Location of a polypeptide sequence within the alpha-subunit of the acetylcholine receptor containing the cholinergic binding site

Abstract

Proteolytic fragments of the alpha-subunit of the acetylcholine receptor of Torpedo electric organ were generated by digestion with Staphylococcus aureus V8 protease, and their ability to bind alpha-bungarotoxin was assessed following resolution on polyacrylamide gels and transfer to nitrocellulose. The position of the smallest fragment (Mr = 17,000) with toxin-binding activity was located within the primary sequence of the alpha-subunit by isolation and chemical characterization. The amino acid sequence at its amino terminus is Val-Asn-Gln-Ile-Val-Glu, which is identical to a unique sequence on the alpha-subunit beginning at Val 46. Based on considerations of the apparent molecular weight of this polypeptide fragment, the enzyme specificity of V8 protease, analysis of the partial amino acid composition, and the position of various identifying landmarks of the primary sequence of the alpha-subunit, the carboxy terminus is restricted to an acidic amino acid residue between Asp 152 and Asp 180. The apparent affinities of the 17-kDa polypeptide for alpha-bungarotoxin (IC50 = 100 nM) and d-tubocurarine (IC50 = 0.4 mM) were not significantly different from the values obtained for the undigested alpha-subunit, suggesting that the 17-kDa polypeptide contains all of the essential elements present on the alpha-subunit that are necessary for the binding of both of these cholinergic ligands. A second toxin-binding fragment (Mr = 19,000) was also identified. Although the amino terminus of this polypeptide fragment has not been determined directly, its position relative to Asn 141, the site of N-glycosylation, was established, and it appears that its amino terminus cannot be located any closer to the amino terminus of the alpha-subunit than Asp 152. If this tentative assignment is correct, then the region of the alpha-subunit involved in binding cholinergic ligands is more closely defined as occurring within the segment of the primary sequence of the alpha-subunit that the 17- and 19-kDa polypeptide fragments have in common, i.e., an overlapping sequence maximally bounded by Asp 152 and Asp 180.