MONOCLONAL-ANTIBODIES FK1 AND WF6 DEFINE 2 NEIGHBORING LIGAND-BINDINGSITES ON TORPEDO ACETYLCHOLINE-RECEPTOR ALPHA-POLYPEPTIDE

Previous studies have identified the sequence region flanking the inva riant vicinal cysteinyl residues at positions 192 and 193 of the nicot inic acetylcholine receptor alpha-subunit as containing major elements of the binding site for acetylcholine and its agonists and antagonist s, including antibody WF6 (Conti-Tronconi, B. M., Diethelm, B. M., Wu, X., Tang, F., Bertazzon, T., Schroder, B., Reinhardt-Maelicke, A., an d Maelicke, A (1991) Biochemistry 30, 2575-2584). Recently we have sho wn that the sequence region flanking lysine alpha 125 contains element s of the binding site for physostigmine and related ligands, including antibody FK1 (Schrattenholz, A., Godovac-Zimmerman, J., Schafer, H.-J ., Albuquerque, E. X, and Maelicke, A. (1993) Eur. J. Biochem. 216, 67 1-677). Here we report the identification by enzyme-linked immunosorbe nt assay techniques, employing fragments of the Torpedo nicotinic acet ylcholine receptor alpha-subunit N-terminal region and a panel of synt hetic peptides matching in sequence preselected portions of this subun it, of the sequence regions alpha 118-145 and alpha 181-216 as contrib uting to the FK1 epitope. Of the synthetic peptides employed, alpha 11 8-137 displayed the highest affinity of FK1 binding. Binding of FK1 an d WF6 to single residue-substituted analogs of the sequence alpha 181- 200 indicated that the two antibodies have different attachment point patterns within this sequence region. These results, and those of liga nd competition studies, suggest that the binding sites for FK1 and phy sostigmine, and those of WF6 and acetylcholine, are within the same ge neral region of the receptor's three-dimensional structure. The sites neighbor each other, with limited overlap in the case of occupation by high molecular weight ligands.