Similarities between the transferrin receptor proteins on human reticulocytes and human placentae

The transferrin receptor of the human reticulocyte was isolated by two different immunoaffinity procedures. These included indirect immunoprecipitation with a transferrin/anti-transferrin complex and direct immunoprecipitation with antiserum to purified transferrin receptor from placentae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the receptor isolated from reticulocytes reveals a polypeptide at Mr = 94,000 identical in molecular weight with that of the placenta. A radioimmunoassay using purified 125I-labeled transferrin receptor from placentae and antiserum to transferrin receptor fails to distinguish any immunological differences between the reticulocyte and placental forms of the protein. In addition, proteolytic digests of both of these polypeptides with Staphylococcus aureus protease show identical proteolytic patterns, indicating similar sequences.     

Regulation of HeLa cell transferrin receptors

HeLa cells were found to have a single class of non-interacting receptors specific for transferrin. Both apotransferrin and diferric transferrin competed equally with 125I-diferric transferrin for receptor binding. Transferrin binding was temperature-dependent and reversible. Binding of transferrin to cells exhibited a KD of 27 nM with a maximum binding capacity of 1.8-3.7 x 10(6) molecules/cell. Cells grown in the presence of diferric transferrin or in the presence of ferric ammonium citrate exhibited a concentration- and time-dependent decrease in 125I-diferric transferrin binding. The decrease in binding activity reflected a reduction in receptor number rather than an alteration in ligand receptor affinity. Growth of cells in saturating concentrations of apotransferrin did not cause a decrease in receptor number. When iron-treated cells were removed to media free of ferric ammonium citrate, the receptor number returned to control values by 40 h. When receptors were removed with trypsin, cells grown and maintained in ferric ammonium citrate-supplemented media demonstrated a rate of receptor reappearance 47% that of control cells grown in ferric ammonium citrate-free media. Cells grown in media supplemented with diferric transferrin or ferric ammonium citrate exhibited an increase in cytosolic iron content. The transferrin receptor number returned to normal after cells were removed to unsupplemented media, despite persistent elevation of cytosolic iron content. Increased iron content did not appear to be the sole factor determining receptor number.     

Complete inhibition of transferrin recycling by monensin in K562 cells

Monensin blocks human transferrin recycling in a dose-dependent and reversible manner in K562 cells, reaching 100% inhibition at a noncytocidal dose of 10(-5) M, whereas transferrin recycling is virtually unaffected by noncytocidal doses of chloroquine. The intracellular pathway of human transferrin in K562 cells, both in the presence and absence of 10(-5) M monensin, was localized by indirect immunofluorescence. Monensin blocks transferrin recycling by causing internalized ligand to accumulate in the perinuclear region of the cell. The effect of 10(-5) M monensin on human transferrin kinetics was quantitatively measured by radioimmunoassay and showed a positive correlation with immunofluorescent studies. Immunoelectron microscopic localization of human transferrin as it cycles through K562 cells reveals the appearance of perinuclear transferrin-positive multivesicular bodies within 3 min of internalization, with subsequent exocytic delivery of the ligand to the cell surface via transferrin-staining vesicles arising from these perinuclear structures within 5 min of internalization. Inhibition of ligand recycling with 10(-5) M monensin causes dilated transferrin-positive multivesicular bodies to accumulate within the cell with no evidence of recycling vesicles. A coordinated interaction between multivesicular bodies and the Golgi apparatus appears to be involved in the recycling of transferrin in K562 cells. Cell-surface-binding sites for transferrin were reduced by 50% with 10(-5) M monensin treatment; however, this effect was not attenuated by 80% protein synthesis inhibition with cycloheximide, supporting the idea that the transferrin receptor is also recycled through the Golgi.     

Mitogenic agents induce redistribution of transferrin receptors from internal pools to the cell surface.

Incubation of serum-growth HeLa cells in serum-free medium causes a rapid (t1/2 3 min) 30-60% decrease in the binding of 125I-diferric transferrin to the cell surface. Addition of fetal bovine serum to cells in serum-free medium results in a rapid (t1/2 3 min) and concentration-dependent increase in binding activity. The loss or gain in ligand binding is a result of changes in surface receptor number rather than an alteration in ligand-receptor affinity. A variety of hormones (insulin, insulin-like growth factor, interleukin 1 and platelet-derived factor) were found to mimic the effect of serum on receptor number. The alteration in surface receptor number was found to be calcium-dependent. Changes in surface receptor number were independent of either receptor biosynthetic rate or the absolute cellular content of receptors. The effect of insulin or serum on Hela cell transferrin receptor distribution was unaffected by the presence of transferrin, demonstrating that receptor distribution in this cell type is ligand-independent. The ability of serum or insulin to modify surface transferrin receptor number was also observed in mouse L-cells, human skin fibroblasts, and J774 macrophage tumour cells. However, transferrin receptors on K562 and Epstein-Barr virus-transformed human lymphoblasts were unaltered by these agents. The quantities of receptors whose distribution is predominantly on the surface (i.e. epidermal growth factor or low density lipoprotein receptor) were unaltered by addition of the mitogenic agents. These results extend our previous studies [H.S. Wiley & J. Kaplan (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7456-7460] demonstrating that mitogenic agents can induce redistribution of receptor pools in selected cell types.     

The role of iron in the growth of human leukemic cell lines

The growth requirements of three human leukemic cell lines (K 562, HEL, U937) have been studied in the absence of serum. For growth in serum-free medium, the cells require insulin, transferrin, and albumin. Two highly water-soluble iron salts, ferric ammonium citrate and ferric ammonium sulfate, may completely replace transferrin for supporting the growth of these cell lines. Similar results were obtained when mitogen-stimulated lymphocytes were grown in serum-free media. Iron containing compounds, such as hemin or hemoglobin, were also able to replace transferrin. Experiments using 42/6 monoclonal antibody strongly suggest that free-iron salts are taken up by the cells by a mechanism that is completely independent from transferrin-receptors.     

Transferrin receptor regulation is coupled to intracellular ferritin in proliferating and differentiating HL60 leukemia cells

Cultured myeloid leukemia cells display transferrin receptors but decrease receptor display after differentiation induction or accumulation of intracellular iron. To determine whether regulation of transferrin receptors and ferritin were linked under these disparate conditions, serum-free and fetal bovine serum (FBS) cultures of HL60 promyelocytic leukemia cells were used to investigate relationships between transferrin receptor display and intracellular ferritin. Using 125I-transferrin binding and immunofluorescence staining for transferrin receptors, HL60 cells cultured in serum-free, transferrin-free medium expressed fewer transferrin receptors and contained increased ferritin when compared to cells cultured with FBS or transferrin supplemented, serum-free medium. When placed in medium containing transferrin, cells previously grown in transferrin-free medium rapidly re-expressed transferrin receptors and decreased their ferritin content. HL60 cells induced to differentiate into granulocytes or macrophages also decreased transferrin receptor display and increased their ferritin content. Transferrin receptor display and ferritin content in both proliferating and differentiating myeloid leukemia cells are inversely related and their regulation is closely linked. Regulation of transferrin receptor display and ferritin synthesis may be important events regulating myeloid cell growth and differentiation.     

Characterization of transferrin receptor released by K562 erythroleukemia cells

A soluble form of transferrin receptor has been detected in human serum and has been shown recently to be a truncated form of the intact membrane bound receptor. Mechanisms governing the release of transferrin receptor by cells are poorly understood and could be better defined by tissue culture. The present investigation was undertaken to characterize the transferrin receptor released by K562 erythroleukemic cells. In contrast with maturing sheep reticulocytes, which have been shown to release transferrin receptor in small vesicles termed exosomes, we demonstrated, with a monoclonal enzyme-linked immunoassay, that less than 30% of the transferrin receptor released by K562 cells in log phase growth was in a particulate form. The relative amounts of soluble and particulate receptor released to the supernatant did not change significantly during 48 hr of incubation. Soluble receptor was purified by immunoaffinity chromatography. On polyacrylamide gel electrophoresis, its mobility was the same (85 kDa) as that of the truncated monomeric form recently identified in human serum. Further evidence that serum and soluble receptors released by K562 cells are identical was provided by amino acid sequence analysis, which demonstrated that 16 of the first 19 residues of the N-terminal sequence of soluble K562 receptor are homologous with the serum receptor. The remaining three were not identifiable. K562 cells provide a useful in vitro model for studying the production of membrane-bound and soluble forms of released transferrin receptor.     

Demonstration of two distinct transferrin receptor recycling pathways and transferrin-independent receptor internalization in K562 cells

The endocytosis and recycling of the human transferrin receptor were evaluated by several experimental modalities in K562 cells perturbed with 10(-5) M monensin. The work presented is an extension of a previous study demonstrating both complete inhibition of release of internalized human transferrin and a 50% reduction in the number of cell surface transferrin binding sites in K562 cells treated with monensin (Stein, B. S., Bensch, K. G., and Sussman, H. H. (1984) J. Biol. Chem. 259, 14762-14772). The data directly reveal the existence of two distinct transferrin receptor recycling pathways. One pathway is monensin-sensitive and is felt to represent recycling of transferrin receptors through the Golgi apparatus, and the other pathway is monensin-resistant and most likely represents non-Golgi-mediated transferrin receptor recycling. A transferrin-free K562 cell culture system was developed and used to demonstrate that cell surface transferrin receptors can be endocytosed without antecedent ligand binding, indicating that there are factors other than transferrin binding which regulate receptor internalization. Evidence is presented suggesting that two transferrin receptor recycling pathways are also operant in K562 cells under ligand-free conditions, signifying that trafficking of receptor into either recycling pathway is not highly ligand-dependent.     

Growth-stimulating effect of transferrin on a hybridoma cell line: Relation to transferrin iron-transporting function

The relation of the growth-stimulating capacity of transferrin to its iron-transporting function was investigated in mouse hybridoma PLV-01 cells cultivated in a chemically defined medium. The cells were precultivated in protein-free medium supplemented either with ferric citrate (cells with a high intracellular iron level) or with iron-saturated transferrin (cells with a low intracellular iron level). Iron uptake was monitored after the application of 59Fe-labeled ferric citrate or pig transferrin. Cultivation of the cells at the optimum growth-stimulating concentration (500 microM) of ferric citrate resulted in an intracellular iron level about 100-fold higher than that of cells cultivated at the optimum transferrin concentration (5 micrograms/ml). Replacement of pig transferrin with bovine transferrin resulted in similar intracellular iron levels, but the growth-stimulating effect of bovine transferrin was more than one order of magnitude lower. Cells with a high intracellular iron level grew equally well when cultivated with iron-saturated transferrin or with apotransferrin + deferoxamine (2 micrograms/ml). On the other hand, cells with a low intracellular iron level required iron-saturated transferrin for further growth and apotransferrin + deferoxamine was ineffective. The results suggest that transferrin can act as a cell growth factor only in the iron-saturated form. However, several findings of this work indicate that supplying cells with iron cannot be accepted as the full explanation of the transferrin growth-stimulating effect.     

Changes in glycosylation alter the affinity of the human transferrin receptor for its ligand

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.     

Identification of the D2--Dopamine Receptor Binding Subunit in Several Mammalian Tissues and Species by Photoaffinity Labeling

Photoaffinity labeling of the D2-dopamine receptor in plasma membrane preparations of various tissues from several mammalian species was performed using the recently developed D2-dopaminergic antagonist probe [125I]N-(p-azidophenethyl)spiperone ([125I]N3-NAPS). In tissues containing D2-receptors such as the corpus striatum from rat, dog, calf, hamster, guinea pig, and rabbit as well as the anterior pituitary of rat, bovine, and hamster, the probe covalently labels a peptide of Mr = 94,000. Specificity of the labeling is typically D2-dopaminergic in character. The covalent labeling is blocked by (+)-butaclamol but not by the inactive (-)isomer. Agonists block incorporation with the order of potency: N-n-propylnorapomorphine greater than apomorphine greater than dopamine. The D2-selective antagonist spiperone blocks labeling of the Mr = 94,000 peptide whereas the D1-selective antagonist SCH-23390 is ineffective. Thus, these results indicate that the ligand binding subunit of the D2-dopamine receptor resides on a Mr = 94,000 peptide in these various tissues from several species. Under conditions where proteolysis is not stringently controlled, peptides of lower Mr (32-38,000) are labeled at the expense of the Mr = 94,000 peptide. The most efficient protease inhibitor tested in these systems was EDTA, suggesting that the generation of these lower Mr receptor fragments might be the result of a metal-dependent proteolysis in the membrane preparations. In the rat neurointermediate lobe, a tissue containing D2-receptors, [125I]N3-NAPS specifically labels a major peptide of Mr approximately equal to 120,000 in addition to the Mr = 94,000 peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of growth of cultured hepatic epithelial cells by transferrin

Late-passage cells of a nontumorigenic and anchorage-dependent hepatic epithelial line (WB-F344), which produce insulinlike growth factor II and transforming growth factor beta constitutively, grow in serum-free medium supplemented only with transferrin. In the presence of transferrin, epidermal growth factor further augments population growth, although epidermal growth factor alone is without effect. Insulin, platelet-derived growth factor, and several inorganic iron salts are also ineffective in supporting cell growth in the absence of transferrin; furthermore, these factors do not augment the action of transferrin. The population growth-promoting effect of transferrin occurs at concentrations of 0.5 nM or greater and the maximal effect is reached with a concentration of approximately 6 nM. A lipophilic iron chelator, ferric pyridoxal isonicotinoyl hydrazone (FePIH), can fully mimic the effect of transferrin on the proliferation of WB-F344 cells, but the molar concentration of transferrin. These results suggest that the critical function of transferrin in the proliferation of WB-F344 cells may be in the delivery of iron to the cells. In the absence of transferrin the proliferation of WB-F344 cells is arrested in serum-free medium in the G0/G1 phase, and a period of protein synthesis after the addition of transferrin is necessary before the cells can proceed to S phase and initiate DNA synthesis. Replacement of transferrin causes quiescent WB-F344 cells to cycle parasynchronously. Epidermal growth factor does not alter the length of the latency period prior to S phase but appears to stimulate the uptake of [3H]thymidine subsequently. Transferrin may act as a "competence" and/or "progression" factor, allowing the replication of these epithelial cell in vitro.     

Stimulation of rat placental cell DNA synthesis by transferrin

The purpose of the present investigation was to evaluate the in vitro requirements for rat placental cell DNA synthesis. A cell line established from the labyrinth region of midgestation rat chorioallantoic placentas was used to examine the actions of various agents. Transferrin was found to stimulate rat placental cell DNA synthesis and cell proliferation. The effects of transferrin on rat placental cell growth paralleled those observed with fetal bovine serum. Rat placental cells were responsive to both rat and human transferrin. Iron-saturated (holo-) transferrin was a more potent stimulator of rat placental cell DNA synthesis than was iron-free (apo-) transferrin. Addition of insulin, epidermal growth factor, or insulin-like growth factor-II to serum-free medium supplemented with rat transferrin did not significantly enhance rat placental cell DNA synthesis beyond that observed with only transferrin. The results demonstrate that a population of cells exists within the rat chorioallantoic placenta that are highly responsive to transferrin.     

Human transferrin receptor contains O-linked oligosaccharides

We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked oligosaccharides of the receptor, we discovered that the receptor contains O-linked oligosaccharides. This report describes the isolation and characterization of these O-linked oligosaccharides. Three different human cell lines--K562, A431, and BeWo--were grown in media containing either [2-3H] mannose or [6-3H]glucosamine. The newly synthesized and radiolabeled transferrin receptors were purified by immunoprecipitation from cell extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor was proteolytically digested or treated directly with mild base/borohydride. The released radiolabeled glycopeptides and oligosaccharides were separated by a variety of chromatographic techniques, and their structures were analyzed. The transferrin receptor from all three cell types contains O-linked oligosaccharides that are released from peptide by mild base/borohydride treatment. The receptor from K562 cells contains at least one O-linked oligosaccharide having two sialic acid residues and a core structure of the disaccharide galactose-N-acetyl-galactosamine. In contrast, the O-linked oligosaccharides in the transferring receptors from both A431 and BeWo cell lines are not as highly sialylated and were identified as both the neutral disaccharide galactose-N-acetylgalactosamine and the neutral monosaccharide N-acetylgalactosamine. In addition, the receptors from all three cell lines contain both complex-type and high mannose-type N-linked oligosaccharides. The complex-type chains in the receptor from A431 cells have properties of blood group A antigens, whereas oligosaccharides in receptors from both BeWo and K562 cells lack these properties. These results are interesting since both A431 and BeWo cells, but not K562 cells, are positive for blood group A antigens. Thus, our results demonstrate that the human transferrin receptor contains O-linked oligosaccharides and that there are differences in the structures of both the O-linked and complex-type N-linked oligosaccharides on the receptors synthesized by different cell types.     

Increased glucocorticoid responsiveness of CD4+ T-cell clonal lines grown in serum-free media

Summary CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for 3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid hormone action may be pursued more precisely in a clearly defined culture medium. This work was conducted in conjunction with the Walls Medical Research Foundation.     

Comparison of the kinetics of cycling of the transferrin receptor in the presence or absence of bound diferric transferrin.

The kinetics of cycling of the transferrin receptor in A431 human epidermoid-carcinoma cells was examined in the presence or absence of bound diferric transferrin. In order to investigate the properties of the receptor in the absence of transferrin, the cells were maintained in defined medium without transferrin. It was demonstrated that Fab fragments of a monoclonal anti-(transferrin receptor) antibody (OKT9) did not alter the binding of diferric 125I-transferrin to the receptor or change the accumulation of [59Fe]diferric transferrin by cells. OKT9 125I-Fab fragments were prepared and used as a probe for the function of the receptor. The first-order rate constants for endocytosis (0.16 +/- 0.02 min-1) and exocytosis (0.056 +/- 0.003 min-1) were found to be significantly lower for control cells than the corresponding rate constants for endocytosis (0.22 +/- 0.02 min-1) and exocytosis (0.065 +/- 0.004 min-1) measured for cells incubated with 1 microM-diferric transferrin (mean +/- S.D., n = 3). The cycling of the transferrin receptor is therefore regulated by diferric transferrin via an increase in both the rate of endocytosis and exocytosis. Examination of the accumulation of OKT9 125I-Fab fragments indicated that diferric transferrin caused a marked decrease in the amount of internalized 125I-Fab fragments associated with the cells after 60 min of incubation at 37 degrees C. Diferric transferrin therefore increases the efficiency of the release of internalized 125I-Fab fragments compared with cells incubated without diferric transferrin. These data indicate that transferrin regulates the sorting of the transferrin receptor at the cell surface and within endosomal membrane compartments.     

Replacement of serum by insulin and transferrin supports growth and differentiation of the human promyelocytic cell line, HL-60

The human promyelocytic leukemia cell line HL-60 can be grown in serum-free synthetic medium supplemented with insulin and transferrin alone. Growth of HL-60 in this defined medium is at a rate approx. 80% of that in medium containing serum. Moreover, the distinct morphological and histochemical myeloid characteristics of HL-60 are maintained in such serum-free medium. The HL-60 promyelocytes are induced by DMSO to differentiate to mature granulocytes equally well in both serum-supplemented and serum-free medium. However, this defined medium does not support colony growth of HL-60 in semi-solid medium such as methylcellulose.     

Biosynthetic regulation of the human transferrin receptor by desferrioxamine in K562 cells

Treatment of K562 cells with the iron chelator desferrioxamine results in the gradual increase in total cell receptors for transferrin. Receptor number rises 2.5-4.5-fold over 24 h and remains at the elevated level if the chelator is continuously present. Preincubation of the chelator with ferric chloride abolishes the effect. The drug has no effect on the 7-h half-life of the receptor. The increased number of receptors can be accounted for by a specific increase in the rate of receptor biosynthesis which reaches 3-4 times that seen in untreated cells by 6 h after the addition of the chelator. Isolation of mRNA from treated cells reveals that, after 8 h in the presence of desferrioxamine, there is a 3-fold increase in the specific translation of transferrin receptor over untreated cells. Total protein synthesis is not changed under these conditions.     

Growth of human malignant lymphoid cell lines in serum-free medium

Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products.