Beta-actin mRNA is localized in the leading lamellae of chicken embryo fibroblasts (CEFs) (Lawrence, J., and R. Singer. 1986. Cell. 45:407-415), close to where actin polymerization in the lamellipodia drives cellular motility. During serum starvation beta-actin mRNA becomes diffuse and non-localized. Addition of FCS induces a rapid (within 2-5 min) redistribution of beta-actin mRNA into the leading lamellae. A similar redistribution was seen with PDGF, a fibroblast chemotactic factor. PDGF-induced beta-actin mRNA redistribution was inhibited by the tyrosine kinase inhibitor herbimycin, indicating that this process requires intact tyrosine kinase activity, similar to actin filament polymerization and chemotaxis. Lysophosphatidic acid, which has been shown to rapidly induce actin stress fiber formation (Ridley, A., and A. Hall. 1992. Cell. 790:389-399), also increases peripheral beta-actin mRNA localization within minutes. This suggests that actin polymerization and mRNA localization may be regulated by similar signaling pathways. Additionally, activators or inhibitors of kinase A or C can also delocalize steady-state beta-actin mRNA in cells grown in serum, and can inhibit the serum induction of peripherally localized beta-actin mRNA in serum-starved CEFs. These data show that physiologically relevant extracellular factors operating through a signal transduction pathway can regulate spatial sites of actin protein synthesis, which may in turn affect cellular polarity and motility.

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 alpha 2 beta gamma delta 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 (Paulson, H.L., and T. Claudio. 1990. J. Cell Biol. 110:1705-1717). We use this phenomenon to further analyze putative assembly-competent intermediates. In nonionic detergent at an assembly-permissive temperature, the majority of alpha, beta, gamma, and delta 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 alpha, contain some disulfide-linked subunits. The coprecipitation of alpha subunit with BiP or GRP78, a resident protein of the ER, further indicates that at least some of these internally sequestered subunits also associated 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 alpha 2 beta gamma delta pentamers.

Assembly of nicotinic acetylcholine receptor (AChR) subunits was investigated using mouse fibroblast cell lines stably expressing either Torpedo (All-11) or mouse (AM-4) alpha, beta, gamma, and delta 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 degrees C. We find that Torpedo AChR assembly efficiency increases 56-fold as the temperature is decreased from 37 to 20 degrees 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 degrees C. With reduced temperatures, we can achieve assembly efficiencies of Torpedo AChR in fibroblasts of 20-35%. Mouse AChR in muscle cells is also approximately 30% and we obtain approximately 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.

Torpedo californica acetylcholine receptor (AChR) alpha-, beta-, gamma-, and delta-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 alpha) to bind alpha-bungarotoxin (BuTx). We demonstrated that the alpha, beta, gamma, and delta 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 Kd for BuTx of 7.8 X 10(-11) M. In contrast, subunits expressed individually were not detected on the surface of fibroblasts and the Kd for BuTx binding to individual alpha polypeptides was only approximately 4 X 10(-7) M. The half-lives of the alpha, gamma, and delta subunits at 37 degrees C were all found to be quite short (approximately 43 min), while the half-life of the beta subunit was found to be even shorter (approximately 12 min). The unique half-life of the beta 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 beta, gamma, and delta subunits simultaneously. When this cell line was infected with a retroviral alpha 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.