The EGF-like domain of chick acetylcholine receptor-inducing activity (ARIA) contains its full biological activity

Abstract

Acetylcholine receptor-inducing activity (ARIA) is a glycoprotein initially purified from chick brain based on its ability to increase the synthesis of acetylcholine receptor (AChR) on cultured myotubes. cDNA encoding ARIA contains different domains and the functions of each domain in ARIA activity are not known. We used molecular genetic methods to construct a chimeric fusion protein, designated ARIA S136–K205-Fc, that contained the leader sequence, the EGF-like domain of chick ARIA (S 136 to K 205) and the Fc region of human immunoglobulin. The ARIA S136–K205-Fc cDNA was transfected into HEK 293 cells and stable cell lines secreting soluble ARIA S136–K205-Fc were obtained. The secreted ARIA S136–K205-Fc has a molecular mass of ∼60 kDa and can be purified by protein G chromatography. The purified ARIA S136–K205-Fc retained its full biological activity of chick ARIA that included: (i) induction of tyrosine phosphorylation of erbB 3 receptor in C2C12 myotubes; and (ii) ∼12-fold stimulation of AChR α-subunit mRNA synthesis when applied onto cultured chick myotubes. This Fc-tagged ARIA could be rapidly purified and provides a very useful ligand for identifying its true receptor(s) on muscle cell surface. © 1997 Federation of European Biochemical Societies.