphostin RG50864 (80 mu M) completely abolished the effect, Initial binding sites for all agrin isoforms were evenly distributed in a punctate manner on the muscle cell surface. After a 14-h incubation, the active isoforms induced AChR clustering, and agrin was enriched at these sites of clustering. Agrin binding to the cell surface and induction of AChR clustering were Ca2+-dependent, as previously shown in other systems. This is the first quantitative characterization of agrin's effects using Xenopus cell culture, providing a basis for further elucidating agrin's role in synaptogenesis using this elegant system.