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 post synaptic differentiation of the neuromuscular junction. As a step towa rd further understanding the mechanisms by which agrin induces the agg regation of acetylcholine receptors (AChRs), we have characterized the activity of purified, full-length chick agrin isoforms on Xenopus mus cle cells. Incubation with agrin isoforms led to the formation of nume rous small AChR clusters primarily on the ventral surface of cells wit h differing efficacies: Y(4)Z(8) > Y(4)Z(11) = Y(4)Z(19), with Y4B0 be ing ineffective. Agrin activity appeared to be tyrosine phosphorylatio n-dependent as the kinase inhibitor tyrphostin RG50864 (80 mu M) compl etely abolished the effect, Initial binding sites for all agrin isofor ms were evenly distributed in a punctate manner on the muscle cell sur face. After a 14-h incubation, the active isoforms induced AChR cluste ring, and agrin was enriched at these sites of clustering. Agrin bindi ng to the cell surface and induction of AChR clustering were Ca2+-depe ndent, as previously shown in other systems. This is the first quantit ative characterization of agrin's effects using Xenopus cell culture, providing a basis for further elucidating agrin's role in synaptogenes is using this elegant system.