Temperature-sensitive expression of all-Torpedo and Torpedo-rat hybrid AChR in mammalian muscle cells.
When the four subunits of the Torpedo californica nicotinic acetylcholine receptor (AChR) are expressed in mammalian fibroblasts, they properly assembly into alpha 2 beta gamma delta pentamers only at temperatures lower than 37 degrees C (Claudio, T., W. N. Green, D. S. Hartman, D. Hayden, H. L. Paulson, F. J. Sigworth, S. M. Sine, and A. Swedlund. 1987. Science (Wash. DC). 238:1688-1694). Experiments here with rat L6 myoblast cell lines indicate that this temperature sensitivity is not specific to fibroblasts, but is intrinsic to Torpedo subunits. A clonal isolate of L6 cells cotransfected with the four Torpedo subunit cDNAs synthesizes the exogenous AChR subunits at 37 degrees and 26 degrees C, but expresses Torpedo AChR complexes only at the lower temperature. When Torpedo alpha alone is expressed in L6 myotubes, hybrid AChRs are formed, again only at temperatures below 37 degrees C. These hybrid AChRs can contain either two Torpedo alpha subunits or one each of rat and Torpedo alpha, proving that the two alpha subunits in an AChR pentamer need not derive from the same polysome. Further analysis of hybrid and all-Torpedo AChR established that there is no internally sequestered pool of AChR at the nonpermissive temperature, and that the AChR, once formed, is thermostable. Two lines of experimentation with alpha subunits expressed in fibroblasts indicate that alpha polypeptides exhibit different conformations at 26 degrees and 37 degrees C, favoring the hypothesis that the temperature-sensitive step occurs before assembly and reflects, at least in part, misfolding of subunits: at 37 degrees C, there is a reduction in the fraction of alpha subunits that (a) bind the AChR antagonist alpha-bungarotoxin with high affinity; and (b) bind a monoclonal antibody that recognizes correctly folded and/or assembled alpha subunit.
Paulson
H L
HL
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Claudio
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D002038:Bungarotoxins / Q000378:metabolism; D002460:Cell Line; D046911:Macromolecular Substances; D009132:Muscles / Q000378:metabolism; D009154:Mutation; D011233:Precipitin Tests; D011487:Protein Conformation; D051381:Rats; D011950:Receptors, Cholinergic / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature; D014101:Torpedo / Q000378:metabolism*; D014162:Transfection
D000911:Antibodies, Monoclonal; D002038:Bungarotoxins; D046911:Macromolecular Substances; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins