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PubMed1989

Human papillomavirus type 16 E7 protein expressed in Escherichia coli and monkey COS-1 cells: immunofluorescence detection of the nuclear E7 protein.

Human papillomavirus type 16 E7 protein, expressed in Escherichia coli as fusion protein lac alpha peptide-E7 (lac-E7), was purified by electrophoresis and used to immunize rabbits to raise antisera. The anti-lac-E7 sera recognized another bacterially expressed fusion protein trpE-E7 by the Western blot method. The antisera were used to identify E7 protein transiently expressed in monkey COS-1 cells from an SV40-derived expression plasmid containing E7 gene. Like the E7 protein from CaSki cells, the majority of 19K E7 protein in the transfected COS-1 cells was found by immunoprecipitation in the soluble cytoplasmic fraction. By immunofluorescence staining, however, the E7 protein was detectable in the nuclei of the transfected COS-1 cells. The results suggest that the E7 protein expressed in monkey cells is nuclear, although it is readily released from nuclei when the cell structure is broken.

Animals↗
PubMed1993

Differential estrogen substrate specificities for transiently expressed human placental 17 beta-hydroxysteroid dehydrogenase and an endogenous enzyme expressed in cultured COS-m6 cells.

The metabolism of estrogens catalyzed by human placental 17 beta-hydroxysteroid dehydrogenase (17HSD) transiently expressed in COS-m6 cells was studied, and the properties of the enzyme were compared with those of an endogenous hydroxysteroid dehydrogenase (HSD) expressed in the cells. In cultured cells, the endogenous HSD had almost exclusively oxidative activity, converting estradiol to estrone (oxidative and reductive activity, 0.84 +/- 0.164 and 0.034 +/- 0.01 nmol/mg protein.h, respectively). This was, nevertheless, opposed to the activity of the transiently expressed human placental 17HSD, as a high reductive activity (0.86 +/- 0.30 nmol/mg protein.h) appeared in the cells after transfection, whereas oxidative activity was not significantly induced. In the different transfections, the reductive activity was induced 13- to 34-fold, and the oxidative activity in the 17HSD-transfected cells was 65-162% of that in the mock-transfected cells. Thus, in cultured cells, these two enzymes preferentially catalyze opposite reactions. When the metabolism of the estrogens was followed up to 20 h, the two enzymes were found to regulate the proportion of estrone to estradiol in the culture medium. The different properties found for the enzymes show that the endogenous HSD expressed in the COS-m6 cells is an additional member of the family of 17HSD enzymes. It is suggested that different 17HSD enzymes exist, with differential estrogen substrate specificities in cultured cells. Thus, in addition to cofactor and substrate availability, the biological activity of estrogens in different cell types may be regulated by the expression of different forms of 17HSD enzymes, resulting in the dominance of either estradiol or estrone production.

17-Hydroxysteroid Dehydrogenases↗
PubMed2004

Fruit extracts antagonize Abeta- or DA-induced deficits in Ca2+ flux in M1-transfected COS-7 cells.

Evidence suggests that there is a selective sensitivity to oxidative stress (OSS) among muscarinic receptor (MAChR) subtypes with M1, M2 and M4 showing > OSS than M3 or M5 subtypes in transfected COS-7 cells. This may be important in determining the regional specificity in neuronal aging and Alzheimer disease (AD). We assessed the effectiveness of blueberry (BB) and other high antioxidant (HA) fruit extracts (boysenberry, BY; cranberry, CB; black currant, BC; strawberry, SB; dried plums, DP; and grape, GR) on the toxic effects of Abeta 25-35 (100 microM, 24 hrs) and DA (1 mM, 4 hrs) on calcium buffering (Recovery) following oxotremorine (750 microM) -induced depolarization in M1AChR-transfected COS-7 cells, and on cell viability following DA (4 hrs) exposure. The extracts showed differential levels of Recovery protection in comparisons to the non-supplemented controls that was dependent upon whether DA or Abeta was used as the pretreatment. Interestingly, assessments of DA-induced decrements in viability revealed that all of the extracts had some protective effects. These findings suggest that the putative toxic effects of Abeta or DA might be reduced by HA fruit extracts.

Alzheimer Disease↗
PubMed2004

Truncated variants of hyaluronan-binding protein 1 bind hyaluronan and induce identical morphological aberrations in COS-1 cells.

Hyaluronan (HA)-binding protein 1 (HABP1) is multifunctional in nature and exists as a trimer through coiled-coil interaction between alpha-helices at its N- and C-termini. To investigate the importance of trimeric assemblage and HA-binding ability of HABP1, we generated and overexpressed variants of HABP1 by truncating the alpha-helices at its termini. Subsequently, these variants were transiently expressed in COS-1 cells to examine the influence of these structural variations on normal cell morphology, as compared with those imparted by HABP1. Substantiating the centrality of coiled-coil interaction for maintaining the trimeric assembly of HABP1, we demonstrate that disruption of trimerization does not alter the affinity of variants towards its ligand HA. Transient expression of HABP1 altered the morphology of COS-1 cells by generating numerous cytoplasmic vacuoles along with disruption of the f-actin network. Interestingly, the truncated variants also imparted identical morphological changes. Characterization of the cytoplasmic vacuoles revealed that most of these vacuoles were autophagic in nature, resembling those generated under stress conditions. The identical morphological changes manifested in COS-1 cells on transient expression of HABP1 or its variants is attributed to their comparable HA-binding ability, which in concert with endogenous HABP1, may deplete the cellular HA pool. Such quenching of HA below a threshold level in the cellular milieu could generate a stress condition, manifested through cytoplasmic vacuoles and a disassembly of the f-actin network.

Alternative Splicing↗
PubMed2004

Transport of dopamine and levodopa and their interaction in COS-7 cells heterologously expressing monoamine neurotransmitter transporters and in monoaminergic cell lines PC12 and SK-N-SH.

The present study investigated the effects of levodopa, a precursor of dopamine (DA) therapeutically used for the treatment of Parkinson's disease, on DA transport in the two different systems, COS-7 cells heterologously expressing rat monoamine transporter cDNA and in monoaminergic cell lines PC12 and SK-N-SH. Levodopa enhanced uptake of [3H]DA and [3H]norepinephrine (NE) but not [3H]serotonin in the transfected COS-7 cells in a concentration-dependent manner. On the other hand, in PC12 and SK-N-SH cells where NET is functionally expressed, levodopa enhanced [3H]DA and [3H]NE uptake at low concentrations and inhibited the uptake at higher concentrations. The effects of levodopa on catecholamine transporters in the opposite direction suggest a different mechanism at the intra- and extracellular sites in a levodopa transport-dependent and independent manner.

Animals↗
PubMed1997

Adenovirus-mediated expression of the secreted form of basic fibroblast growth factor (FGF-2) induces cellular proliferation and angiogenesis in vivo.

Blood supply through collateral arteries is of critical importance in occlusive arterial diseases such as coronary atherosclerosis. Induction of angiogenic growth factor within either the narrowing arteries or jeopardized myocardium may promote angiogenesis in vivo, leading to salvage of ischemic myocardium. We constructed a replication-defective adenovirus (AdCAsFGF-2) coding for human basic fibroblast growth factor (FGF)-2 that is modified, so that its secretion will be facilitated, by tagging a signal sequence derived from FGF-4. A large quantity of FGF-2 was detected in both the cell lysate and culture medium of COS cells infected with AdCAsFGF-2, indicating that FGF-2 was secreted at least partly from the infected cells. The conditioned medium from the infected COS cells stimulated DNA synthesis in and induced cellular proliferation of arterial smooth muscle cells. These effects were eliminated by adenovirus-mediated overexpression of a dominant-negative truncated FGF-receptor type 1. Implantation of a gel of basement membrane proteins containing fibroblasts infected with AdCAsFGF-2 into the ventral subcutaneous space of mice induced extensive cellular proliferation and the formation of functional arterioles. Cells surrounding the vessels were positively immunostained with antibodies recognizing either smooth muscle-specific alpha-actin or factor VIII antigen as a marker for endothelium. These results suggest that AdCAsFGF-2 may be useful for delivering functional FGF-2 into tissues and may lead to therapeutic angiogenesis in vivo.

Actins↗
PubMed1997

A minimal region on the integrin beta4 subunit that is critical to its localization in hemidesmosomes regulates the distribution of HD1/plectin in COS-7 cells.

The integrin alpha6 beta4 is a major component of hemidesmosomes, in which it mediates firm adhesion to laminin 5. Previous studies have shown that the incorporation of alpha6 beta4 into hemidesmosomes requires a 303 amino acid stretch of the cytoplasmic domain of beta4, comprising part of the first fibronectin type III (FNIII) repeat, the second FNIII repeat and the segment that connects the second to the third FNIII repeat (connecting segment). Now, we have further defined sequences within beta4 that are critical for its localization in hemidesmosomes and we demonstrate that these sequences also induce the redistribution of HD1/plectin into junctional complexes containing the integrin alpha6 beta4 in COS-7 cells, transfected with cDNAs encoding alpha6A and beta4. Truncation of the cytoplasmic domain of beta4 after amino acids 1,382 or 1,355 in the connecting segment, by which a potential tyrosine activation motif (TAM) is removed, does not prevent the localization of alpha6 beta4 in hemidesmosomes in the rat bladder carcinoma cell line 804G and neither did it eliminate the ability of alpha6 beta4 to change the subcellular distribution of HD1/plectin in COS-7 cells. In contrast, beta4 subunits in which the entire connecting segment had been deleted or which were truncated after amino acid 1,328, which removes almost the complete segment, had lost both of these functions. Furthermore, when beta4 subunits with either a deletion of the second FNIII repeat or a small deletion in this repeat were co-expressed with alpha6, the integrins were not localized in hemidesmosomes and did not induce the redistribution of HD1/plectin in COS-7 cells. Finally, the fourth FNIII repeat of beta4 could not replace the second in either of these activities. These findings establish that a region in beta4, which encompasses the second FNIII repeat and a stretch of 27 amino acids (1,329-1,355) of the connecting segment, is critical for the localization of alpha6beta4 in hemidesmosomes and that it regulates the distribution of HD1/plectin.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of HEK, the gene encoding a receptor tyrosine kinase expressed by human lymphoid tumor cell lines.

We describe the molecular cloning of a receptor tyrosine kinase from a cell line (LK63) derived from a case of human pre-B-cell leukemia. We have previously shown that a monoclonal antibody (IIIA4) raised against LK63 recognized a glycosylated, cell-surface 135-kDa molecule (HEK), which displayed tyrosine kinase activity in vitro. The HEK protein was purified by using a IIIA4 antibody column and both N-terminal and internal amino acid sequences were obtained. A 51-mer degenerate oligonucleotide based on the internal amino acid sequence was used to screen an LK63-derived lambda gt10 cDNA library under low-stringency hybridization conditions. One clone of 2.5 kilobases (kb) was isolated and characterized and used to rescreen the library under more-stringent hybridization conditions. A 4.5-kb clone containing the entire HEK coding region was isolated and its complete DNA sequence was determined. The 4.5-kb insert was subcloned into the expression vector CDM8 and transfected into COS cells. COS cells transfected with the sense HEK/CDM8 construct stained specifically with the IIIA4 antibody, thereby confirming that the antigen recognized by the IIIA4 antibody and the expressed protein product of the HEK cDNA clone were identical. DNA sequence analysis revealed that HEK is a newly discovered member of the EPH/ELK family of receptor tyrosine kinases. Northern blot analysis of a number of cell lines demonstrated the expression of 5.5- to 6.0-kb HEK transcripts in LK63 and the T-cell lines JM and HSB-2. Southern blot analysis of DNA from LK63 suggested that the HEK gene was neither amplified nor rearranged in the LK63 tumor.

Amino Acid Sequence↗
PubMed1999

Mutations in the env gene of human immunodeficiency virus type 1 NDK isolates and the use of African green monkey CXCR4 as a co-receptor in COS-7 cells.

A previous report from this laboratory described the isolation of the first CD4-independent human immunodeficiency virus type 1 isolate, m7NDK. This independence of CD4 is due to seven mutations located in the C2, V3 and C3 regions of the gp120 protein. The present report describes the entry features of the m5NDK virus, which contains five of the seven m7NDK mutations, located in the V3 loop and C3 region. The entry of this virus is strictly CD4-dependent but it can fuse with African green monkey (agm) COS-7 cells bearing human CD4 (h-CD4). This fusion is directly due to the five mutations in the envgene. It has also been shown that entry of m7NDK is CD4-independent in COS-7 cells. Since the wild-type NDK and m7NDK viruses use the human CXCR4 protein as co-receptor, agm-CXCR4 was cloned and used in transfection and fusion inhibition experiments to show that this receptor can be used by the m5 and m7NDK viruses. The wild-type NDK virus, which does not enter COS-7 cells, can use agm-CXCR4, but only when the receptor is transfected into target cells. Although co-receptor nature and expression levels are still major determinants of virus entry, this is the first case where a few mutations in the env gene can overcome this restriction.

Amino Acid Sequence↗
PubMed1988

Isolation of a human cDNA of urokinase and its expression in COS-1 cells.

The cDNA encoding human urokinase (UK) has been isolated from a cDNA library prepared from human normal fibroblast (WI38) cells, which had been stimulated by endothelial cell growth factor and heparin. This cDNA was sequenced and found to contain a few silent substitutions, thus encoding the same amino acids as deduced from the published genomic sequence of UK. After modification, the cDNA of UK was inserted into a transient expression vector and used to transfect COS-1 cells. The recombinant UK protein (rUK) in the serum-free medium of transfected COS-1 cells was characterized by biochemical and functional assays. These studies indicated that rUK from COS-1 cells is glycosylated, enzymatically active, and very similar to native single-chain plasminogen activator (scuPA). Therefore, such rUK can be a convenient source of scuPA for any further studies.

Amino Acid Sequence↗
PubMed2001

Simultaneous expression of guinea pig UDP-glucuronosyltransferase 2B21 and 2B22 in COS-7 cells enhances UDP-glucuronosyltransferase 2B21-catalyzed morphine-6-glucuronide formation.

Although UDP-glucuronosyltransferases (UGTs) act as an important detoxification system for many endogenous and exogenous compounds, they are also involved in the metabolic activation of morphine to form morphine-6-glucuronide (M-6-G). The cDNAs encoding guinea pig liver UGT2B21 and UGT2B22, which are intimately involved in M-6-G formation, have been cloned and characterized. Although some evidence suggests that UGTs may function as oligomers, it is not known whether hetero-oligomer formation leads to differences in substrate specificity. In this work, evidence for a functional hetero-oligomer between UGT2B21 and UGT2B22 is provided by studies on the glucuronidation of morphine in transfected COS-7 cells. Cells transfected with UGT2B21 cDNA catalyzed mainly morphine-3-glucuronide formation although M-6-G was also formed to some extent. In contrast, cells transfected with UGT2B22 cDNA did not show any significant activity toward morphine. When UGT2B21 and UGT2B22 were expressed simultaneously in different ratios in COS-7 cells, extensive M-6-G formation was observed. This stimulation of M-6-G formation was not observed, however, when microsomes containing UGT2B21were mixed with those containing UGT2B22 in the presence of detergent. Furthermore, this effect was not very marked when human UGT1A1 and UGT2B21 were coexpressed in COS-7 cells. This is the first report suggesting that UGT hetero-oligomer formation leads to altered substrate specificity.

Amino Acid Sequence↗
PubMed1991

The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4.

NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate.

Animals↗
PubMed2001

Binding of microsomal triglyceride transfer protein to lipids results in increased affinity for apolipoprotein B: evidence for stable microsomal MTP-lipid complexes.

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are known to interact with each other. We evaluated the effect of different lipids on the protein-protein interactions between MTP and apoB100 or its C-terminally truncated forms. Negatively charged lipids decreased protein-protein interactions between apoB and MTP. In contrast, zwitterionic phospholipids enhanced (2-4-fold) the binding of apoB100 to MTP by increasing affinity (1.5-3-fold) between these proteins without affecting the number of binding sites. Similarly, phospholipids augmented (1.5-4-fold) the binding of various C-terminally truncated apoB peptides to MTP. The increased binding was greater for apoB peptides containing lipid-binding domains, such as apoB28 and apoB42. Surprisingly, preincubation of apoB28 with lipid vesicles had no effect on MTP binding. In contrast, incubation of MTP with lipid vesicles resulted in a stable association of MTP with vesicles, and MTP-lipid vesicles bound better (5-fold increase) to LDL than did lipid-free MTP. To determine whether MTP exists stably associated with lipids in cells, microsomal contents from COS cells expressing MTP, HepG2 cells, and mouse liver were ultracentrifuged, and MTP was visualized in different density fractions. MTP was found associated and unassociated with lipids. In contrast, apoB17 and apoB:270-570 were present unassociated with lipids in COS cells. These studies show that the binding of MTP to lipids results in increased affinity for apoB and that stable MTP-lipid complexes exist in the lumen of the endoplasmic reticulum. Protein-protein interactions between apoB and MTP may juxtapose lipids associated with MTP to lipid-binding domains of apoB and facilitate hydrophobic interactions leading to enhance affinity. We speculate that MTP-lipid complexes may serve as nuclei to form "primordial lipoproteins" and may also play a role in the bulk addition of lipids during the "core expansion" of these lipoproteins.

Apolipoproteins B↗
PubMed1998

A G protein beta gamma dimer-mediated pathway contributes to mitogen-activated protein kinase activation by thyrotropin-releasing hormone receptors in transfected COS-7 cells.

Activation of mitogen-activated protein kinase (MAPK) is induced by adding thyrotropin-releasing hormone (TRH) to COS-7 cells cotransfected with TRH receptors and an epitope-tagged MAPK. Long term treatment of the cells with pertussis toxin has no effect on TRH-induced MAPK activation. Incubation of the cells with the protein kinase C (PKC) inhibitor GF109203X causes an almost complete inhibition of MAPK activation by the PKC activator phorbol-12-myristate-13-acetate. In contrast, only approximately 50% of the TRH-induced MAPK activity is inhibited by GF109203X, indicating that activation of MAPK by TRH is only partially dependent on PKC. The inhibitory effect of GF109203X is additive with that of p21(N17ras), a dominant negative mutant of p21(ras) that exerts little effect on PKC-dependent MAPK activation by phorbol-12-myristate-13-acetate. The TRH-induced activation of MAPK also is inhibited partially by overexpression of transducin alpha subunits (alpha t), an agent known to sequester free G protein beta gamma dimers. However, the inhibitory potency of alpha t on TRH-induced activation is about half of that obtained in cells transfected with m2 muscarinic receptors, which activate MAPK exclusively through beta gamma dimers. The effect of alpha t is also additive with that of GF109203X but not with that of p21(N17ras). MAPK activation is not induced by the constitutively active form of G alpha q due to an inhibitory effect of its expression at a step downstream of that at which PKC-dependent and -independent routes to MAPK converge. Our results demonstrate that TRH receptors activate MAPK by a pathway only partially dependent on PKC activity. Furthermore, they indicate that beta gamma dimers of a pertussis and cholera toxin-insensitive G protein are involved in the PKC-independent fraction of the dual signaling route to MAPK initiated in the TRH receptor.

Animals↗
PubMed1996

Expression of OBCAM-related cDNA clones in Cos 1 cells: evidence for a phosphatidylinositol linkage to the cell membrane.

Previously, our laboratory purified and isolated the cDNA for OBCAM (opioid binding cell adhesion molecule) from bovine brain, as well as highly homologous rat brain cDNA clones, SG13 and DUZ-1. Structural similarities with members of the immunoglobulin superfamily suggest a possible role for OBCAM in cell adhesion and recognition, while studies in our own laboratory suggest that OBCAM is important in the regulation of opioid binding and signal transduction. However, OBCAM lacks a putative transmembrane domain, and its possible mode of linkage to the cellular membrane has not been studied. Upon transfection of Cos 1 cells with SG13 and DUZ-1 cDNAs, the OBCAM-homologous proteins were expressed on the surface of the Cos 1 cells. These proteins were released from the membrane of the Cos 1 cells upon digestion with phosphatidylinositol-specific phospholipase C (PI-PLC), demonstrating that they are linked to the membrane via a phosphatidylinositol (PI) linkage. These results are consistent with a role for OBCAM in cell recognition and adhesion, as well as in cellular signaling.

Animals↗
PubMed2006

Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells.

Nitric oxide (NO) may block apoptosis by inhibiting caspases via S-nitrosylation of cysteines. Here, we investigated whether effector caspases might cleave and thereby inhibit endothelial nitric oxide synthase (eNOS). Exposure of eNOS-transfected COS-7 cells and bovine aortic endothelial cells to staurosporine resulted in significant loss of 135-kDa eNOS protein and activity, and appearance of a 60-kDa eNOS fragment; effects were inhibited by the general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp[OMe]-fluoromethyl ketone (zVAD-fmk). In eNOS-transfected COS-7 cells, staurosporine-induced activation of caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage coincided with increased eNOS degradation and decreased activity. Loss of eNOS activity was greater than the degree of proteolysis. Incubation of immunoprecipitated eNOS with caspase-3, caspase-6 or caspase-7 resulted in eNOS cleavage. Staurosporine, a general protein kinase inhibitor, also reduced phosphorylation and decreased calmodulin binding, an effect that may explain the reduction in activity. eNOS, therefore, is both an inhibitor of apoptosis and a target of apoptosis-associated proteolysis.

Amino Acid Chloromethyl Ketones↗
PubMed1996

G alpha q family members couple parathyroid hormone (PTH)/PTH-related peptide and calcitonin receptors to phospholipase C in COS-7 cells.

The PTH/PTH-related peptide (PTHrP) receptor and the calcitonin receptor mediate the action of their physiological ligands by activating two different effectors, adenylyl cyclase and phospholipase C. Whereas regulation of adenylyl cyclase via both receptors is thought to involve the G protein G(s), it is not known whether activation of phospholipase C results from coupling of the receptors to G(q) family members or whether beta gamma-subunit released from receptor-activated G(s) lead to phospholipase C activation. To elucidate the mechanism of this type of dual signaling, we reconstituted the signal transduction of the PTH/PTHrP and the calcitonin receptor in COS-7 and HEK293 cells. In COS-7 cells expressing the receptor alone, addition of the respective ligands resulted in the accumulation of cAMP and inositol phosphates. When cells were cotransfected with the cDNAs of receptor and different alpha-subunits of the Gq family (G alpha q, G alpha 11, G alpha 14, G alpha 15, and G alpha 16, a severalfold increase in the ligand-dependent inositol phosphate production could be observed, indicating that the receptors functionally interacted with all alpha-subunits of the G alpha q family. Additionally, whereas PTH treatment of HEK293 cells coexpressing both the PTH/PTHrP receptor and G alpha q increased both second messengers, the same treatment in cells expressing the PTH/PTHrP receptor alone increased only cAMP. Under all conditions tested, activation of phospholipase C via the PTH/PTHrP and calcitonin receptor required higher ligand concentrations than receptor-mediated adenylyl cyclase activation. Our data strongly support the idea that dual signaling of the PTH/PTHrP and calcitonin receptors is due to the a activation of different G proteins belonging to the G(s) and G(q) families.

Adenylyl Cyclases↗
PubMed2004

[Cloning of human Nanog gene and its expression in COS-7L cells].

AIM: To clone human Nanog gene and express it in mammalian cells. METHODS: LA-PCR was used to amplify human Nanog gene from genomic DNA of HEK 293 cells. The Nanog gene was inserted into the Flag-tagged pCMV vector by gene recombination technique. After being confirmed by sequencing, the pFlag-Nanog was transfected into COS-7L cells. Western blot and indirect immunofluorescent staining against the Flag tag were used to detect the expression of Nanog protein. RESULTS: The sequence of the cloned Nanog was confirmed to be correct by sequence analysis. The COS-7L cells transfected with pFlag-Nanog could efficiently express Nanog protein. CONCLUSION: The cloning of human Nanog gene and the construction of its eukaryotic expression vector were successful. The study will lay the foundation for further research on the function of Nanog and its role in neural stem cells.

Animals↗