Monoclonal antibodies FK1 and WF6 define two neighboring ligand binding sites on Torpedo acetylcholine receptor alpha-polypeptide.
Previous studies have identified the sequence region flanking the invariant vicinal cysteinyl residues at positions 192 and 193 of the nicotinic acetylcholine receptor alpha-subunit as containing major elements of the binding site for acetylcholine and its agonists and antagonists, including antibody WF6 (Conti-Tronconi, B. M., Diethelm, B. M., Wu, X., Tang, F., Bertazzon, T., Schröder, B., Reinhardt-Maelicke, A., and Maelicke, A. (1991) Biochemistry 30, 2575-2584). Recently we have shown that the sequence region flanking lysine alpha 125 contains elements of the binding site for physostigmine and related ligands, including antibody FK1 (Schrattenholz, A., Godovac-Zimmerman, J., Schäfer, H.-J., Albuquerque, E. X., and Maelicke, A. (1993) Eur. J. Biochem. 216, 671-677). Here we report the identification by enzyme-linked immunosorbent assay techniques, employing fragments of the Torpedo nicotinic acetylcholine receptor alpha-subunit N-terminal region and a panel of synthetic peptides matching in sequence preselected portions of this subunit, of the sequence regions alpha 118-145 and alpha 181-216 as contributing to the FK1 epitope. Of the synthetic peptides employed, alpha 118-137 displayed the highest affinity of FK1 binding. Binding of FK1 and 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 ligand competition studies, suggest that the binding sites for FK1 and physostigmine, and those of WF6 and acetylcholine, are within the same general 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.
Schröder
B
B
Laboratory of Molecular Neurobiology, Johannes-Gütenberg University Medical School, Mainz, Germany.
Reinhardt-Maelicke
S
S
Schrattenholz
A
A
McLane
K E
KE
Kretschmer
A
A
Conti-Tronconi
B M
BM
Maelicke
A
A
D000109:Acetylcholine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010830:Physostigmine / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014101:Torpedo
D000911:Antibodies, Monoclonal; D008024:Ligands; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; D010830:Physostigmine; D000109:Acetylcholine