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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.

Abstract. Assembly of nicotinic acetylcholine receptor (AChR) subunits was investigated using mouse fibroblast cell lines stably expressing either Torpedo or mouse (AM-4) a, ~, % and ~ AChR subunits. Both cell lines produce fully functional cell surface AChRs. We find that two independent treatments, lower temperature and increased intracellular cAMP can increase AChR expression by increasing the efficiency of subunit assembly. Previously, we showed that the rate of degradation of individual subunits was decreased as the temperature was lowered and that Torpedo AChR expression was acutely temperature sensitive, requiring temperatures lower than 37°C. We find that Torpedo AChR assembly efficiency increases 56-fold as the temperature is decreased from 37 to 20°C. To determine how much of this is a temperature effect on degradation, mouse AChR assembly efficiencies were determined and found to be only approximately fourfold more efficient at 20 than at 37°C.With reduced temperatures, we can achieve assembly efficiencies of Torpedo AChR in fibroblasts of 20-35%. Mouse AChR in muscle cells is also ~30% and we obtain ",,30% assembly efficiency of mouse AChR in fibroblasts (with reduced temperatures, this value approaches 100%). Forskolin, an agent which increases intracellular cAMP levels, increased subunit assembly efficiencies twofold with a corresponding increase in cell surface AChR. Pulse-chase experiments and immunofluorescence microscopy indicate that oligomer assembly occurs in the ER and that AChR oligomers remain in the ER until released to the cell surface. Once released, AChRs move rapidly through the Golgi membrane to the plasma membrane. Forskolin does not alter the intracellular distribution of AChR. Our results indicate that cell surface expression of AChR can be regulated at the level of subunit assembly and suggest a mechanism for the cAMP-induced increase in AChR expression.