Fibroblasts transfected with Torpedo acetylcholine receptor beta-, gamma-, and delta-subunit cDNAs express functional receptors when infected with a retroviral alpha recombinant.

Abstract

Abstract. Torpedo californica acetylcholine receptor (AChR) tx-, ~-, 3'-, and tS-subunit cDNAs were each stably introduced into muscle and/or fibroblast cell lines using recombinant retroviral vectors and viral infection, or using SV-40 vectors and DNA-mediated cotransfection. The expressed proteins were characterized in terms of their molecular mass, antigenicity, posttranslational processing, cell surface expression, stability in fibroblasts, stability in differentiated and undifferentiated muscle cells, and ability (of a) to bind o~-bungarotoxin (BuTx). We demonstrated that the ct, ~, .,/, and t5 polypeptides acquired one, one, two, and three units of oligosaccharide, respectively. If all four subunits were expressed in the same cell, fully functional cell surface AChRs were produced which had a K~ for BuTx of 7.8 x 10-" M. In contrast, subunits expressed individually were not detected on the surface of fibroblasts and the Kd for BuTx binding to individual o~ polypeptides was only '~4 x 10 -7 M. The half-lives of the or, 3', and t5 subunits at 37°C were all found to be quite short (,x,43 min), while the half-life of the ~ subunit was found to be even shorter ('~12 min). The unique half-life of the/3 subunit suggests that it might perform a key regulatory role in the process of AChR subunit assembly. One stable fibroblast cell line was established by transfection that expressed ~, -y, and t5 subunits simultaneously. When this cell line was infected with a retroviral o~ recombinant, fully functional cell surface AChRs were produced. The successful expression of this pentameric protein complex combining transfection and infection techniques demonstrates one strategy for stably introducing the genes of a heterologous multisubunit protein complex into cells.

Endoplasmic Reticulum Chaperones Stabilize Nicotinic Receptor Subunits and Regulate Receptor Assembly

“…ERAD of Torpedo ␣ subunits is essentially fit by single exponential decay, as shown by the line through the data points and in this case has a decay constant () of 55 min. This is consistent with previous measurements of the turnover of single nAChR subunits, where the data were well fit by single exponentials with values of ϳ40 min (43)(44)(45). Similar results (Table 1) were obtained for the degradation of ␤, ␥, and ␦ subunits when transfected individually and assayed using the same protocol.…”

N-Linked Glycosylation Is Required for Nicotinic Receptor Assembly but Not for Subunit Associations with Calnexin

“…This is because CN is extremely metabolically stabile compared with the AChR subunits. The half-life of CN is greater than 24 h (34) compared with a half-life of 30 -60 min at 37°C for Torpedo AChR subunits (39). Because of its stability, the CN protein is not metabolically labeled well and is not easily observed when co-immunoprecipitated with other proteins.…”

Surface Expression of the AMPA Receptor Subunits GluR1, GluR2, and GluR4 in Stably Transfected Baby Hamster Kidney Cells

“…A further important finding in the present work is that fully glycosylated GluR subunits are all trafficked with roughly equal efficiency to the cell surface and that heteromeric combinations are not trafficked more efficiently than homomeric combinations. The subunits of certain other ionotropic receptors, such as NMDA-type glutamate receptors (Mcllhinney et al, 1996) and nicotinic acetylcholine receptors (Claudio et al, 1991), are trafficked much more efficiently to the cell surface as heteromeric assemblies than as homomeric assemblies. In cases such as these, where efficient receptor trafficking is dependent on proper subunit stoichiometry, the number of subunit combinations that might potentially yield functional receptors is somewhat restricted.…”