Proteins In Chinese Hamster Ovary Cells Research Articles

Identification of cellular target proteins for signaling cyclic phosphates

Cyclic glycerophosphates and their deoxy analogs were previously found to induce intracellular tyrosine and threonine phosphorylation in Chinese hamster ovary (CHO) cells. Further studies have indicated that these compounds induce neuronal outgrowth in PC-12 cells, as well as elevation of the state of cellular differentiation in human breast cancer cell lines. The mechanism by which these cyclic phosphates operate is not yet fully delineated. Using an affinity labeling approach we probed for possible cyclic phosphate target proteins in CHO cells. A 170 kDa protein that was labeled by an affinity cyclic phosphate reagent was identified by mass spectrometry as the largest subunit of the eukaryotic initiation factor 3 (eIF3). Using In-Gel kinase assays allowed the detection of a ∼70 kDa target kinase directly activated by cyclic phosphates. Identification of these proteins may provide a basis for deciphering the mechanisms, by which cyclic phosphates exert their effects.

A balanced expression of two chains of heterodimer protein, the human interleukin-12, improves high-level expression of the protein in CHO cells

Interleukin-12 (IL-12) comprises of p40 and p35 subunits that are encoded by genes on separate chromosomes and form p70 heterodimer, a bioactive protein, and free p40, an antagonist of IL-12. Balance expression of two subunits within cells would be the key for high-level of production of bioactive IL-12. Thinking about different expression efficiencies of two genes (p40 gene with higher efficiency), we selected two expression vectors with different efficiencies and inserted genes of p40 and p35 into them separately and co-transfected them into Chinese hamster ovary (CHO) cells. The high-level expression of IL-12 was obtained when p40 cDNA was inserted into pcDNA3 (lower expression vector) and p35 cDNA was inserted into pEE14 (higher expression vector), but using pEE14 for p40 cDNA and pcDNA3 for p35 cDNA, which was opposite to the optimal design, or pEE14 or pcDNA3 for both p35 cDNA and p40 cDNA did not obtain high-level of production of p70 heterodimer, the bioactive IL-12. We also observed that using two chemical reagents in combination, as a pressure selection method or amplification for the two vectors, markedly enhanced the IL-12 production, when compared with any one selection chemical. Our results indicated that the balance expressions of two chains of hetrodimer protein, such as p40 and p35 of IL-12, would be a better choice to obtain high-level of production of the proteins.

GCNF-dependent repression of BMP-15 and GDF-9 mediates gamete regulation of female fertility.

To determine the function of germ cell nuclear factor (GCNF) in female reproduction, we generated an oocyte-specific GCNF knockout mouse model (GCNF(fl/fl)Zp3Cre(+)). These mice displayed hypofertility due to prolonged diestrus phase of the estrous cycle and aberrant steroidogenesis. These reproductive defects were secondary to a primary defect in the oocytes, in which expression of the paracrine transforming growth factor-beta signaling molecules, bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9), were up-regulated in GCNF(fl/fl)Zp3Cre(+) females at diestrus. This was a direct effect of GCNF, as molecular studies showed that GCNF bound to DR0 elements within the BMP-15 and GDF-9 gene promoters and repressed their reporter activities. Consistent with these findings, abnormal double-oocyte follicles, indicative of aberrant BMP-15/GDF-9 expression, were observed in GCNF(fl/fl)Zp3Cre(+) females. The Cre/loxP knockout of GCNF in the oocyte has uncovered a new regulatory pathway in ovarian function. Our results show that GCNF directly regulates paracrine communication between the oocyte and somatic cells by regulating the expression of BMP-15 and GDF-9, to affect female fertility.

Subunit-Specific Modulation of KCNQ Potassium Channels by Src Tyrosine Kinase

We studied regulation by c-Src tyrosine kinase (Src) of KCNQ1-5 channels heterologously expressed in Chinese hamster ovary (CHO) cells and of native M current in rat sympathetic neurons. Using whole-cell patch clamp, we found that Src modulates currents from KCNQ3, KCNQ4, and KCNQ5 homomultimers, KCNQ2/3 heteromultimers and native M current, but not currents from KCNQ1 or KCNQ2 homomultimers. Src overexpression had two effects: a decrease of current amplitude (4- to 15-fold for cloned channels and approximately 3-fold for M current) and a slowing of activation kinetics by 2-fold. Both Src actions were mostly reversed by bath application of the Src inhibitors erbstatin (20 microm) and PP2 (200 nm), and mimicked by the tyrosine phosphatase inhibitor sodium vanadate (100 microm). Immunoprecipitation and immunoblot analysis showed Src-dependent phosphotyrosine signals associated with KCNQ3, KCNQ4, and KCNQ5 but not with KCNQ1 or KCNQ2 that may be tyrosine phosphorylation of the channel subunits. Expression of a dominant negative Src that cannot phosphorylate substrates had no effect on the current and did not induce phosphotyrosine signals associated with KCNQ3-5 subunits, further indicating that Src actions on KCNQ currents are mediated by tyrosine phosphorylation. Immunostaining and confocal analysis showed no effect of Src overexpression on the abundance of KCNQ3 protein in CHO cells. Finally, experiments using cloned KCNQ2/3 channels, Src and M(1) muscarinic receptors, and sympathetic neurons demonstrated that the actions on KCNQ channels by Src and by muscarinic agonists use distinct mechanisms.

Antibodies to Heterologous Proteins in Hemophilia A Patients Receiving Recombinant Factor VIII (Recombinate™)

As a consequence of the manufacturing process, trace quantities of Chinese hamster ovary cell protein, bovine serum albumin and murine immunoglobulin G are present in Recombinate recombinant human factor VIII (rhFVIII). The development of antibodies (Abs) to these heterologous proteins was evaluated during long-term rhFVIII therapy of hemophilia A in 68 previously treated and 73 previously untreated patients. Ab prevalence was also assessed in 157 non-hemophilic subjects. Abs against heterologous proteins could be detected in varying percentages of patients and non-hemophilic subjects. Abs arose in patients sporadically, and levels were typically low. There were no adverse events associated with development or presence of anti-heterologous protein Abs. These data indicate that sustained immune responses to trace levels of heterologous proteins are very infrequent during long-term rhFVIII therapy.

Expression of Streptococcus mutans wall-associated protein A gene in Chinese hamster ovary cells: prospect for a dental caries DNA vaccine.

The Streptococcus mutans strain GS-5 wall-associated protein A (Wap-A) is a precursor to the extracellular antigen A (AgA), a recognized candidate dental caries vaccine. The full-length wapA gene (wapA-E) and a C-terminal truncated version (wapA-G) encoding the AgA were cloned into the mammalian expression vector pcDNA 3.1/V5/His-TOPO. The resulting constructs were propagated in the Escherichia coli Top10. To investigate the expression of the S. mutans genes in mammalian cells, the above constructs were used to transfect Chinese hamster ovary (CHO) cells in the presence of the cationic lipid pfx-8. Transient expression of the wapA-E and wapA-G genes was observed at 24 h post-transfection, as shown by Western immunoblot analysis using a rabbit antiserum to S. mutans cell wall. Immunochemical staining of the transfected CHO cells showed expression of WapA mainly in the cells and budding vesicles, whereas AgA was found mainly in the transfected cells and extracellular medium. The expression of S. mutans proteins in CHO cells, in either vesicles or soluble form, suggested an antibody response to the above DNA constructs. Work is under way to test the efficacy of these as DNA vaccines against S. mutans.

Genetic engineering of CHO cells producing human interferon-gamma by transfection of sialyltransferases.

Natural human interferon-gamma (hIFN-gamma) contains mainly biantennary complex-type sugar chains. We previously remodeled the branch structures of N-glycans on hIFN-gamma in Chinese hamster ovary (CHO) cells by overexpressing UDP-N-acetylglucosamine: alpha1,6-D-mannoside beta1,6-N-acetylglucosaminyltransferase (GnT-V). Normal CHO cells primarily produced hIFN-gamma having biantennary sugar chains, whereas a CHO clone, designated IM4/Vh, transfected with GnT-V, primarily produced hIFN-gamma having GlcNAcbeta1-6 branched triantennary sugar chains when sialylation was incomplete and an increase in poly-N-acetyllactosamine (Galbeta1-4GlcNAcbeta1-3)n was observed. In the present study, we introduced mouse Galbeta1-3/4GlcNAc-R alpha2,3-sialyltransferase (ST3Gal IV) and/or rat Galbeta1-4GlcNAc-R alpha2,6-sialyltransferase (ST6Gal I) cDNAs into the IM4/Vh cells to increase the extent of sialylation and to examine the effect of sialyltransferase (ST) type on the linkage of sialic acid. Furthermore, we speculated that sialylation extent might affect the level of poly-N-acetyllactosamine. We isolated four clones expressing different levels of alpha2,3-ST and/or alpha2,6-ST. The extent of sialylation of hIFN-gamma from the IM4/Vh clone was 61.2%, which increased to about 80% in every ST transfectant. The increase occurred regardless of the type of overexpressed ST, and the proportion of alpha2,3- and alpha2,6-sialic acid corresponded to the activity ratio of alpha2,3-ST to alpha2,6-ST. Furthermore, the proportion of N-glycans containing poly-N-acetyllactosamine was significantly reduced (less than 10%) in the ST transfectants compared with the parental IM4/Vh clone (22.9%). These results indicated that genetic engineering of STs is highly effective for regulating the terminal structures of sugar chains on recombinant proteins in CHO cells.

A possible function of DNA curvature in a vector system that allows efficient expression of heterologous proteins in CHO cells.

A cis-acting element derived from the nontranscribed spacer region of murine rDNA allows efficient expression of heterologous proteins in transformed Chinese hamster ovary (CHO) cells. Analysis of the structural conformation of this cis-acting element revealed that it has a DNA curvature. Whether the DNA curvature plays any roles in regulating expression of heterologous proteins or not has been studied.

Chinese Hamster Ovary Cells Require the Coexpression of Microsomal Triglyceride Transfer Protein and Cholesterol 7α-Hydroxylase for the Assembly and Secretion of Apolipoprotein B-containing Lipoproteins

Due to the absence of microsomal triglyceride transfer protein (MTP), Chinese hamster ovary (CHO) cells lack the ability to translocate apoB into the lumen of the endoplasmic reticulum, causing apoB to be rapidly degraded by an N-acetyl-leucyl-leucyl-norleucinal-inhibitable process. The goal of this study was to examine if expression of MTP, whose genetic deletion is responsible for the human recessive disorder abetalipoproteinemia, would recapitulate the lipoprotein assembly pathway in CHO cells. Unexpectedly, expression of MTP mRNA and protein in CHO cells did not allow apoB-containing lipoproteins to be assembled and secreted by CHO cells expressing apoB53. Although expression of MTP in cells allowed apoB to completely enter the endoplasmic reticulum, it was degraded by a proteolytic process that was inhibited by dithiothreitol (1 mM) and chloroquine (100 microM), but resistant to N-acetyl-leucyl-leucyl-norleucinal. In marked contrast, coexpression of the liver-specific gene product cholesterol 7alpha-hydroxylase with MTP resulted in levels of MTP lipid transfer activity that were similar to those in mouse liver and allowed intact apoB53 to be secreted as a lipoprotein particle. These data suggest that, although MTP-facilitated lipid transport is not required for apoB translocation, it is required for the secretion of apoB-containing lipoproteins. We propose that, in CHO cells, MTP plays two roles in the assembly and secretion of apoB-containing lipoproteins: 1) it acts as a chaperone that facilitates apoB53 translocation, and 2) its lipid transfer activity allows apoB-containing lipoproteins to be assembled and secreted. Our results suggest that the phenotype of the cell (e.g. expression of cholesterol 7alpha-hydroxylase by the liver) may profoundly influence the metabolic relationships determining how apoB is processed into lipoproteins and/or degraded.

Mixed Agonist–Antagonist Properties of Clozapine at Different Human Cloned Muscarinic Receptor Subtypes Expressed in Chinese Hamster Ovary Cells

We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarinic receptor subtype. In the present study, we investigated whether the drug could elicit similar effects at the cloned human m1, m2, and m3 muscarinic receptor subtypes expressed in the Chinese hamster ovary (CHO) cells. Clozapine elicited a concentration-dependent stimulation of [3H]inositol phosphates accumulation in CHO cells expressing either the m1 or the m3 receptor subtype. Moreover, clozapine inhibited forskolin-stimulated cyclic AMP accumulation and enhanced [35S] GTPγS binding to membrane G proteins in CHO cells expressing the m2 receptor. These agonist effects of clozapine were antagonized by atropine. The intrinsic activity of clozapine was lower than that of the full cholinergic agonist carbachol, and, when the compounds were combined, clozapine potently reduced the receptor responses to carbachol. These data indicate that clozapine behaves as a partial agonist at different muscarinic receptor subtypes and may provide new hints for understanding the receptor mechanisms underlying the antipsychotic efficacy of the drug.

Analysis of isoprenoid biosynthesis in peroxisomal-deficient Pex2 CHO cell lines

ZR-78 and ZR-82 cells are two peroxisomal-deficient Chinese hamster ovary (CHO) cell mutants. These cells lack normal peroxisomes and show reduced levels of plasmalogen synthesis and other peroxisomal functions attributed to the deficiency of peroxisomal matrix enzymes. As we have recently identified two HMG-CoA reductase proteins in CHO cells, a 97 kDa reductase localized in the ER and a 90 kDa reductase protein localized in peroxisomes, this enabled us to study the two reductase proteins for the first time in peroxisomal-deficient CHO cells. In this study we report the results of a detailed analysis of the isoprenoid biosynthetic pathway in the peroxisomal-deficient CHO cell lines ZR-78 and ZR-82. We demonstrate that total HMG-CoA reductase activity is significantly reduced in the peroxisomal-deficient cells as compared to the wild type cells. Analysis of the two reductase proteins in permeabilized cells indicated that in the ZR-78 and ZR-82 cells the 90 kDa peroxisomal reductase protein was mainly localized to the cytosol. We further show that the rates of both sterol (cholesterol) and non-sterol (dolichols) biosynthesis were significantly lower in the peroxisomal-deficient cells, when either [3H] acetate or [3H] mevalonate was used as substrate. In contrast, the rate of dolichol biosynthesis in the peroxisomal-deficient cells was similar to that of the wild type cells when incubated with [3H] farnesol. In addition, we demonstrate that the peroxisomal-deficient cells exhibited increased rates of lanosterol biosynthesis as compared to wild type cells.▪ The results of this study provide further evidence for the essential requirement of peroxisomes for cholesterol biosynthesis as well as for dolichol production.— Aboushadi, N., and S. K. Krisans. Analysis of isoprenoid biosynthesis in peroxisomal-deficient Pex2 CHO cell lines.

The gut-enriched Krüppel-like factor (GKLF) suppresses the promoter activity of the cytochrome P450 1A1 (CYP1A1) gene in a SP1-dependent fashion

Background: GKLF is a recently identified transcription factor that contains 3 C2H 2 zinc fingers with homology to the Drosophila Kriippel protein [Shields, Christy and Yang (1996) J. Biol. Chem. 271, 20009; Shields and Yang (1997) J. Biol. Chem. 277~2 18504]. Expression of GKLF is enriched in the gut epithelium and is associated with growth arrest. Recent studies have identified a consensus DNA sequence to which GKLF binds [Shields and Yang (1998) Nucl. Acids Res., in press]. This sequence is very similar to the basal transcription dement (BTE) this is essential for the promoter activity of many genes in the cytochrome P450 family, including CYP1A1. Aim: The aim of the present study is to investigate whether GKLF is involved in the regulation of the CYP1A1 promoter through the BTE. Methods: The binding of GKLF to BTE was analyzed by methylation interference and electrophoretic mobility shift assays (EMSA) using partially purified recombinant GKLF and a radioactively labeled double-stranded oligonucleotide containing the GKLF consensus binding sequence (named TDA) or BTE. The effect of GKLF on CYP1A1 promoter activity was examined by cotransfection experiments in Chinese hamster ovary (CHO) cells with an eukaryotic expression construct containing full-length or truncated GKLF and a chloramphenicol acetyl transferase (CAT) reporter gene driven by the CYP1A1 Y-flanking sequence including or not BTE. Results: EMSA of labeled TDA and BTE using recombinant GKLF demonstrated the formation of a single, identical DNA-protein complex. The minimal essential DNA sequences within TDA and BTE that bound GKLF were identical as defined by base-specific mutagenesis and methylation interference assays. In transiently transfected CHO cells, an expression construct containing full-length but not truncated GKLF was able to suppress, in a dose-dependent fashion, the activity of the CAT reporter driven by the wild-type CYP1A1 promoter but not by a mutated promoter in which the BTE was specifically deleted. As a deletion of the BTE from the CYP1A1 promoter also resulted in a significant decrease in the basal promoter activity, nuclear extracts from CHO cells were prepared and analyzed by EMSA. The results indicate that the primary protein in CHO cells binding to BTE was Spl as demonstrated by antibody super-shift experiments. Cotransfection of the CYP1A1 promoter-reporters with an expression construct containing Spl showed that Spl transactivated the CYP1A1 promoter in a BTE-dependent fashion. The further addition of GKLF to the above transfection condition resulted in a dose-dependent abrogation of the ability of Spl to activate CYP1A1 promoter. Conclusion: The results indicate that GKLF is an GASTROENTEROLOGY Vol. 114, No. 4

Purification and Characterization of a GDP-fucose:Polypeptide Fucosyltransferase from Chinese Hamster Ovary Cells

A GDP-fucose:polypeptide fucosyltransferase was purified 5000-fold to homogeneity from Chinese hamster ovary cell extracts in the absence of detergent. The purification procedure included two affinity chromatographic steps using the acceptor substrate, a recombinant factor VII EGF-1 domain, and the donor substrate analog, GDP-hexanolamine, as ligands. The purified enzyme migrates as a single band of 44,000 daltons on SDS-polyacrylamide gel electrophoresis and is itself a glycoprotein with more than one high mannose type oligosaccharide chain with a total molecular weight of 4000. The Km values for factor VII EGF-1 domain and GDP-fucose are 15 and 6 microM, respectively. The Vmax is 2.5 micromol.min-1.mg-1. The presence of 50 mM Mn2+ increased the enzyme activity 17-fold, but Mn2+ was not absolutely required, since the enzyme exhibited some activity even in the presence of EDTA. The acceptor substrate specificity was studied using site-directed mutagenesis of human factor IX EGF domain. Only one of several differently folded species could serve as acceptor substrate, although they all had the same molecular weight as determined by liquid chromatography on-line with mass spectrometry. This indicates that the enzyme requires proper folding of the epidermal growth factor domain for its activity.

Bivalence of EGF-like ligands drives the ErbB signaling network.

Signaling by epidermal growth factor (EGF)-like ligands is mediated by an interactive network of four ErbB receptor tyrosine kinases, whose mechanism of ligand-induced dimerization is unknown. We contrasted two existing models: a conformation-driven activation of a receptor-intrinsic dimerization site and a ligand bivalence model. Analysis of a Neu differentiation factor (NDF)-induced heterodimer between ErbB-3 and ErbB-2 favors a bivalence model; the ligand simultaneously binds both ErbB-3 and ErbB-2, but, due to low-affinity of the second binding event, ligand bivalence drives dimerization only when the receptors are membrane anchored. Results obtained with a chimera and isoforms of NDF/neuregulin predict that each terminus of the ligand molecule contains a distinct binding site. The C-terminal low-affinity site has broad specificity, but it prefers interaction with ErbB-2, an oncogenic protein acting as a promiscuous low-affinity subunit of the three primary receptors. Thus, ligand bivalence enables signal diversification through selective recruitment of homo- and heterodimers of ErbB receptors, and it may explain oncogenicity of erbB-2/HER2.

Uptake of recombinant myeloperoxidase, free or fused to Fc gamma, by macrophages enhances killing activity toward micro-organisms.

A chimeric antibody-like molecule consisting of the human myeloperoxidase (rMPO) fused to the second and third constant-sequence (CH2 and CH3) Fc domains of human immunoglobulin G-1 has been constructed and expressed in Chinese hamster ovary (CHO) cells. This fusion molecule was designed to combine the binding specificity of Fc with the antimicrobial properties of rMPO. The rMPO-Fc fusion dimerized through the Fc fragment, while retaining the enzymatic activity of rMPO. The chimeric molecule was glycosylated and most of the propeptide was eliminated, indicating a better processing of the polypeptide than for rMPO alone. Both rMPO and rMPO-Fc bound to and were internalized by macrophage-like U937 promonocytic cells. Unexpectedly, the chimera failed to bind to the Fc receptor but interacted with a higher affinity than rMPO with the same binding sites. The presence of the Fc fragment in the chimera, in addition, did not extend the plasma half-life of the fusion protein. In vitro, rMPO-Fc exhibited a stronger killing effect than rMPO toward Candida albicans in the presence of either H202 alone or human macrophages. In vivo, rMPO-Fc similarly conferred a better protection than rMPO in a lethal model of murine cowdriosis. These properties could be related to the Fc-induced dimerization of the fusion protein in CHO cells.

Pertussis toxin-catalyzed ADP-ribosylation of Gi-2 and Gi-3 in CHO cells is modulated by inhibitors of intracellular trafficking

In previous studies, an in vitro ADP-ribosylation assay was developed to quantitatively evaluate the in vivo ADP-ribosylation of eukaryotic target proteins in intact Chinese hamster ovary (CHO) cells by pertussis toxin (PT). Immunoblot analysis identified the two PT-sensitive target proteins in CHO cells as Gi-2 and Gi-3. In this in vitro ADP-ribosylation assay, the ability of PT and ADP-ribosylate Gi-2 and Gi-3 intact CHO cells was not inhibited by cytochalasin D but was inhibited by chloroquine, monensin, and nocodazole. These data implicated the involvement of a cytochalasin D-independent endocytic mechanism, a pH-sensitive step, and microtubules in the ADP-ribosylation of Gi-2 and Gi-3 by PT in intact CHO cells. Preincubation of CHO cells with cycloheximide, at concentrations that reduced protein synthesis by > 95%, did not inhibit the ability of PT to ADP-ribosylate Gi-2 and Gi-3. Control experiments showed that these agents did not affect either the ability of PT to directly ADP-ribosylate the heterotrimeric G protein, Gt, or the binding of PT to CHO cells, except that monensin slightly inhibited the binding of PT to CHO cells. These results are consistent with a model in which PT is internalized by receptor-mediated endocytosis, probably via a cytochalasin D-independent pathway, which involves intracellular trafficking through late endosomes and the Golgi apparatus. An alternative model predicts the presence of a eukaryotic factor that traffics within cells via this pathway and is required by PT to ADP-ribosylate Gi proteins.

Regulation and Immunolocalization of Acyl-Coenzyme A:Cholesterol Acyltransferase in Mammalian Cells as Studied with Specific Antibodies

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesterol esters in various tissues. We recently reported the cloning and expression of human macrophage ACAT cDNA. In the current study, we report the production of specific polyclonal antibodies against ACAT by immunizing rabbits with the recombinant fusion protein composed of glutathione S-transferase and the first 131 amino acids of ACAT protein. Immunoblot analysis showed that the antibodies cross-reacted with a 50-kDa protein band from a variety of human cell lines. These antibodies immunodepleted more than 90% of detergent-solubilized ACAT activities from six different human cell types, demonstrating that the 50-kDa protein is the major ACAT catalytic component in these cells. In multiple human tissues examined, the antibodies recognized protein bands with various molecular weights. These antibodies also cross-reacted with the ACAT protein in Chinese hamster ovary cells. Immunoblot analysis showed that the ACAT protein contents in human fibroblast cells, HepG2 cells, or Chinese hamster ovary cells were not affected by sterol in the medium, demonstrating that the main mechanism for sterol-dependent regulation of ACAT activity in these cells is not change in ACAT protein content. As revealed by indirect immunofluorescent microscopy, the ACAT protein in tissue culture cells was located in the endoplasmic reticulum. This finding, along with earlier studies, suggests that cholesterol concentration in the endoplasmic reticulum may be the major determinant for regulating ACAT activity in the intact cells.

Modulation of brain Na+ channels by a G-protein-coupled pathway.

Na+ channels in acutely dissociated rat hippocampal neurons and in Chinese hamster ovary (CHO) cells transfected with a cDNA encoding the alpha subunit of rat brain type IIA Na+ channel (CNaIIA-1 cells) are modulated by guanine nucleotide binding protein (G protein)-coupled pathways under conditions of whole-cell voltage clamp. Activation of G proteins by 0.2-0.5 mM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a nonhydrolyzable GTP analog, increased Na+ currents recorded in both cell types. The increase in current amplitude was caused by an 8- to 10-mV negative shift in the voltage dependence of both activation and inactivation. The effects of G-protein activators were blocked by treatment with pertussis toxin or guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), a nonhydrolyzable GDP analog, but not by cholera toxin. GDP[beta S] (2 mM) alone had effects opposite those of GTP[gamma S], shifting Na(+)-channel gating 8-10 mV toward more-positive membrane potentials and suggesting that basal activation of G proteins in the absence of stimulation is sufficient to modulate Na+ channels. In CNaIIA-1 cells, thrombin, which activates pertussis toxin-sensitive G proteins in CHO cells, caused a further negative shift in the voltage dependence of Na(+)-channel activation and inactivation beyond that observed with GTP alone. The results in CNaIIA-1 cells indicate that the alpha subunit of the Na+ channel alone is sufficient to mediate G protein effects on gating. The modulation of Na+ channels via a G-protein-coupled pathway acting on Na(+)-channel alpha subunits may regulate electrical excitability through integration of different G-protein-coupled synaptic inputs.

Transfected platelet-activating factor receptor activates mitogen-activated protein (MAP) kinase and MAP kinase kinase in Chinese hamster ovary cells.

The platelet-activating factor (PAF) was seen to potently activate mitogen-activated protein (MAP) kinase and MAP kinase kinase through the cloned guinea pig PAF receptor stably expressed in Chinese hamster ovary (CHO) cells. Both 42- and 44-kDa MAP kinases were activated and tyrosine-phosphorylated in response to PAF. The PAF receptor also triggered the production of inositol phosphates and the release of arachidonic acid and inhibited cyclic AMP accumulation. Differential inhibitory effects of pertussis toxin (PTX) on these signals suggested that the PAF receptor couples to both PTX-sensitive and -insensitive G proteins in CHO cells. MAP kinase and MAP kinase activations were partially regulated by PTX-sensitive G proteins. The PAF receptor did not trigger any detectable increase in the GTP form of Ras under the conditions in which the human insulin receptor expressed in the same parent CHO cells potently increased the level. Since these agonists induced comparable MAP kinase activations through cognate receptors, Ras seems to play different roles in MAP kinase activation by the two different classes of receptors. The activation of MAP kinase by the cloned PAF receptor may explain part of the mechanisms underlying PAF-induced differentiation and proliferation in non-inflammatory cells.

Inhibition of degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vivo by cysteine protease inhibitors

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulatory enzyme of cholesterol biosynthesis and is located in the endoplasmic reticulum (ER). A fusion protein, HMGal, consisting of the membrane domain of HMG-CoA reductase fused to Escherichia coli beta-galactosidase and expressed in Chinese hamster ovary (CHO) cells from the SV40 promoter, was previously constructed and was found to respond to regulatory signals for degradation in a similar fashion to the intact HMG-CoA reductase. Degradation of both HMG-CoA reductase and HMGal in CHO cells was enhanced by addition of mevalonate or low density lipoprotein (LDL). In this report we show that 2 cysteine protease inhibitors, N-acetyl-leucyl-leucyl-norleucinal (ALLN) and N-acetyl-leucyl-leucyl-methioninal (ALLM), completely inhibit the mevalonate- or LDL-accelerated degradation of HMG-CoA reductase and HMGal and also block the basal degradation of these enzymes. It has been shown that in vitro these protease inhibitors inhibit the activities of Ca(2+)-dependent neutral proteases as well as lysosomal proteases, including cathepsin L, cathepsin b, and cathepsin D. However, the mevalonate-accelerated degradation of HMG-CoA reductase and HMGal is not affected by lysosomotropic agents, suggesting that the site of action of these inhibitor peptides in preventing the degradation is not the cathepsins. In brefeldin A-treated cells, where protein export from the ER is blocked, ALLN is still effective in inhibiting the degradation of HMG-CoA reductase and HMGal. These results indicate the involvement of non-lysosomal Ca(2+)-dependent proteases in the basal and the accelerated degradation of HMG-CoA reductase and HMGal. Enzymatic assays in vitro and immunoblot analyses have revealed calpain- and calpastatin-like proteins in CHO cells. The activities and the amount of these proteins do not change under conditions of enhanced degradation, indicating that the levels of these proteins are not subject to mevalonate regulation.