TE671 cells express an abundance of a partially mature acetylcholine receptor alpha subunit which has characteristics of an assembly intermediate.
A partially mature form of the nicotinic acetylcholine receptor alpha subunit was found to be expressed in the human cell line TE671. We found that 40-50% of the alpha-bungarotoxin-binding sites in detergent extracts of these cells corresponds to this unassembled alpha subunit. These unassembled alpha subunits are not found in the surface membrane. The unassembled alpha subunits in extracts from TE671 cells appear, like mature receptors, to have a disulfide bond between Cys-192 and Cys-193 near the acetylcholine-binding site. The unassembled alpha subunit binds alpha-bungarotoxin with high affinity, but its dissociation constant is still 5-fold higher than the native assembled acetylcholine receptor. The cholinergic ligands d-tubocurarine and carbamylcholine have negligible affinity for the immature alpha subunit. Similarly, Xenopus oocytes injected with RNA transcripts for the TE671 alpha subunit express an alpha-bungarotoxin-binding component which is insensitive to carbamylcholine and has a sedimentation coefficient on sucrose gradients of 5.0 S. Oocytes injected with RNA for the Torpedo alpha subunit did not have alpha-bungarotoxin binding activity under similar conditions, suggesting a possible differential efficiency in the maturation of this alpha subunit. We examined the binding of monoclonal antibodies specific to the main immunogenic region and found that this epitope on the unassembled alpha subunit was formed, but was not in a fully mature conformation because although these antibodies bound, they bound with lower affinity than to native acetylcholine receptors. Antibodies in myasthenia gravis patient sera also bound to the unassembled alpha subunits, but with an average 14-fold lower titer.
Conroy
W G
WG
Salk Institute for Biological Studies, San Diego, California 92138.
Saedi
M S
MS
Lindstrom
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism; D002217:Carbachol / Q000378:metabolism; D002451:Cell Compartmentation; D002460:Cell Line; D003001:Cloning, Molecular; D004220:Disulfides; D015870:Gene Expression; D006801:Humans; D046911:Macromolecular Substances; D009157:Myasthenia Gravis / Q000276:immunology; D011233:Precipitin Tests; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D014403:Tubocurarine / Q000378:metabolism; D014981:Xenopus
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D001323:Autoantibodies; D002038:Bungarotoxins; D004220:Disulfides; D046911:Macromolecular Substances; D011498:Protein Precursors; D011978:Receptors, Nicotinic; D002217:Carbachol; D014403:Tubocurarine