共查询到20条相似文献,搜索用时 8 毫秒
1.
A L Schwartz S E Fridovich B B Knowles H F Lodish 《The Journal of biological chemistry》1981,256(17):8878-8881
The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells. 相似文献
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Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents 总被引:69,自引:0,他引:69
A Ciechanover A L Schwartz A Dautry-Varsat H F Lodish 《The Journal of biological chemistry》1983,258(16):9681-9689
Growing HepG2 cells contain 50,000 functional surface transferrin-binding sites (Ciechanover, A., Schwartz, A.L., and Lodish, H.F. (1983) Cell 32,267-275) and 100,000 intracellular sites. At saturating concentrations of [59Fe]transferrin, and under conditions in which protein synthesis is blocked, iron uptake is linear for several hours at a rate of 9,500 transferrin molecules/cell/min. Thus, each receptor must recycle a ligand, on the average, each 15.8 min. Surface-bound transferrin is rapidly endocytosed (t1/2 = 3.5 min). All of the iron remains within the cell, while the apotransferrin is rapidly (t1/2 = 5.0 min) secreted into the medium. Previously, we showed (Dautry-Varsat, A., Ciechanover, A., and Lodish, H.F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2258-2262) that exposure of a ferrotransferrin-receptor complex to medium of pH less than 5.0 results in dissociation of iron, but that apotransferrin remains bound to its receptor. If the pH is raised to 7.0, such as would occur when an acidic intracellular vesicle fuses with the plasma membrane, apotransferrin is very rapidly dissociated (t1/2 = 17 s at 37 degrees C) from its receptor. Taken together, these results indicate that transferrin remains bound to its receptor throughout the endocytic cycle. In the present study, we have directly measured all the kinetic parameters involved in the transferrin receptor cycle. They are similar to those of the asialoglycoprotein receptor in the same cell line, and can be described by a simple kinetic model. In the presence of lysosomotropic agents, ferrotransferrin binds to its surface receptor and is internalized normally. However, iron is not dissociated from transferrin, and ferrotransferrin recycles back to the cell surface and is secreted into the medium. We conclude that the low pH in endocytic vesicles is essential for the dissociation of iron from transferrin and its delivery to the cell, but is not required for recycling of transferrin, and presumably of its receptor. 相似文献
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Asialoglycoprotein receptor phosphorylation and receptor-mediated endocytosis in hepatoma cells. Effect of phorbol esters 总被引:6,自引:0,他引:6
The asialoglycoprotein (ASGP) receptor on Hep G2 cells undergoes constitutive recycling and ligand endocytosis in the presence of phorbol dibutyrate, at a 50% reduced rate relative to control cells (Fallon, R. J., and Schwartz, A. L. (1986) J. Biol. Chem. 261, 15081-15089). The relevance of receptor phosphorylation to these events was investigated by selective immunoprecipitation of surface receptors with polyclonal anti-human ASGP antiserum and pulse-chase labeling with [32P]orthophosphate to identify subcellular locations of initial receptor phosphorylation events as well as the eventual fate of phosphorylated receptor during recycling. The surface immunoprecipitation method recovers greater than 95% of surface ASGP receptors and only 5% or less of intracellular (brief[35S]methionine pulse-labeled) receptors. With this assay we detected low levels of ASGP receptor phosphorylation at the cell surface in control cells (0.1 mol of P/mol of R) which were rapidly (less than 1 min) stimulated 20-fold by 400 nM phorbol dibutyrate addition (1.7 mol of P/mol of R). Staurosporine, a protein kinase C inhibitor, blocks this stimulation by phorbol. Receptor phosphorylation at early time points in the presence of phorbol esters was restricted to the plasma membrane. Subsequent chase in the presence of excess unlabeled phosphate and phorbol esters lowered [32P] ATPi specific activity by 68% at 1 h. Surface immunoprecipitation during this chase period showed the phosphorylated ASGP receptors were rapidly lost from the cell surface (t1/2 = 20 min). In contrast, examination of intracellular receptor during the pulse-chase experiment in phorbol dibutyrate-treated cells showed the presence of phosphorylated pool(s) of ASGP receptors which were detectable for 6 h of chase. Since no labeled receptor can be detected at the cell surface at this time, the described intracellular phosphorylated receptors are in a non-recycling pool. 相似文献
6.
Phosphofructokinase 2 and glycolysis in HT29 human colon adenocarcinoma cell line. Regulation by insulin and phorbol esters.
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Kinetic properties of phosphofructokinase 2 (PFK2) and regulation of glycolysis by phorbol 12-myristate 13-acetate (PMA) and insulin were investigated in highly glycolytic HT29 colon cancer cells. PFK2 was found to be inhibited by citrate and, to a lesser extent, by phosphoenolpyruvate and ADP, but to be insensitive to inhibition by sn-glycerol phosphate. From these kinetic data, PFK2 from HT29 cells appears different from the liver form, but resembles somewhat the heart isoenzyme. Fructose 2,6-bisphosphate (Fru-2,6-P2) levels, glucose consumption and lactate production are increased in a dose-dependent manner in HT29 cells treated with PMA or insulin. The increase in Fru-2,6-P2 can be related to an increase in the Vmax. of PFK2, persisting after the enzyme has been precipitated with poly(ethylene glycol), without change in the Km for fructose 6-phosphate. The most striking effects of PMA and insulin on Fru-2,6-P2 production are observed after long-term treatment (24 h) and are abolished by actinomycin, cycloheximide and puromycin, suggesting that protein synthesis is involved. Furthermore, the effects of insulin and PMA on glucose consumption, lactate production, Fru-2,6-P2 levels and PFK2 activity are additive, and the effect of insulin on Fru-2,6-P2 production is not altered by pre-treatment of the cells with the phorbol ester. This suggests that these effects are exerted by separate mechanisms. 相似文献
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Targeting of immunological effector cells to tumor cells could be an efficient strategy of adoptive immunotherapy. The success of this strategy depends on the specificity of the effector cells and their availability in sufficient numbers. The aim of this study was to target the human natural killer cell line YT specifically to tumor cells. The cell line was modified by transfection with the cDNA of the human asialoglycoprotein receptor (ASGPR). This C-type lectin recognizes carbohydrates containing terminal galactosyl (Gal) residues, including the beta1-Gal bearing Thomsen-Friedenreich (TF) antigen, which is found on tumor cells. Binding assays revealed that the ASGPR-gene-transfected YT cell line binds significantly higher to tested target tumor cell lines than the mock-transfected control cells. Cytolytic activity against the tumor cell lines Raji, Jurkat and the TF-positive KG1 subline was increased. Genetic modification of YT cells could provide a useful tool for tumor targeting in immunotherapy. 相似文献
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Angiotensinogen is the precursor of biologically active peptide angiotensin II and its synthesis is increased in the liver during acute inflammation. We have used radiolabeled human angiotensinogen cDNA to study the effect of hepatocyte stimulating factor (HSF), a protein synthesized in differentiating monocytes which increases the synthesis of various hepatic proteins during inflammation, on angiotensinogen mRNA levels in human hepatoma cells (HepG2). Our results indicate that angiotensinogen mRNA is present in human hepatoma (HepG2) cells and its levels are decreased when treated with hepatocyte stimulating factor. Although dexamethasone elevated angiotensinogen mRNA levels, HSF reduced this increase. These results suggest that a factor other than HSF may be involved in elevating the angiotensinogen mRNA levels in the liver during inflammation. 相似文献
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Inhibition of insulin receptor binding by phorbol esters 总被引:16,自引:0,他引:16
P Thomopoulos U Testa M F Gourdin C Hervy M Titeux W Vainchenker 《European journal of biochemistry》1982,129(2):389-393
Phorbol esters inhibit the binding of insulin to its receptors on U-937 monocyte-like and HL-60 promyelocytic leukemia human cell lines. Within 20-30 min, exposure of these cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) at 37 degrees C results in a 50% reduction of the specific binding of 125I-insulin. Half-maximal inhibition occurs at 1 nM TPA. Other tumor-promoting phorbol esters also inhibit 125I-insulin binding in a dose-dependent manner which parallels their known promoting activity in vivo. TPA does not alter the degradation of the hormone nor does it induce any shedding of its receptors in the medium. The effect of phorbol esters is dependent on temperature and cell type. It is less prominent at 22 degrees C than at 37 degrees C. It is reversible within 2 h at 37 degrees C. TPA reduces the binding of insulin predominantly by increasing its dissociation rate. This effect results in an accelerated turnover of the hormone on its receptors. 相似文献
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Changes in glycosylation alter the affinity of the human transferrin receptor for its ligand 总被引:2,自引:0,他引:2
When transferrin receptors of human erythroleukemic cells were pulse-labeled with [35S]methionine and then chased in the absence of radioactive precursor, the first detectable immunoprecipitable form of the receptor had a molecular mass of 85 kDa. This form of the receptor was converted to the mature form of 93 kDa with a half-time of about 40-60 min. Both the immature (85 kDa) and mature (93 kDa) receptors associated as dimers, the native form of the receptor. The 85-kDa, as well as the 93-kDa, receptors bound to a monoclonal antibody raised against the transferrin receptor or to transferrin-Sepharose. In order to determine whether glycosylation was necessary for ligand binding, purified receptors were isolated from cells grown in the presence of tunicamycin. When K562 cells were grown in the presence of tunicamycin, an 80-kDa nonglycosylated form of the receptor was synthesized. This nonglycosylated receptor was also capable of dimer formation; however, much less of it reached the cell surface than the fully glycosylated form, although both untreated and tunicamycin-grown cells appeared to synthesize transferrin receptors at similar rates. Although the number of receptor molecules/cell was similar in control and tunicamycin-treated cells, the nonglycosylated receptors exhibited a much lower affinity for transferrin than those of untreated cells; in contrast, when receptors were purified by immunoprecipitation and digested with bacterial alkaline phosphatase, no difference was observed between the affinity of these receptors and undigested immunoprecipitated receptors. These results suggest that glycosylation is not necessary for specific binding of transferrin to its receptor, but the affinity of this binding can be influenced greatly by the presence or absence of carbohydrate residues. 相似文献
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In human beings, serum transferrin levels increase during iron deficiency and decrease with iron overload. Yet, whether or not iron levels actually affect the synthesis of transferrin in human liver cells is not known. In previous studies, iron was shown to suppress the expression of chimeric human transferrin genes in livers of transgenic mice. The goal of this study was to determine if iron suppresses intact endogenous human transferrin synthesis by testing the effects of changes in iron levels on synthesis of transferrin in a human hepatoma cell line HepG2. In HepG2 cells, normalized(35)S-metabolically labeled transferrin synthesis was consistently less following iron treatment with hemin or ferric citrate, than following treatment with an iron-chelator deferroxamine. Thus, this study provides new evidence that iron can regulate synthesis of intact endogenous human transferrin. 相似文献
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Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line 总被引:31,自引:19,他引:12
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Acidification of endocytic vesicles has been implicated as a necessary step in various processes including receptor recycling, virus penetration, and the entry of diphtheria toxin into cells. However, there have been few accurate pH measurements in morphologically and biochemically defined endocytic compartments. In this paper, we show that prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of approximately 5.4. (We previously reported that similar vesicles in mouse fibroblasts have a pH of 5.0.) The pH values were obtained from the fluorescence excitation profile after internalization of fluorescein labeled asialo-orosomucoid (ASOR). To make fluorescence measurements against the high autofluorescence background, we developed digital image analysis methods for estimating the pH within individual endocytic vesicles or lysosomes. Ultrastructural localization with colloidal gold ASOR demonstrated that the pH measurements were made when ligand was in tubulovesicular structures lacking acid phosphatase activity. Biochemical studies with 125I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37 degrees C. At 23 degrees C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue. These results demonstrate that acidification of endocytic vesicles, which causes ligand dissociation, occurs without fusion of endocytic vesicles with lysosomes. Methylamine and monensin raise the pH of endocytic vesicles and cause a ligand-independent loss of receptors. The effects on endocytic vesicle pH are rapidly reversible upon removal of the perturbant, but the effects on cell surface receptors are slowly reversible with methylamine and essentially irreversible with monensin. This suggests that monensin can block receptor recycling at a highly sensitive step beyond the acidification of endocytic vesicles. Taken together with other direct and indirect estimates of endocytic vesicle pH, these studies indicate that endocytic vesicles in many cell types rapidly acidify below pH 5.5, a pH sufficiently acidic to allow receptor-ligand dissociation and the penetration of some toxin chains and enveloped virus nucleocapsids into the cytoplasm. 相似文献
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Treatment of H4 hepatoma cells with the lectin wheat germ agglutinin (WGA) in the concentration range of 10-25 micrograms/ml increased 125I-insulin binding fivefold as compared to control binding in untreated cells. The increased insulin binding was rapid, readily reversible, and correlated with a 10-fold increase in the binding affinity of the receptor for insulin. Kinetic studies indicate that this increased affinity resulted from a decrease in the dissociation rate. The effect was specifically mediated by the lectin since it was reversed by simultaneous incubation with the monosaccharide N-acetyl-D-glucosamine (50 mM) or the disaccharide N,N'-diacetylchitobiose (1 mM). The WGA-mediated increase in insulin binding was not caused by inhibited insulin degradation. While WGA (5 micrograms/ml) mimicked insulin to induce stimulated uptake of [3H]aminoisobutyrate, the lectin failed to enhance the biological sensitivity of H4 hepatoma cells to insulin. At higher concentrations of WGA (125 micrograms/ml), interference with the insulin-mediated response was observed. Trypsin treatment of H4 hepatoma cells prior to measuring binding of 125I-insulin in the presence of increasing concentrations of native insulin, led to a leftward shift of the competition curve, indicating an increased affinity of the receptor. No further increase was observed when the trypsin-treated cells were subsequently exposed to WGA. These results suggest that trypsin treatment and WGA exposure may increase the affinity of the receptor by a similar mechanism. The results are consistent with the concept that WGA and trypsin decrease interaction between insulin binding and receptor affinity regulating components in the plasma membrane, leading to an increase in the affinity of the receptor for insulin. 相似文献
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Andersen PS Geisler C Buus S Mariuzza RA Karjalainen K 《The Journal of biological chemistry》2001,276(36):33452-33457
Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3 (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency of the SEC3 variants correlated with enhanced binding without any optimum in the binding range covered by native TCR ligands. Comparable studies using anti-TCR antibodies of known affinity confirmed these observations. By comparing the biological potency of the two sets of ligands, we found a significant correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength. 相似文献
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The rat liver asialoglycoprotein receptor consists of two typesof subunits, a predominant polypeptide designated rat hepaticlectin 1 (RHL-1) and a minor polypeptide, RHL-2/3, that comesin two differentially glycosylated forms. The exact stoichiometryand arrangement of the subunits in the RHL oligomer are notknown. The carbohydrate-recognition domain of RHL-2/ has beenprepared by limited proteolysis of the liver receptor so thatits properties can be compared with those of the correspondingdomain of RHL-1 previously produced in a bacterial expressionsystem. Binding studies indicate that while RHL-1 binds N-acetylgalactosaminewith approximately 60-fold higher affinity than it binds galactose,RHL-2/ has only 2-fold selectivity for N-acetylgalactosamine.In general, the pattern of monosaccharide-binding specificityfor RHL-2/ is similar to RHL-1, but the discrimination of varioussugars relative to galactose is reduced substantially. Limitedproteolysis and crosslinking studies demonstrate that RHL- 2/is easily removed from the RHL oligomer in detergent solutionand that RHL-1 remains at least trimeric following removal ofRHL-2/. These studies suggest that RHL-1 forms a ligand-bindingcore while RHL-2/ acts more as an accessory subunit contributingto selective binding of certain oligosaccharide structures. asialoglycoprotein receptor binding carbohydrate recognition lectin proteolysis 相似文献
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Phosphorylation of histones is stimulated by phorbol esters in quiescent Reuber H35 hepatoma cells 总被引:5,自引:0,他引:5
Histones isolated from Reuber H35 rat hepatoma cells treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined for possible alterations in phosphorylation. Incorporation of 32P orthophosphate into individual acid-extracted histones was monitored by autoradiography and scintillation counting of polyacrylamide gels or by reverse-phase high performance liquid chromatography. Treatment of quiescent H35 cells (arrested by serum starvation) with submicromolar doses of TPA resulted in a rapid and specific increase in phosphorylation of histones H2B and H1(0). Smaller increases in phosphorylation were observed for H4. No significant change in phosphorylation of the major H1 histones or H2A were observed after 1 h of treatment. The phosphorylation was TPA dose-dependent, with a maximum increase of approximately 14-fold for H2B, 11-fold for H1(0), and 2-fold for H4 achieved at 0.8 M TPA. The nonpromoting parent compound phorbol did not induce any of these changes. Furthermore, the mitogenic hormone insulin did not cause a similar pattern of histone phosphorylation, suggesting that the effect observed was not due to a general mitogenic response in the H35 hepatoma cells. Addition of 8-Br-cAMP also failed to reproduce the effect of TPA on histone phosphorylation, suggesting that cAMP-dependent protein kinases are not likely to be involved in mediating this response to TPA. 相似文献
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N Berry K Ase U Kikkawa A Kishimoto Y Nishizuka 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(5):1407-1413
Activation of protein kinase C (PKC), by the phorbol ester PMA, or the membrane-permeable diacylglycerol 1-oleoyl 2-acetylglycerol (OAG), had different effects on the proliferation-associated responses of a more than 99% pure population of human T cells. Treatment with PMA or OAG caused down-regulation of the TCR-CD3 complex, but only PMA, in combination with ionomycin, was capable of stimulating IL-2R expression and proliferation. Immunocytochemical staining with antisera specific for the PKC subspecies alpha, beta I, beta II, and gamma showed that untreated resting T cells normally coexpress alpha, beta I, and beta II PKC subspecies, which are distributed diffusely throughout the cell, with some localization around the periphery of the nucleus. There was no difference between the responses of these PKC subspecies to OAG and PMA, redistributing, after 10 min of treatment, to a discrete focal area within the cell. Treatment with OAG resulted in transient redistribution of PKC, maximal at 10 min, while in PMA-stimulated cells, the PKC redistribution was prolonged, persisting for at least 24 h. The results suggest that the difference in cellular response to treatment with PMA and OAG is not a consequence of differential activation of various PKC subspecies. 相似文献