THE EGF-LIKE DOMAIN OF CHICK ACETYLCHOLINE RECEPTOR-INDUCING ACTIVITY(ARIA) CONTAINS ITS FULL BIOLOGICAL-ACTIVITY

Acetylcholine receptor-inducing activity (ARIA) is a glycoprotein init ially purified from chick brain based on its ability to increase the s ynthesis of acetylcholine receptor (AChR) on cultured myotubes. cDNA e ncoding ARIA contains different domains and the functions of each doma in in ARIA activity are not known, We used molecular genetic methods t o 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(S 136)-(K205)-Fc cDNA was transfected into HEK 293 cells and stable cell lines secreting soluble ARIA(S136-K205)-FC were obtained, The secrete d ARIA(S136-K205)-Fc has a molecular mass of similar to 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) similar to 12-fold stimulation of AChR alpha-subun it mRNA synthesis when applied onto cultured chick myotubes. This Fc-t agged ARIA could be rapidly purified and provides a very useful ligand for identifying its true receptor(s) on muscle cell surface. (C) 1997 Federation of European Biochemical Societies.