AN AMINO-TERMINAL EXTENSION IS REQUIRED FOR THE SECRETION OF CHICK AGRIN AND ITS BINDING TO EXTRACELLULAR-MATRIX

Agrin is an extracellular matrix (ECM) protein with a calculated relat ive molecular mass of more than 200 kD that induces the aggregation of acetylcholine receptors (AChRs) at the neuromuscular junction. This a ctivity has been mapped to its COOH terminus. In an attempt to identif y the functions of the NH2-terminal end, we have now characterized ful l-length chick agrin. We show that chick agrin encoded by a previously described cDNA is not secreted from transfected cells, Secretion is a chieved with a construct that includes an additional 350 bp derived fr om the 5' end of chick agrin mRNA. Recombinant agrin is a heparan sulf ate proteoglycan (HSPG) of more than 400 kD with glycosaminoglycan sid e chains attached only to the NH2-terminal half. Endogenous agrin in t issue homogenates also has an apparent molecular mass of >400 kD. Whil e the amino acid sequence encoded by tile 350-bp extension has no homo logy to published rat agrin, it includes a stretch of 15 amino acids t hat is 80% identical to a previously identified bovine HSPG. The exten sion is required for binding of agrin to ECM. AChR aggregates induced by recombinant agrin that includes the extension are considerably smal ler than those induced by agrin fragments, suggesting that binding of agrin to ECM modulates the size of receptor clusters. In addition, we found a site encoding seven amino acids at the NH2-terminal end of agr in that is alternatively spliced, While motor neurons express the spli ce variant with the seven amino acid long insert, muscle cells mainly synthesize isoforms that lack this insert. In conclusion, the cDNAs de scribed here code for chick agrin that has all the characteristics pre viously allocated to endogenous agrin.