Fibroblasts transfected with Torpedo acetylcholine receptor β-, γ-, and δ-subunit cDNAs express functional receptors when infected with a retroviral α recombinant

Abstract

Torpedo californica acetylcholine receptor (AChR) α-, β-, γ-, and δ-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 α) to bind α-bungarotoxin (BuTx). We demonstrated that the α, β, γ, and δ 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(d) for BuTx of 7.8 x 10-11 M. In contrast, subunits expressed individually were not detected on the surface of fibroblasts and the K(d) for BuTx binding to individual α polypeptides was only ~4 x 10-7 M. The half-lives of the α, γ, and δ subunits at 37°C were all found to be quite short (~43 min), while the half-life of the β subunits was found to be even shorter (~12 min). The unique half-life of the β 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 β, γ, and δ subunits simultaneously. When this cell line was infected with a retroviral α 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.