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 that 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 is not fully exposed on the surface when the receptor is in the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
D000109:Acetylcholine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D004557:Electric Organ / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D007106:Immune Sera; D007202:Indicators and Reagents; D007700:Kinetics; D046911:Macromolecular Substances; D010455:Peptides / Q000138:chemical synthesis; D011485:Protein Binding; D011863:Radioimmunoassay; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014101:Torpedo
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007106:Immune Sera; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D010455:Peptides; D011950:Receptors, Cholinergic; D000109:Acetylcholine