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 acetylcholine receptors by skeletal muscle. ARIA is derived from a transmembrane precursor (pro-ARIA) via proteolytic cleavage of the ectodomain. We studied requirements in the amino acid sequence at the cleavage site with various substitution and deletion mutations. Wild type (WT) and mutant proteins were transiently expressed in COS cells, and release of ARIA into the conditioned medium was measured by tyrosine phosphorylation of its receptor, p185, in L6 cells. Removal of all potential cleavage sites between the extracellular epidermal growth factor domain and the transmembrane domain by substitution and small deletions (<11 amino acid residues out of 21) did not significantly reduce ARIA release, whereas larger deletions abolished it. We propose that cleavage occurs independently of amino acid sequence at a short distance from the epidermal growth factor domain, unless sterically hindered by the nearby secondary structure. A mutant with shorter cytoplasmic domain ("c" isoform) released significantly less ARIA than the WT ("a" isoform), suggesting that the c isoform may be suitable for signaling through direct cell-cell contact. Alternatively, proteolytic conversion of the a isoform to the c isoform may rapidly down-regulate release of ARIA.
Han
B
B
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Fischbach
G D
GD
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017384:Sequence Deletion; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D009419:Nerve Tissue Proteins; D014443:Tyrosine; D004815:Epidermal Growth Factor