Propylbenzilylcholine mustard labels an acidic residue in transmembrane helix 3 of the muscarinic receptor

Muscarinic acetylcholine receptors were purified from rat forebrain and labeled with [3H]N-(2-chloroethyl)N-(2',3'-[3H2]propyl)-2-aminoethylbenzilate. Cleavage of the labeled muscarinic acetylcholine receptors with a lysine-specific protease yielded labeled, glycosylated peptides about 130 and 200 residues in length, which came from different receptor sequences. The probable cleavage sites are in the second intracellular loop and in the second extracellular or third intracellular loop. The N-terminal 130 residues are disulfide-bonded to another part of the receptor structure, supporting the presence of a link between the second and third extracellular loops. The [3H]propylbenzilylcholine mustard-receptor link is cleaved by nucleophiles, acids, and bases under denaturing conditions, suggesting modification of an acidic residue. Cyanogen bromide cleavage points to transmembrane helix 3 as the site of label attachment.