Evidence for unpredicted transmembrane domains in acetylcholine receptor subunits.

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Abstract
Two monoclonal antibodies (mAbs 236 and 237) against a synthetic peptide composed of the same amino acid residues as the sequence 152-167 of the .alpha. subunit of the acetylcholine receptor were obtained, and their crossreaction with the synthetic peptide, .alpha. subunit and solubilized receptor was demonstrated. Crossreaction with the synthetic peptide .alpha.159-169 was less by a factor of 104, suggesting that the mAbs bind primarily to the sequence .alpha.152-159. Cholinergic ligands did not inhibit mAb binding. No crossreaction was observed with the receptor in native membranes, but the MAbs could bind to receptor reconstituted into liposomes in which 50% of the receptors have their cytoplasmic surface oriented outside. When native membranes were permeabilized with saponin, mAbs directed against cytoplasmic determinants of the receptor could bind to them, but mAbs 236 and 237 could not. After treatments that removed peripheral proteins from the cytoplasmic surface, binding of both mAbs was observed. Further evidence for the cytoplasmic localization of this sequence was provided by observation of partial competition for binding between mAbs 236 and 237 and mAbs previously demonstrated to bind to the cytoplasmic surface of the receptor. To account for these findings, a model for the organization of the polypeptide chains in receptor subunits is proposed that has a total of 7 transmembrane domains in each subunit, 2 of which are amphipathic and one of which is not .alpha.-helical.