Characterization of the Interaction between Diferric Transferrin and Transferrin Receptor 2 by Functional Assays and Atomic Force Microscopy

Transferrin receptor 2 (TfR2), a homologue of the classical transferrin receptor 1 (TfR1), is found in two isoforms, α and β. Like TfR1, TfR2α is a type II membrane protein, but the β form lacks transmembrane portions and therefore is likely to be an intracellular protein. To investigate the functional properties of TfR2α, we expressed the protein with FLAG tagging in transferrin-receptor-deficient Chinese hamster ovary cells. The association constant for the binding of diferric transferrin (Tf) to TfR2α is 5.6 × 10⁶ M^− 1, which is about 50 times lower than that for the binding of Tf to TfR1, with correspondingly reduced rates of iron uptake. Evidence for Tf internalization and recycling via TfR2α without degradation, as in the TfR1 pathway, was also found. The interaction of TfR2α with Tf was further investigated using atomic force microscopy, a powerful tool used for investigating the interaction between a ligand and its receptor at the single-molecule level on the living cell surface. Dynamic force microscopy reveals a difference in the interactions of Tf with TfR2α and TfR1, with Tf-TfR1 unbinding characterized by two energy barriers, while only one is present for Tf-TfR2. We speculate that this difference may reflect Tf binding to TfR2α by a single lobe, whereas two lobes of Tf participate in binding to TfR1. The difference in the binding properties of Tf to TfR1 and TfR2α may help account for the different physiological roles of the two receptors.     

Transferrin receptor 2 mediates a biphasic pattern of transferrin uptake associated with ligand delivery to multivesicular bodies

The physiological role of transferrin (Tf) receptor 2 (TfR2), a homolog of the well-characterized TfR1, is unclear. Mutations in TfR2 result in hemochromatosis, indicating that this receptor has a unique role in iron metabolism. We report that HepG2 cells, which endogenously express TfR2, display a biphasic pattern of Tf uptake when presented with ligand concentrations up to 2 µM. The apparently nonsaturating pathway of Tf endocytosis resembles TfR1-independent Tf uptake, a process previously characterized in some liver cell types. Exogenous expression of TfR2 but not TfR1 induces a similar biphasic pattern of Tf uptake in HeLa cells, supporting a role for TfR2 in this process. Immunoelectron microscopy reveals that while Tf, TfR1, and TfR2 are localized in the plasma membrane and tubulovesicular endosomes, TfR2 expression is associated with the additional appearance of Tf in multivesicular bodies. These combined results imply that unlike TfR1, which recycles apo-Tf back to the cell surface after the release of iron, TfR2 promotes the intracellular deposition of ligand. Tf delivered by TfR2 does not appear to be degraded, which suggests that its delivery to this organelle may be functionally relevant to the storage of iron in overloaded states. iron transport; HepG2 cells     

Src regulates Tyr(20) phosphorylation of transferrin receptor-1 and potentiates breast cancer cell survival

Transferrin receptor 1 (TfR1) is a ubiquitous type II membrane receptor with 61 amino acids in the N-terminal cytoplasmic region. TfR1 is highly expressed in cancer cells, particularly under iron deficient conditions. Overexpression of TfR1 is thought to meet the increased requirement of iron uptake necessary for cell growth. In the present study, we used transferrin (Tf), a known ligand of TfR1, and gambogic acid (GA), an apoptosis-inducing agent and newly identified TfR1 ligand to investigate the signaling role of TfR1 in breast cancer cells. We found that GA but not Tf induced apoptosis in a TfR1-dependent manner in breast cancer MDA-MB-231 cells. Estrogen receptor-positive MCF-7 cells lack caspase-3 and were not responsive to GA treatment. GA activated the three major signaling pathways of the MAPK family, as well as caspase-3, -8, and Poly(ADP-ribose)polymerase apoptotic pathway. Interestingly, only Src inhibitor PP2 greatly sensitized the cells to GA-mediated apoptosis. Further investigations by confocal fluorescence microscopy and immunoprecipitation revealed that Src and TfR1 are constitutively bound. Using TfR1-deficient CHO TRVB cells, point mutation studies showed that Tyr(20) within the (20)YTRF(23) motif of the cytoplasmic region of TfR1 is the phosphorylation site by Src. TfR1 Tyr(20) phosphomutants were more sensitive to GA-mediated apoptosis. Our results indicate that, albeit its iron uptake function, TfR1 is a signaling molecule and tyrosine phosphorylation at position 20 by Src enhances anti-apoptosis and potentiates breast cancer cell survival.     

Transferrin receptor 2: evidence for ligand-induced stabilization and redirection to a recycling pathway

Transferrin receptor 2 (TfR2) is a homologue of transferrin receptor 1 (TfR1), the protein that delivers iron to cells through receptor-mediated endocytosis of diferric transferrin (Fe(2)Tf). TfR2 also binds Fe(2)Tf, but it seems to function primarily in the regulation of systemic iron homeostasis. In contrast to TfR1, the trafficking of TfR2 within the cell has not been extensively characterized. Previously, we showed that Fe(2)Tf increases TfR2 stability, suggesting that trafficking of TfR2 may be regulated by interaction with its ligand. In the present study, therefore, we sought to identify the mode of TfR2 degradation, to characterize TfR2 trafficking, and to determine how Fe(2)Tf stabilizes TfR2. Stabilization of TfR2 by bafilomycin implies that TfR2 traffics to the lysosome for degradation. Confocal microscopy reveals that treatment of cells with Fe(2)Tf increases the fraction of TfR2 localizing to recycling endosomes and decreases the fraction of TfR2 localizing to late endosomes. Mutational analysis of TfR2 shows that the mutation G679A, which blocks TfR2 binding to Fe(2)Tf, increases the rate of receptor turnover and prevents stabilization by Fe(2)Tf, indicating a direct role of Fe(2)Tf in TfR2 stabilization. The mutation Y23A in the cytoplasmic domain of TfR2 inhibits its internalization and degradation, implicating YQRV as an endocytic motif.     

Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel transferrin receptor

A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH-Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron.     

The molecular mechanism for receptor-stimulated iron release from the plasma iron transport protein transferrin

Human transferrin receptor 1 (TfR) binds iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes where iron is released in a TfR-facilitated process. Consistent with our hypothesis that TfR binding stimulates iron release from Fe-Tf at acidic pH by stabilizing the apo-Tf conformation, a TfR mutant (W641A/F760A-TfR) that binds Fe-Tf, but not apo-Tf, cannot stimulate iron release from Fe-Tf, and less iron is released from Fe-Tf inside cells expressing W641A/F760A-TfR than cells expressing wild-type TfR (wtTfR). Electron paramagnetic resonance spectroscopy shows that binding at acidic pH to wtTfR, but not W641A/F760A-TfR, changes the Tf iron binding site > or =30 A from the TfR W641/F760 patch. Mutation of Tf histidine residues predicted to interact with the W641/F760 patch eliminates TfR-dependent acceleration of iron release. Identification of TfR and Tf residues critical for TfR-facilitated iron release, yet distant from a Tf iron binding site, demonstrates that TfR transmits long-range conformational changes and stabilizes the conformation of apo-Tf to accelerate iron release from Fe-Tf.     

Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment

Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tf10) which was prepared by chemical cross- linking of transferrin (Tf). Tf10 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tf10 was investigated using fluorescence and electron microscopy. Tf10 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tf10 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf10. The presence of Tf10 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf10 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf10 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tf10 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.     

Mysteries of the transferrin-transferrin receptor 1 interaction uncovered

How does the iron (Fe) binding protein, transferrin (Tf), bind to the transferrin receptor 1 (TfR1) to donate Fe to cells? In this issue of Cell, Cheng et al., describe the molecular structure of the human TfR1-Tf complex, This atomic model shows that Tf binds laterally to the TfR1 dimer and extends into the gap between the bottom of the receptor ectodomain and the membrane.     

Unconventional endocytosis and trafficking of transferrin receptor induced by iron

The posttranslational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH₄Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knockdown experiments showed that TfR1 lysosomal degradation is independent of 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-ferritin; and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional posttranslational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis, often not considering its function in iron homeostasis.     

The role of the transferrin receptor for the activation of human lymphocytes

The proliferative response of peripheral blood mononuclear cells (PBMC) in synthetic serum-free media depends on the presence of sufficient amounts of transferrin (Tf). In the present communication we show that the reduction of Tf concentration in culture media results in a decreased proliferation, whereas lymphokine production and the expression of activation markers (IL-2 receptor; transferrin receptor, (TfR); HLA class II) remain unchanged. To examine whether this effect is due to iron depletion we added iron chelates (ferric citrate, FeCi; ferric nitrilotriacetic acid, FeNTA) which can be internalized by cells without the requirement for Tf. The iron chelates could fully restore the proliferative response even in complete absence of Tf, suggesting that the observed inhibitory effect was indeed caused by iron depletion. Addition of a monoclonal TfR antibody, J 64, also caused a marked inhibition of proliferation of PBMC in regular serum-containing medium as well as in Tf-free synthetic medium; this effect could not be overcome by any of the tested iron chelates. Therefore, growth inhibition caused by J 64 cannot simply be attributed to iron starvation. These data suggest that J 64 may interfere with processes others than iron uptake and that the TfR might confer a necessary promoting signal for lymphocyte proliferation.     

Mechanism for multiple ligand recognition by the human transferrin receptor

Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.     

Transferrin-based radiolabeled probe predicts the sensitivity of human renal cancer cell lines to ferroptosis inducer erastin

Ferroptosis induction has been recognized as a novel cancer therapeutic strategy. To effectively apply ferroptosis-targeting cancer therapy to individual patients, a diagnostic indicator for selecting this therapeutic strategy from a number of molecular targeting drugs is needed. However, to date, methods that can predict the efficacy of ferroptosis-targeting treatment have not been established yet. In this study, we focused on the iron metabolic pathway to develop a nuclear imaging technique for diagnosing the susceptibility of cancer cells to ferroptosis. As a nuclear probe, human transferrin (Tf) was labeled with Gallium-68 (⁶⁸Ga) using 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) as a chelator (⁶⁸Ga-NOTA-Tf). Western blot assay and clonogenic survival assay with human renal cancer cell lines A498 and 786-O revealed that the protein expression level of transferrin receptor1 (TfR1) and sensitivity to a ferroptosis inducer, erastin, were correlated. A cellular uptake assay with ⁶⁸Ga-NOTA-Tf revealed that the cancer cells sensitive to erastin highly internalized the ⁶⁸Ga-NOTA-Tf. Furthermore, treatment with the TfR1 inhibitor ferristatin II reduced the cellular uptake of ⁶⁸Ga-NOTA-Tf, indicating that the intracellular uptake of the probe was mediated by TfR1. These results suggest that ⁶⁸Ga-NOTA-Tf can be useful in predicting the sensitivity of cancer cells to ferroptosis inducers.     

Exploring transferrin-receptor interactions at the single-molecule level

Interaction between the iron transporter protein transferrin (Tf) and its receptor at the cell surface is fundamental for most living organisms. Tf receptor (TfR) binds iron-loaded Tf (holo-Tf) and transports it to endosomes, where acidic pH favors iron release. Iron-free Tf (apo-Tf) is then brought back to the cell surface and dissociates from TfR. Here we investigated the Tf-TfR interaction at the single-molecule level under different conditions encountered during the Tf cycle. An atomic force microscope tip functionalized with holo-Tf or apo-Tf was used to probe TfR. We tested both purified TfR anchored to a mica substrate and in situ TfR at the surface of living cells. Dynamic force measurements showed similar results for TfR on mica or at the cell surface but revealed striking differences between holo-Tf-TfR and apo-Tf-TfR interactions. First, the forces necessary to unbind holo-Tf and TfR are always stronger compared to the apo-Tf-TfR interaction. Second, dissociation of holo-Tf-TfR complex involves overcoming two energy barriers, whereas the apo-Tf-TfR unbinding pathway comprises only one energy barrier. These results agree with a model that proposes differences in the contact points between holo-Tf-TfR and apo-Tf-TfR interactions.     

Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites.

The transferrin receptor (TfR) binds two proteins critical for iron metabolism: transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. Previous results demonstrated that Tf and HFE compete for binding to TfR, suggesting that Tf and HFE bind to the same or an overlapping site on TfR. TfR is a homodimer that binds one Tf per polypeptide chain (2:2, TfR/Tf stoichiometry), whereas both 2:1 and 2:2 TfR/HFE stoichiometries have been observed. In order to more fully characterize the interaction between HFE and TfR, we determined the binding stoichiometry using equilibrium gel-filtration and analytical ultracentrifugation. Both techniques indicate that a 2:2 TfR/HFE complex can form at submicromolar concentrations in solution, consistent with the hypothesis that HFE competes for Tf binding to TfR by blocking the Tf binding site rather than by exerting an allosteric effect. To determine whether the Tf and HFE binding sites on TfR overlap, residues at the HFE binding site on TfR were identified from the 2.8 A resolution HFE-TfR co-crystal structure, then mutated and tested for their effects on HFE and Tf binding. The binding affinities of soluble TfR mutants for HFE and Tf were determined using a surface plasmon resonance assay. Substitutions of five TfR residues at the HFE binding site (L619A, R629A, Y643A, G647A and F650Q) resulted in significant reductions in Tf binding affinity. The findings that both HFE and Tf form 2:2 complexes with TfR and that mutations at the HFE binding site affect Tf binding support a model in which HFE and Tf compete for overlapping binding sites on TfR.     

Iodination significantly influences the binding of human transferrin to the transferrin receptor

The human transferrin receptor (TfR) and its ligand, the serum iron carrier transferrin, serve as a model system for endocytic receptors. Although the complete structure of the receptor's ectodomain and a partial structure of the ligand have been published, conflicting results still exist about the magnitude of equilibrium binding constants, possibly due to different labeling techniques. In the present study, we determined the equilibrium binding constant of purified human TfR and transferrin. The results were compared to those obtained with either iodinated TfR or transferrin. Using an enzyme-linked assay for receptor-ligand interactions based on the published direct calibration ELISA technique, we determined an equilibrium constant of Kd=0.22 nM for the binding of unmodified human Tf to surface-immobilized human TfR. In a reciprocal experiment using soluble receptor and surface-bound transferrin, a similar constant of Kd=0.23 nM was measured. In contrast, covalent labeling of either TfR or transferrin with 125I reduced the affinity 3-5-fold to Kd=0.66 nM and Kd=1.01 nM, respectively. The decrease in affinity upon iodination of transferrin is contrasted by an only 1.9-fold decrease in the association rate constant, suggesting that the iodination affects rather the dissociation than the association kinetics. These results indicate that precautions should be taken when interpreting equilibrium and rate constants determined with covalently labeled components.     

Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE

The transferrin receptor (TfR) interacts with two proteins important for iron metabolism, transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. A second receptor for Tf, TfR2, was recently identified and found to be functional for iron uptake in transfected cells (Kawabata, H., Germain, R. S., Vuong, P. T., Nakamaki, T., Said, J. W., and Koeffler, H. P. (2000) J. Biol. Chem. 275, 16618-16625). TfR2 has a pattern of expression and regulation that is distinct from TfR, and mutations in TfR2 have been recognized as the cause of a non-HFE linked form of hemochromatosis (Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., and Gasparini, P. (2000) Nat. Genet. 25, 14-15). To investigate the relationship between TfR, TfR2, Tf, and HFE, we performed a series of binding experiments using soluble forms of these proteins. We find no detectable binding between TfR2 and HFE by co-immunoprecipitation or using a surface plasmon resonance-based assay. The affinity of TfR2 for iron-loaded Tf was determined to be 27 nm, 25-fold lower than the affinity of TfR for Tf. These results imply that HFE regulates Tf-mediated iron uptake only from the classical TfR and that TfR2 does not compete for HFE binding in cells expressing both forms of TfR.     

Structural and functional studies on the stalk of the transferrin receptor

Transferrin (Tf) is an iron carrier protein that consists of two lobes, the N- and C-lobes, which can each bind a Fe³⁺ ion. Tf binds to its receptor (TfR), which mediates iron delivery to cells through an endocytotic pathway. Receptor binding facilitates iron release from the Tf C-lobe, but impedes iron release from the N-lobe. An atomic model of the Tf-TfR complex based on single particle electron microscopy (EM) indicated that receptor binding is indeed likely to hinder opening of the N-lobe, thus interfering with its iron release. The atomic model also suggested that the TfR stalks could form additional contacts with the Tf N-lobes, thus potentially further slowing down its iron release. Here, we show that the TfR stalks are unlikely to make strong interactions with the Tf N-lobes and that the stalks have no effect on iron release from the N-lobes of receptor-bound Tf.     

Structural allostery and binding of the transferrin*receptor complex

The structural allostery and binding interface for the human serum transferrin (Tf)*transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions (Cheng, Y., Zak, O., Aisen, P., Harrison, S. C. & Walz, T. (2004) Structure of the human transferrin receptor.transferrin complex. Cell 116, 565-576). The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.     

Inhibition of heme synthesis decreases transferrin receptor expression in mouse erythroleukemia cells.

The coordination of transferrin receptor (TfR) expression and heme synthesis was investigated in mouse erythroleukemia (MEL) cells of line 707 treated with heme synthesis inhibitors or in a variant line Fw genetically deficient in heme synthesis. Cells of line 707 were induced for differentiation by 5 mM hexamethylene bisacetamide (HMBA). TfR expression increased in the course of induction, as judged by increased TfR mRNA synthesis, increased cytoplasmic TfR mRNA level, and by the increased number of cellular 125I-Tf binding sites. Addition of 0.1 mM succinylacetone (SA) decreased cellular TfR to the level comparable with the uninduced cells. The decrease was reverted by the iron chelator desferrioxamine (DFO) but not by exogenous hemin. In short-term (1-2 hours) incubation, SA inhibited 59Fe incorporation from transferrin into heme, whereas total cellular 59Fe uptake was increased. A decrease in TfR mRNA synthesis was apparent after 2 hours of SA treatment. Conversely, glutathione peroxidase mRNA synthesis, previously shown to be inducible by iron, was increased by SA treatment. Cells of heme deficient line Fw did not increase the number of Tf binding sites after the induction of differentiation by 5 mM sodium butyrate. SA had no effect on TfR expression in Fw cells. The results suggest that the depletion of cellular non-heme iron due to the increase in heme synthesis maintains a high level of transferrin receptor expression in differentiating erythroid cells even after the cessation of cell division.