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.     

Effect of pH and iron content of transferrin on its binding to reticulocyte receptors

The effect of pH on the binding of apotransferrin and diferric transferrin to reticulocyte membrane receptors was investigated using rabbit transferrin and rabbit reticulocyte ghosts, intact cells and a detergent-solubilized extract of reticulocyte membranes. The studies were performed within the pH range 4.5–8.0. The binding of apotransferrin to ghosts and membrane extracts and its uptake by intact reticulocytes was high at pH levels below 6.5 but decreased to very low values as the pH was raised above 6.5. By contrast, diferric transferrin showed a high level of binding and uptake between pH 7.0 and 8.0 in addition to binding only slightly less than did apotransferrin at pH values below 6.5. It is proposed that the high affinity of apotransferrin for its receptor at lower pH values and low affinity at pH 7.0 or above allow transferrin to remain bound to the receptor when it is within acidic intracellular vesicles, even after loss of its iron, but also allow ready release from the cell membrane when it is exteriorized by exocytosis after iron uptake. The binding of transferrin to the receptor throughout the endocytosis-exocytosis cycle may protect it from proteolytic breakdown and aid in its recycling to the outer cell membrane     

The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes.

Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 X 10(6)M-1, for monoferric (C-terminal iron) 2.5 X 10(7)M-1, for monoferric (N-terminal iron) 2.8 X 10(7)M-1 and for diferric transferrin, 1.1 X 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin.     

Uptake of ferritin and iron bound to ferritin by rat hepatocytes: modulation by apotransferrin, iron chelators and chloroquine

Rat liver ferritin is an effective donor of iron to rat hepatocytes. Uptake of iron from ferritin by the cells is partially inhibited by including apotransferrin in the culture medium, but not by inclusion of diferric transferrin. This inhibition is dependent on the concentration of apotransferrin, with a 30% depression in iron incorporation in the cells detected at apotransferrin concentrations above 40 micrograms/ml. However, apotransferrin does not interfere with uptake of 125I-labeled ferritin, suggesting that apotransferrin decreases retention of iron taken up from ferritin by hepatocytes by sequestering a portion of released iron before it has entered the metabolic pathway of the cells. The iron chelators desferrioxamine (100 microM), citrate (10 mM) and diethylenetriaminepentaacetate (100 microM) reduce iron uptake by the cells by 35, 25 and 8%, respectively. In contrast, 1 mM ascorbate increases iron accumulation by 20%. At a subtoxic concentration of 100 microM, chloroquine depresses ferritin and iron uptake by hepatocytes by more than 50% after 3 h incubation. Chloroquine presumably acts by retarding lysosomal degradation of ferritin and recycling of ferritin receptors.     

Release of iron from the two iron-binding sites of transferrin by cultured human cells: modulation by methylamine

We have investigated the effect of increasing concentrations of methylamine (5, 10, and 25 mM) on the removal of iron from the two iron-binding sites of transferrin during endocytosis by human erythroleukemia (K562) cells. The molecular forms of transferrin released from the cells were analyzed by polyacrylamide gel electrophoresis in 6 M urea. Endocytosis of diferric transferrin was efficient since greater than 10% of surface-bound protein escaped endocytosis and was released in the diferric form. Although transferrin exocytosed from control cells had been depleted of 80% of its iron and contained 65-70% apotransferrin, iron-bearing species were also released (15% C-terminal monoferric; 10% N-terminal; 10% diferric). The ratio of the two monoferric species (C/N) was 1.32 +/- 0.12 (mean +/- SD; n = 4), suggesting that iron in the N-terminal site was more accessible to cells. In the presence of methylamine there was a concentration-dependent increase in the proportion of diferric transferrin release (less than 80% at 25 mM) and a concomitant decrease in apotransferrin. Small amounts of the iron-depleted species, especially apotransferrin, appeared before diferric transferrin, suggesting that these were preferentially released from the cells. The discrepancy between the proportions of the monoferric transferrin species noted with control cells was enhanced at all concentrations of methylamine, most markedly at 10 mM when the C/N ratio was 2.4. The N-terminal site of transferrin loses its iron at a higher pH than the C-terminal site, and so by progressively perturbing the pH of the endocytic vesicle we have increased the difference between the two sites observed with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium chelators induce association with the detergent-insoluble cytoskeleton and functional inactivation of the transferrin receptor in reticulocytes

Incubation of reticulocytes with EDTA, EGTA (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), but not with desferrioxamine B, at temperatures above 20 degrees C resulted in the loss of their ability to take up iron in a temperature-, time- and concentration-dependent manner. No inhibition of transferrin or iron uptake occurred if the incubations were performed at 20 degrees C or below. At higher temperatures, the inhibition was attributable to loss of functional transferrin receptors, not to altered affinity or endocytosis of the remaining receptors. The changes could not be reversed by washing the cells and reincubation in the presence of Ca2+, Mg2+ or Zn2+. However, they could be completely prevented by performing the initial incubation with chelators in the presence of diferric transferrin and partly prevented by the use of apotransferrin. Incubation with the chelators resulted in much less reduction in the ability of the cells to bind anti-transferrin receptor immunoglobulin than transferrin. The fate of the receptor was studied by polyacrylamide gel electrophoresis of reticulocyte membrane proteins before and after extraction with Triton X-100, and by immunological staining of Western blots for the transferrin receptor. Treatment of the cells with EDTA led to a loss of the ability of Triton X-100 to solubilize the receptor and its retention in the Triton-insoluble cytoskeletal matrix of the cells. It is concluded that incubation of reticulocytes with the chelators at temperatures above 20 degrees C causes an altered interaction of the transferrin receptor with the cytoskeleton. This change, which is probably due to chelation of Ca2+ in the cell membrane, is accompanied by an irreversible loss of the receptor's ability to bind transferrin.     

Effect of iron chelators on the transferrin receptor in K562 cells

Delivery of iron to K562 cells by diferric transferrin involves a cycle of binding to surface receptors, internalization into an acidic compartment, transfer of iron to ferritin, and release of apotransferrin from the cell. To evaluate potential feedback effects of iron on this system, we exposed cells to iron chelators and monitored the activity of the transferrin receptor. In the present study, we found that chelation of extracellular iron by the hydrophilic chelators desferrioxamine B, diethylenetriaminepentaacetic acid, or apolactoferrin enhanced the release from the cells of previously internalized 125I-transferrin. Presaturation of these compounds with iron blocked this effect. These chelators did not affect the uptake of iron from transferrin. In contrast, the hydrophobic chelator 2,2-bipyridine, which partitions into cell membranes, completely blocked iron uptake by chelating the iron during its transfer across the membrane. The 2,2-bipyridine did not, however, enhance the release of 125I-transferrin from the cells, indicating that extracellular iron chelation is the key to this effect. Desferrioxamine, unlike the other hydrophilic chelators, can enter the cell and chelate an intracellular pool of iron. This produced a parallel increase in surface and intracellular transferrin receptors, reaching 2-fold at 24 h and 3-fold at 48 h. This increase in receptor number required ongoing protein synthesis and could be blocked by cycloheximide. Diethylenetriaminepentaacetic acid or desferrioxamine presaturated with iron did not induce new transferrin receptors. The new receptors were functionally active and produced an increase in 59Fe uptake from 59Fe-transferrin. We conclude that the transferrin receptor in the K562 cell is regulated in part by chelatable iron: chelation of extracellular iron enhances the release of apotransferrin from the cell, while chelation of an intracellular iron pool results in the biosynthesis of new receptors.     

The role of transferrin in heme transport.

Porphyrin accumulation by proliferating cells, e.g., those associated with cancers or wounds, tends to correlate with increased transferrin receptor density. To determine whether transferrin might be implicated in porphyrin transport, fluorescence and absorption spectroscopy were used to study the interaction of porphyrins with transferrin. A single high-affinity binding site for heme and other porphyrins (Kd approximately 20-25 nM) was detected by fluorescence spectroscopy. Difference spectroscopy revealed three additional heme-binding sites. These sites were distinct from the iron-binding sites: 1) Apotransferrin and diferric transferrin bound porphyrins with equal affinity; 2) 59Fe was not displaced from transferrin by porphyrins. Murine erythroleukemia cells incubated with [59Fe]hemin-[125I]transferrin internalized both labels concomitantly. Accumulation of [59Fe]hemin could be blocked by a 100-fold excess of diferric transferrin but not by apotransferrin. These results indicate that cells can internalize exogenous heme, and possibly porphyrins, bound to transferrin via its receptor.     

The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes

The endocytosis of diferric transferrin and accumulation of its iron by freshly isolated rabbit reticulocytes was studied using 59Fe-125I-transferrin. Internalized transferrin was distinguished from surface-bound transferrin by its resistance to release during treatment with Pronase at 4 degrees C. Endocytosis of diferric transferrin occurs at the same rate as exocytosis of apotransferrin, the rate constants being 0.08 min-1 at 22 degrees C, 0.19 min-1 at 30 degrees C, and 0.45 min-1 at 37 degrees C. At 37 degrees C, the maximum rate of transferrin endocytosis by reticulocytes is approximately 500 molecules/cell/s. The recycling time for transferrin bound to its receptor is about 3 min at this temperature. Neither transferrin nor its receptor is degraded during the intracellular passage. When a steady state has been reached between endocytosis and exocytosis of the ligand, about 90% of the total cell-bound transferrin is internal. Endocytosis of transferrin was found to be negligible below 10 degrees C. From 10 to 39 degrees C, the effect of temperature on the rate of endocytosis is biphasic, the rate increasing sharply above 26 degrees C. Over the temperature range 12-26 degrees C, the apparent activation energy for transferrin endocytosis is 33.0 +/- 2.7 kcal/mol, whereas from 26-39 degrees C the activation energy is considerably lower, at 12.3 +/- 1.6 kcal/mol. Reticulocytes accumulate iron atoms from diferric transferrin at twice the rate at which transferrin molecules are internalized, implying that iron enters the cell while still bound to transferrin. The activation energies for iron accumulation from transferrin are similar to those of endocytosis of transferrin. This study provides further evidence that transferrin-iron enters the cell by receptor-mediated endocytosis and that iron release occurs within the cell.     

Transferrin receptors and transferrin iron uptake by cultured human blood monocytes

Transferrin receptors have been previously found on human macrophages and it has also been shown that transferrin iron is taken up by these cells. It has therefore been inferred that the uptake is receptor mediated and involves an endocytic pathway. The subject was addressed directly in the present study in which the transferrin-iron-receptor interaction was characterized in cultured human blood monocytes. Specific, saturable diferric transferrin binding was demonstrated, with a kDa of 3.6 X 10(-8) M and a calculated receptor density of 1.25-2.5 X 10(5) receptors per cell. Incubation at 4 degrees C markedly reduced transferrin binding and completely inhibited iron uptake. Chase experiments confirmed progressive cellular loading of iron, with concomitant loss of transferrin. Inhibitors of endocytic vesicle acidification (ammonium chloride and 2,4-dinitrophenol) inhibited iron unloading from endocytosed diferric transferrin, while microtubular inhibitors (colchicine and vindesine) and a microfilament inhibitor (cytochalasin B) reduced diferric transferrin uptake but had little effect on the iron unloading pathway. A similar effect was noted with a calcium ion antagonist (verapamil) and with 2 calmodulin antagonists (chlorpromazine and imipramine). These latter findings suggest the importance of cytoskeleton-membrane interactions via a calcium, calmodulin and protein kinase C mediated system. Endocytosed iron accumulated progressively as ferritin within the cultured monocytes.     

A variant of human transferrin with abnormal properties.

Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.     

Evidence for the functional equivalence of the iron-binding sites of rat transferrin

The role of the two iron-binding sites of rat transferrin in the exchange of iron with cells has been assessed using urea polyacrylamide gel electrophoresis to separate and quantitate the four possible molecular species of transferrin generated during the incubation of ¹²⁵I-labelled transferrin with rat reticulocytes and hepatocytes. Addition of diferric transferrin to reticulocytes led directly to the appearance of apotransferrin together with small and comparable amounts of the two monoferric transferrins. After 2 h 44.8% of the iron had been removed by the cells, and of the iron-depleted transferrin 71.8% was apotransferrin, the remainder being monoferric transferrin, 16.1% with N-terminal iron and 12.1% with C-terminal iron. A similar pattern emerged with hepatocytes, but the rate of iron removal was slower and the proportion of apotransferrin generated was lower. After 4 h 10.9% of the iron had been removed from the transferrin and the distribution of the iron-depleted protein was: apotransferrin 26.9% and monoferric (N-terminal) 39.2%, (C-terminal) 33.9%. The appearance of apotransferrin during each incubation and the generation of both monoferric transferrins suggest that both cell types are able to remove iron from differic transferrin in pairwise fashion and that they do not appreciably distinguish between the two iron-binding sites of the protein. Release of iron from hepatocytes to apotransferrin lead to the appearance of both monferric species and then to increasing amounts of diferric transferrin. The process of iron release did not seem to distinguish between the vacant iron-binding sites of transferrin.     

NADH diferric transferrin reductase in liver plasma membrane

Evidence is presented that rat liver plasma membranes contain a distinct NADH diferric transferrin reductase. Three different assay procedures for demonstration of the activity are described. The enzyme activity is highest in isolated plasma membrane, and activity in other internal membranes is one-eighth or less than in plasma membrane. The activity is inhibited by apotransferrin and antitransferrin antibodies. Trypsin treatment of the membranes leads to rapid loss of the transferrin reductase activity as compared with NADH ferricyanide reductase activity. Erythrocyte plasma membranes, which lack transferrin receptors, show no diferric transferrin reductase activity, although NADH ferricyanide reductase is present. The transferrin reductase is inhibited by agents that inhibit diferric transferrin reduction by intact cells and is activated by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate) detergent. Inhibitors of mitochondrial electron transport have no effect on the activity. We propose that the NADH diferric transferrin reductase in plasma membranes measures the activity of the enzyme that causes the reduction of diferric transferrin by intact cells. This transmembrane electron transport system requires the transferrin receptor for diferric transferrin reduction. Because the transmembrane electron transport has been shown to stimulate cell growth, the reduction of diferric transferrin at the cell surface may be an important function for diferric transferrin in stimulation of cell growth, in addition to its role in iron transport.     

Regulation of ferritin and transferrin receptor mRNAs

Iron regulates the synthesis of two proteins critical for iron metabolism, ferritin and the transferrin receptor, through novel mRNA/protein interactions. The mRNA regulatory sequence (iron-responsive element (IRE)) occurs in the 5'-untranslated region of all ferritin mRNAs and is repeated as five variations in the 3'-untranslated region of transferrin receptor mRNA. When iron is in excess, ferritin synthesis and iron storage increase. At the same time, transferrin receptor synthesis and iron uptake decrease. Location of the common IRE regulatory sequence in different noncoding regions of the two mRNAs may explain how iron can have opposite metabolic effects; when the IRE is in the 5'-untranslated region of ferritin mRNA, translation is enhanced by excess iron whereas the presence of the IREs in the 3'-untranslated region of the transferrin receptor mRNA leads to iron-dependent degradation. How and where iron actually acts is not yet known. A soluble 90-kDa regulatory protein which has been recently purified to homogeneity from liver and red cells specifically blocks translation of ferritin mRNA and binds IRE sequences but does not appear to be an iron-binding protein. The protein is the first specific eukaryotic mRNA regulator identified and confirms predictions 20 years old. Concerted regulation by iron of ferritin and transferrin receptor mRNAs may also define a more general strategy for using common mRNA sequences to coordinate the synthesis of metabolically related proteins.     

The effect of iron binding on the conformation of transferrin. A small angle x-ray scattering study.

Distance distribution functions, p(r), radii of gyration, Rg, and radii of gyration of cross section, Rq, of apotransferrin, monoferric transferrin, and diferric transferrin have been compared. The alteration of Rg and Rq upon iron binding has been determined by a difference method. An unusual feature of the stepwise structural changes of transferrin upon iron saturation is that binding of the first ferric ion is responsible for more than half of the whole change in Rq, whereas Rg alters significantly only after the binding of the second ferric ion.     

Interaction of gastrin with transferrin: effects of ferric ions

The sedimentation behavior of 125I-labeled gastrin has been studied as a function of Fe3+ ion concentration and pH. Both sedimentation velocity and sedimentation equilibrium experiments indicated that high-molecular-weight Fe3+-gastrin complexes were formed at pH 5.0 and pH 7.4. Self-association of gastrin alone was observed at pH values below 5.0. 125I-labeled gastrin bound to human serum apotransferrin at pH 7.4. Scatchard analysis of the gastrin-apotransferrin complex gave a Kd of approximately 6.4 microM at 37 degrees C, with two binding sites per molecule of apotransferrin. No significant binding of gastrin to diferric transferrin was observed under the same conditions. The binding of gastrin to apotransferrin was inhibited by NaCl. The results are consistent with the hypothesis that gastrin and transferrin act synergistically in the uptake of dietary iron by the gastrointestinal tract.     

The interaction of transferin with isolated hepatocytes

Freshly isolated rat heptocytes display about 36 700 high-affinity sites to which deferric transferrin may bind with an apparent association constant of 1.62·10⁷ 1·mol^?1.Uptake of iron from diferric transferrin by hepatocytes is linear with time and is accelerated at increased differric transferrin concentrations.Apotransferrin is able to decrease net iron uptake by hepatocytes from diferric transferrin by a process not dependent on the apotransferrin concentrations used or on the rate at which the cells take up iron. Immunoprecipitation of the apotransferrin during these incubations indicates that iron is being released from the cells to apotransferrin at the same time as iron is being taken up from diferric transferrin. The simultaneous uptake and release of iron, and the insensitivity to apotransferrin concentration, suggest that the processes of iron uptake and release occur via separate mechanisms. The effect of apotransferrin on net retention of iron may be one way in which the in vivo distribution of iron between sites of storage and utilization is controlled.     

Inhibition of transplasma membrane electron transport by monoclonal antibodies to the transferrin receptor

Reduction of iron in diferric transferrin is inhibited by monoclonal antibodies to the transferrin receptor which bind at sites other than the high affinity transferrin binding site. These antibodies include B3/25, GB16 and GB22. Two antibodies which bind at the high affinity site for transferrin, 42/6 and GB18, do not inhibit iron reduction by transplasma membrane electron transport. The results are consistent with the proposal that differric transferrin reduction or stimulation of transmembrane NADH oxidase activity involves a site different from the high affinity diferric transferrin binding site. A synergistic action of antibodies with epitopes at the tight binding site involved in iron uptake and the antibodies which inhibit electron transport, B3/25 and GB16, can explain the increased inhibition of growth observed when both 42/6 and B3/25 are added to proliferating cells.     

Preferential utilization in vitro of iron bound to diferric transferrin by rabbit reticulocytes.

59Fe uptake by rabbit reticulocytes from human transferrin-bound iron was studied by using transferrin solutions (35, 50, 65, 80 and 100% saturated with iron) whose only common characteristic was their content of diferric transferrin. During the early incubation period, 59Fe uptake from each preparation by reticulocytes was identical despite wide variations in amounts of total transferrin, total iron, monoferric transferrin and apotransferrin in solution. During the later phase of incubation, rate of uptake declined and was proportional to each solution's monoferric transferrin content. Uptake was also studied in a comparative experiment which used two identical, partially saturated transferrin preparations, one uniformly 59Fe-labelled and the other tracer-labelled with [59Fe]diferric transferrin. In both experiments, iron uptake by reticulocytes corresponded to utilization of a ferric ion from diferric transferrin before utilization of iron from monoferric transferrin.     

Iron uptake and heme synthesis by isolated rat liver mitochondria. Diferric transferrin as iron donor and the effect of pyrophosphate

Isolated rat liver mitochondria accumulate iron from fully saturated transferrin at neutral pH. With 5 microM iron as diferric transferrin, accumulation at 30 degrees C amounts to approx. 40 pmol/mg protein per h. With access to a suitable porphyrin substrate, 70-80% of the amount of iron accumulated is recovered in heme. Mobilization of iron and synthesis of heme both depend on a functioning respiratory chain. Vacant iron-binding sites on mono- and apotransferrin compete with the mitochondria for iron mobilized from transferrin. Pyrophosphate at concentrations in the range 10-50 microM enhances mobilization of iron, counterbalances the inhibitory effect of mono- and apotransferrin and enhances metallochelatase activity. The results emphasize the putative suitability of pyrophosphate as an intracellular iron-transport ligand in situ.