Localization of ␤-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3 untranslated region of the mRNA, including a 54-nucleotide element which has been termed the "zipcode" (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441-451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize ␤-actin mRNA.

A b s t r a c t . B-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 B-actin mRNA becomes diffuse and nonlocalized. Addition of FCS induces a rapid (within 2-5 min) redistribution of B-actin mRNA into the leading lamellae. A similar redistribution was seen with PDGF, a fibroblast chemotactic factor. PDGFinduced B-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 B-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 B-actin mRNA in cells grown in serum, and can inhibit the serum induction of peripherally localized B-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.

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.

Abstract. We have investigated the mechanisms regulating the clustering of nicotinic acetylcholine receptor (AChR) on the surface of cultured embryonic chick muscle cells. Treatment of these cells with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, was found to cause a rapid dispersal of AChR clusters, as monitored by fluorescence microscopy of cells labeled with tetramethylrhodamine-conjugated tz-bungarotoxin. The loss of AChR clusters was not accompanied by an appreciable change in the amount of AChR on the surface of these cells, as measured by the specific binding of ['251]Bgt. Analysis of the phosphorylation pattern of immunoprecipitable AChR subunits showed that the ?-and 5-subunits are phosphorylated by endogenous protein kinase activity in the intact muscle cells, and that the 8-subunit displays increased phosphorylation in response to TPA. Structural analogues of TPA which do not stimulate protein kinase C have no effect on AChR surface topography or phosphorylation. Exposure of chick myotubes to the cholinergic agonist carbamylcholine was found to cause a dispersal of AChR clusters with a time course similar to that of TPA. Like TPA, carbamylcholine enhances the phosphorylation of the 5-subunit of AChR. The carbamylcholine-induced redistribution and phosphorylation of AChR is blocked by the nicotinic AChR antagonist d-tubocurarine. TPA and carbamylcholine have no effect on cell morphology during the time-course of these experiments. These findings indicate that cell surface topography of AChR may be regulated by phosphorylation of its subunits and suggest a mechanism for dispersal of AChR clusters by agonist activation.