Native folding of an acetylcholine receptor alpha subunit expressed in the absence of other receptor subunits.
An alpha subunit cDNA of the mouse nicotinic acetylcholine receptor under transcriptional control of the Rous Sarcoma virus long terminal repeat was transfected into and expressed in a quail fibroblast cell line. The biosynthesis and post-translational modification of the alpha subunit protein made in this heterologous system have been studied using immunoprecipitation and ligand binding assays. The polypeptide is present at high steady-state levels and inserted in the correct transmembrane orientation. However, in the absence of assembly with other subunits the alpha subunit is confined to an intracellular membrane compartment and is not transported to the plasma membrane. Twenty percent of the newly synthesized alpha subunit acquired high affinity alpha bungarotoxin binding in a time-dependent process within 20 min of translation. Sucrose gradient fractionation demonstrated that both the polypeptide and toxin binding forms of the alpha subunit have a sedimentation coefficient of 5 s suggesting the absence of stable homo-oligomers. Quantitative binding assays demonstrated that the apparent affinity and rate of association of alpha bungarotoxin to the unassembled alpha subunit are greater than for native receptor. On the other hand, the affinities for the small ligands D-tubocurarine and gallamine are 10(3) lower than for native receptor; no detectable binding was observed for decamethonium, hexamethonium, or carbamylcholine. Thus, the acetylcholine receptor alpha subunit, independent of other subunits of the receptor, acquires a mature conformation and high affinity alpha bungarotoxin binding when expressed in a quail fibroblast cell line.
Blount
P
P
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Merlie
J P
JP
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002038:Bungarotoxins / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000032:analysis; D005703:Gallamine Triethiodide / Q000378:metabolism; D005786:Gene Expression Regulation; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011784:Quail; D011950:Receptors, Cholinergic / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D014403:Tubocurarine / Q000378:metabolism
D002038:Bungarotoxins; D046911:Macromolecular Substances; D011950:Receptors, Cholinergic; D004247:DNA; D005703:Gallamine Triethiodide; D014403:Tubocurarine