Monoclonal antibodies as probes of the alpha-bungarotoxin and cholinergic binding regions of the acetylcholine receptor.
We have probed the acetylcholine receptor (AcChR) molecule with six anti-AcChR monoclonal antibodies (mAbs) whose binding to the AcChR is inhibited or blocked by alpha-bungarotoxin (alpha BgTx). mAbs bound with a maximum stoichiometry of either one mAb (387D, 247G) or two mAbs (383C, 572C, 370C, 249E) per AcChR monomer, and the extent to which they inhibited alpha BgTx binding directly correlated with their stoichiometry of binding. The effect of mAbs on the alpha BgTx and cholinergic ligand binding properties of the AcChR molecule defined three major categories of mAbs: those that block alpha BgTx and carbamylcholine (agonist) binding, but do not block d-tubocurarine (antagonist) binding (383C, 572C, 370C and 249E); mAb 387D, which blocks agonist binding and partially blocks alpha BgTx and d-tubocurarine binding; and mAb 247G, which does not affect agonist binding, blocks at most 50% of the alpha BgTx binding sites, and decreases the affinity of the high affinity component of d-tubocurarine binding (Mihovilovic, M., and Richman, D. P. (1984) J. Biol. Chem. 259, 15051-15059). Except for mAb 247G, these mAbs strongly competed with each other for binding to the AcChR. In contrast, mAb 247G blocks about 50% of the binding of all the other mAbs. The results demonstrate the ability of mAbs to stabilize different conformational states of the AcChR and to probe cholinergic epitopes of functional importance. They also indicate the nonequivalence of the two alpha-toxin binding regions of the AcChR molecule and suggest that it is possible to identify epitopes within the alpha BgTx binding region that when bound produce differential effects on the binding of the agonist (carbamylcholine) and the antagonist (d-tubocurarine).
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism*; D002217:Carbachol / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004797:Enzyme-Linked Immunosorbent Assay; D008970:Molecular Weight; D011950:Receptors, Cholinergic / Q000032:analysis*; D014101:Torpedo; D014403:Tubocurarine / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D002038:Bungarotoxins; D011950:Receptors, Cholinergic; D002217:Carbachol; D014403:Tubocurarine