Analysis of Early Events in Acetylcholine Receptor Assembly

• Published in: The Journal of cell biology 1991-06, Vol.113 (6), p.1371-1384
• Main Authors: Paulson, Henry L., Ross, Anthony F., Green, William N., Claudio, Toni
• Format: Article
• Language: English
• Subjects: 3T3 cells; acetylcholine; Animals; Biological and medical sciences; biological membranes; biosynthesis; Cell aggregates; Cell Differentiation; Cell Line; Cell lines; cell membranes; Cell structures and functions; Cells; Centrifugation, Density Gradient; Complementary DNA; Dimers; Disulfides - metabolism; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Fibroblasts - cytology; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; Gels; Immunoblotting; L cells; Marine; Molecular and cellular biology; Molecular Weight; Monomers; Muscles - cytology; Muscles - metabolism; Precipitin Tests; proteins; Receptors, Cholinergic - biosynthesis; Torpedo; Torpedo californica
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.113.6.1371

Mammalian cell lines expressing nicotinic acetylcholine receptor (AChR) subunit cDNAs from Torpedo californica were used to study early events in AChR assembly. To test the hypothesis that individual subunits form homooligomeric intermediates before assembling into α2β γ δ pentamers, we analyzed the sedimentation on sucrose density gradients of each subunit expressed separately in cell lines. We have shown previously that the acute temperature sensitivity of Torpedo AChR subunit assembly is due, in part, to misfolding of the polypeptide chains. We use this phenomenon to further analyze putative assembly-competent intermediates. In nonionic detergent at an assembly-permissive temperature, the majority of α, β, γ, and δ subunits sediment neither as 3-4S monomers nor as 9S complexes, but rather as 6S species whether synthesized in fibroblasts, myoblasts, or differentiated myosyncytia. Several results indicate that the 6S species are complexes comprised predominantly of incorrectly folded subunit polypeptides. The complexes represent homoaggregates which form rapidly within the cell, are stable to mild SDS treatment and, in the case of α, contain some disulfide-linked subunits. The coprecipitation of α subunit with BiP or GRP78, a resident protein of the ER, further indicates that at least some of these internally sequestered subunits also associate with an endogenous protein implicated in protein folding. The majority of subunits expressed in these cell lines appear to be aggregates of subunits which are not assembly intermediates and are not assembly-competent. The portion which migrates as monomer, in contrast, appears to be the fraction which is assembly competent. This fraction increases at temperatures more permissive for assembly, further indicating the importance of the monomer as the precursor to assembly of α2β γ δ pentamers.