EVIDENCE THAT THE ACETYLCHOLINE BINDING-SITE IS NOT FORMED BY THE SEQUENCE ALPHA-127-143 OF THE ACETYLCHOLINE-RECEPTOR

The sequence .alpha.127-143 of the .alpha. subunit of the acetylcholine receptor has been proposed to contain several important features: (1) the acetylcholine binding site, (2) the only N-glycosylation site of the .alpha. subunit, at asparagine-.alpha.141, and (3) two cysteine residues, at .alpha.128 and .alpha.142, that may participate in a disulfide bond known to be near the binding site. We tested these hypotheses by using antisera to receptor and its subunits and monoclonal antibodies to the synthetic peptide .alpha.127-143 cyclized by a disulfide bond between .alpha.128 and .alpha.142. Antisera to receptor and its .alpha. subunit were able to immunoprecipitate the iodinated peptide, and this reaction was inhibited by soluble receptor, but not by membrane-bound receptor. .alpha.-Bungarotoxin did not inhibit antiserum binding to solubilized receptor. Similarly, cholinergic ligands had little or no effect on binding to immobilized receptors of anti-peptide monoclonal antibodies. In addition, these monoclonal antibodies, when bound to the receptor, did not affect toxin binding kinetics. By contrast, preincubation with concanavalin A did inhibit monoclonal antibody binding. Reduction of the receptor significantly decreased the binding of three of the monoclonal antibodies, but subsequent alkylation with N-ethylmaleimide or the affinity labeling reagent bromoacetylcholine had no additional effect on binding. A dithiothreitol concentration about 100-fold higher than the one needed to reduce the disulfide near the acetylcholine binding site was necessary to inhibit monoclonal antibody binding. We conclude that the sequence .alpha.127-143 (1) is not fully exposed on the surface when the receptor is in the membrane, (2) is probably glycosylated at asparagine-.alpha.141, (3) may have cysteines-.alpha.128 and -.alpha.142 forming a disulfide bond which is not related to the disulfide known to be close to the cholinergic binding sites, and (4) is not involved in the formation of the binding site for cholinergic agonists or antagonists.