Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) alpha subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind alpha-bungarotoxin (alpha-BTX) and several alpha-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the alpha subunit, and bind to AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corresponded to the complete sequence of Torpedo AChR alpha subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to alpha 166-203, were synthesized. alpha-BTX bound to a peptide containing the sequence alpha 181-200 and also, albeit to a lesser extent, to a peptide containing the sequence alpha 55-74. WF6 bound to alpha 181-200 and to a lesser extent to alpha 55-74 and alpha 134-153. The two other mAbs predominantly bound to alpha 55-74, and to a lesser extent to alpha 181-200. Peptides alpha 181-200 and alpha 55-74 both inhibited binding of 125I-alpha-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound alpha-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR alpha subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].
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