ACETYLCHOLINE RECEPTOR-AGGREGATING ACTIVITY OF AGRIN ISOFORMS AND MAPPING OF THE ACTIVE-SITE

Agrin is a basal lamina protein that induces aggregation of acetylchol ine receptors (AChRs) and other molecules at the developing neuromuscu lar junction. Alternative splicing of chick: agrin mRNA at two sites, A and B, gives rise to eight possible isoforms of which five are expre ssed in vivo. Motor neurons express high levels of isoforms with inser ts 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 EC(50) (effecti ve concentration to induce half-maximal AChR clustering) of each agrin isoform and of truncal:ion mutants. On chick myotubes, EC(50) of the COOH-terminal, 95-kD fragment of agrin(A4B8) was similar to 35 pM, of agrin(A4B19) similar to 110 pM and of agrin(A4B11) similar to 5 nM. Wh ile some AChR clusters were observed with 64 nM of agrin(A4B0), no act ivity was detected for agrin(A0B0). Recombinant full-length chick agri n and a 100-kD fragment of ray agrin showed similar EC(50) values. A 4 5-kD, COOH-terminal fragment of agrin(A4B8) retained high activity (EC (50) congruent to 130 pM) and a 21-kD fragment was still active, but r equired higher concentrations (EC congruent to 13 nM). Unlike the 45-k D fragment, the 21-kD fragment neither bound to heparin nor did hepari n inhibit its capability to induce AChR aggregation. These data show q uantitatively that agrin(A4B8) and agrin(A4B19), expressed in motor ne urons, are most active, while no activity is detected in agrin(A0B0), the dominant isoform synthesized by muscle cells. Furthermore, our res ults show that a fragment comprising site B-8 and the most COOH-termin al G-like domain is sufficient for this activity,: and that agrin doma ins required for binding to heparin and those for AChR aggregation are distinct from each other.