Acetylcholine receptor-aggregating activity of agrin isoforms and mapping of the active site

Abstract

Agrin is a basal lamina protein that induces aggregation of acetylcholine receptors (AChRs) and other molecules at the developing neuromuscular junction. Alternative splicing of chick agrin mRNA at two sites, A and B, gives rise to eight possible isoforms of which five are expressed in vivo. Motor neurons express high levels of isoforms with inserts at sites A and B, muscle cells synthesize isoforms that lack amino acids at the B-site. To obtain further insights into the mechanism of agrin-induced AChR aggregation, we have determined the EC50 (effective concentration to induce half- maximal AChR clustering) of each agrin isoform and of truncation mutants. On chick myotubes, EC50 of the COOH-terminal, 95-kD fragment of agrin(A4B8) was ~35 pM, of agrin(A4B19) ~110 pM and of agrin(A4B11) ~5 nM. While some AChR clusters were observed with 64 nM of agrin(A4B0), no activity was detected for agrin(A0B0). Recombinant full-length chick agrin and a 100-kD fragment of ray agrin showed similar EC50 values. A 45-kD, COOH-terminal fragment of agrin(A4B8) retained high activity (EC50 ≃ 130 pM) and a 21- kD fragment was still active, but required higher concentrations (EC50 ≃ 13 nM). Unlike the 45-kD fragment, the 21-kD fragment neither bound to heparin nor did heparin inhibit its capability to induce AChR aggregation. These data show quantitatively that agrin(A4B8) and agrin(A4B19), expressed in motor neurons, are most active, while no activity is detected in agrin(A0B0), the dominant isoform synthesized by muscle cells. Furthermore, our results show that a fragment comprising site B8 and the most COOH- terminal G-like domain is sufficient for this activity, and that agrin domains required for binding to heparin and those for AChR aggregation are distinct from each other.