The release of acetylcholine receptor inducing activity (ARIA) from its transmembrane precursor in transfected fibroblasts

Acetylcholine receptor inducing activity (ARIA) is made by motoneurons and is released at the neuromuscular synapse to stimulate the synthesis of acet ylcholine receptors by skeletal muscle. ARIA is derived from a transmembran e precursor (pro-ARIA) via proteolytic cleavage of the ectodomain. We studi ed requirements in the amino acid sequence at the cleavage site with variou s substitution and deletion mutations. Wild type (WT) and mutant proteins w ere transiently expressed in COS cells, and release of ARIA into the condit ioned medium was measured by tyrosine phosphorylation of its receptor, p185 , in L6 cells. Removal of all potential cleavage sites between the extracel lular epidermal growth factor domain and the transmembrane domain by substi tution and small deletions (<11 amino acid residues out of 21) did not sign ificantly reduce ARIA release, whereas larger deletions abolished it. We pr opose that cleavage occurs independently of amino acid sequence at a short distance from the epidermal growth factor domain, unless sterically hindere d by the nearby secondary structure. A mutant with shorter cytoplasmic doma in ("c" isoform) released significantly less ARIA than the WT ("a" isoform) , suggesting that the c isoform may be suitable for signaling through direc t cell-cell contact. Alternatively, proteolytic conversion of the a isoform to the c isoform may rapidly down-regulate release of ARIA.