The hemochromatosis protein HFE competes with transferrin for binding to the transferrin receptor.

HFE is a class I major histocompatibility complex (MHC)-related protein that is mutated in patients with the iron overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR), the receptor used by cells to obtain iron in the form of diferric transferrin (Fe-Tf). Previous studies demonstrated that HFE and Fe-Tf can bind simultaneously to TfR to form a ternary complex, and that membrane-bound or soluble HFE binding to cell surface TfR results in a reduction in the affinity of TfR for Fe-Tf. We studied the inhibition by soluble HFE of the interaction between soluble TfR and Fe-Tf using radioactivity-based and biosensor-based assays. The results demonstrate that HFE inhibits the TfR:Fe-Tf interaction by binding at or near the Fe-Tf binding site on TfR, and that the Fe-Tf:TfR:HFE ternary complex consists of one Fe-Tf and one HFE bound to a TfR homodimer.     

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.     

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.     

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.     

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.     

Binding to the transferrin receptor is required for endocytosis of HFE and regulation of iron homeostasis

HFE, the protein that is mutated in hereditary haemochromatosis, binds to the transferrin receptor (TfR). Here we show that wild-type HFE and TfR localize in endosomes and at the basolateral membrane of a polarized duodenal epithelial cell line, whereas the primary haemochromatosis HFE mutant, and another mutant with impaired TfR-binding ability accumulate in the ER/Golgi and at the basolateral membrane, respectively. Levels of the iron-storage protein ferritin are greatly reduced and those of TfR are slightly increased in cells expressing wild-type HFE, but not in cells expressing either mutant. Addition of an endosomal-targeting sequence derived from the human low-density lipoprotein receptor (LDLR) to the TfR-binding-impaired mutant restores its endosomal localization but not ferritin reduction or TfR elevation. Thus, binding to TfR is required for transport of HFE to endosomes and regulation of intracellular iron homeostasis, but not for basolateral surface expression of HFE.     

The transferrin receptor binding site on HFE, the class I MHC-related protein mutated in hereditary hemochromatosis.

HFE is a class I major histocompatibility complex (MHC)-related protein that is mutated in patients with the iron storage disease hereditary hemochromatosis. HFE binds tightly to transferrin receptor (TfR), the receptor that mediates uptake of iron-loaded transferrin. The binding affinities for TfR of HFE mutants, designed using the HFE crystal structure, were measured using biosensor assays. The results allow localization of the TfR binding site on HFE to the C-terminal portion of the alpha1 domain helix and an adjacent loop, a region distinct from the ligand binding sites on class I MHC and related proteins. A biosensor-derived pH-dependent affinity profile for the HFE-TfR interaction is discussed in terms of HFE's hypothesized role in intracellular trafficking.     

HFE association with transferrin receptor 2 increases cellular uptake of transferrin-bound iron

Mutations in either HFE or transferrin receptor 2 (TfR2) cause decreased expression of the iron regulatory hormone hepcidin and hemochromatosis. HFE and TfR2 were recently discovered to form a stable complex at the cell membrane when co-expressed in heterologous cell lines. We analyzed the functional consequences of the co-expression of these proteins using transfected TRVb cells, a Chinese hamster ovary derived cell line without endogenous HFE or transferrin receptor. The co-expression of TfR2 in TRVb cells expressing HFE led to accelerated HFE biosynthesis and late-Golgi maturation, suggesting interaction prior to cell surface localization. The co-expression of HFE in cells expressing TfR2 led to increased affinity for diferric transferrin, increased transferrin-dependent iron uptake, and relative resistance to iron chelation. These observations indicate that HFE influences the functional properties of TfR2, and suggests a model in which the interaction of these proteins might influence signal transduction to hepcidin.     

HFE modulates transferrin receptor 2 levels in hepatoma cells via interactions that differ from transferrin receptor 1-HFE interactions

Mutations in the transmembrane glycoproteins transferrin receptor 2 (TfR2) and HFE are associated with hereditary hemochromatosis. Interactions between HFE and transferrin receptor 1 (TfR1) have been mapped to the alpha1- and alpha2-helices in HFE and to the helical domain of TfR1. Recently, TfR2 was also reported to interact with HFE in transfected mammalian cells. To test whether similar HFE residues are important for both TfR1 and TfR2 binding, a mutant form of HFE (W81AHFE) that has an approximately 5,000-fold lower affinity for TfR1 than HFE was employed. As expected, W81AHFE does not interact with TfR1. However, we found that the same mutation in HFE does not affect the TfR2/HFE interaction. This finding indicates that the TfR2/HFE and TfR1/HFE interactions are distinct. We further observed that, unlike TfR1/HFE, Tf does not compete with HFE for binding to TfR2 and that binding is independent of pH (pH 6-7.5). TfR2-TfR1 and HFE-HLA-B7 chimeras were generated to map the domains of the TfR2/HFE interaction. TfR1 and HLA-B7 were chosen because of their similar overall structures with TfR2 and HFE, respectively. We mapped the interacting domains to the putative stalk and protease-like domains of TfR2 located between residues 104 and 250 and to the alpha3 domain of HFE, both of which differ from the TfR1/HFE interacting domains. Furthermore, we found that HFE increases TfR2 levels in hepatic cells independent of holo-Tf.     

Release of the soluble transferrin receptor is directly regulated by binding of its ligand ferritransferrin

The human transferrin receptor (TfR) is shed by an integral metalloprotease releasing a soluble form (sTfR) into serum. The sTfR reflects the iron demand of the body and is postulated as a regulator of iron homeostasis via binding to the hereditary hemochromatosis protein HFE. To study the role of transferrin in this process, we investigated TfR shedding in HL60 cells and TfR-deficient Chinese hamster ovary cells transfected with human TfR. Independent of TfR expression, sTfR release decreases with increasing ferritransferrin concentrations, whereas apo-transferrin exhibits no inhibitory effect. To investigate the underlying mechanism, we generated several TfR mutants with different binding affinities for transferrin. Shedding of TfR mutants in transfected cells correlates exactly with their binding affinity, implying that the effect of ferritransferrin on TfR shedding is mediated by a direct molecular interaction. Analysis of sTfR release from purified microsomal membranes revealed that the regulation is independent from intracellular trafficking or cellular signaling events. Our results clearly demonstrated that sTfR does not only reflect the iron demand of the cells but also the iron availability in the bloodstream, mirrored by iron saturation of transferrin, corroborating the important potential function of sTfR as a regulator of iron homeostasis.     

Identification of the segments of the mouse transferrin receptor 1 required for mouse mammary tumor virus infection

Most enveloped viruses enter cells through binding of virion surface envelope proteins to receptors found on the plasma membrane of the cell. The beta retrovirus mouse mammary tumor virus (MMTV) uses transferrin receptor 1 (TfR1) to enter cells in a pH-dependent mechanism, probably co-trafficking with TfR1 to an acidic compartment where virus entry occurs. We have shown here that, although mouse and rat TfR1 function as entry receptors, cat, dog, hamster, or human TfR1s do not support MMTV infection. We also demonstrated that MMTV entry is independent of transferrin, iron, and the TfR1 cofactor hereditary hematochromatosis HFE protein. Using chimeric mouse/human hybrid TfR1 constructs, we determined the site of interaction with MMTV and found that it maps to two segments physically disparate from the TfR and HFE binding sites. Thus, MMTV has apparently evolved to enter cells independently of the iron status of the host.     

Pumping iron: the strange partnership of the hemochromatosis protein, a class I MHC homolog, with the transferrin receptor

People suffering from hereditary hemochromatosis (HH) can not regulate the uptake of iron properly and gradually accumulate iron in their body over their lifetime. The protein involved in HH, HFE, has been recently identified as a class I major histocompatibility complex (MHC) homolog. The wild-type HFE associates and co-traffics with the transferrin receptor (TfR). The mutation responsible for 83% of HH (C260Y) results in the failure of HFE to form a critical disulfide bond, bind β₂ microglobulin, bind TfR, and traffic to the cell surface. In non-polarized cells, the partnership of HFE and TfR results in decreased iron uptake into cells. The mechanism whereby a class I MHC homolog modifies the function of a membrane receptor and how this dynamic complex of molecules regulates iron transport across intestinal epithelial cells is the subject of this review.     

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.     

Transferrin [corrected] receptor association and endosomal localization of soluble HFE are not sufficient for regulation of cellular iron homeostasis

Iron uptake and storage are tightly regulated to guarantee sufficient iron for essential cellular processes and to prevent the production of damaging free radicals. A non-classical class I MHC molecule, the hemochromatosis factor HFE, has been shown to regulate iron metabolism, potentially via its direct interaction with the transferrin receptor (TfR). In this study, we demonstrate that a soluble beta2microglobulin-HFE monochain (sHFE) folds with beta2microglobulin (beta2m) and associates with the TfR, indicating that the transmembrane and cytoplasmic domains are not necessary for assembly and trafficking through the ER-Golgi network. We also demonstrate human TfR-specific uptake and accumulation of extracellular sHFE by treated cells. The sHFE localized to the endosomal compartment albeit we observed variation in the time taken for endosomal trafficking between different cell types. The sHFE monochain was effective in reducing Tf uptake into cells, however this did not correlate to any changes in TfR or ferritin synthesis, in contrast to the HFE-induced increase and decrease of TfR and ferritin, respectively. These findings of incongruent sHFE activity suggest that either variation in affinity binding of sHFE to TfR prevents efficient modulation of iron-regulated proteins or that HFE has multiple functions some of which may be independent of TfR but dependent on interactions within the endosomal compartment for effective modulation of iron metabolism.     

Regulation of K562 cell transferrin receptors by exogenous iron

Single-cell analysis of K562 human erythroleukemia cells by flow cytometry was used to demonstrate the specific role of iron in regulating transferrin receptors (TfRs) and to establish that TfR expression does not necessarily correlate with growth rate. Exogenous iron concentration in culture was manipulated by supplementing the medium with sera having different iron concentrations over the range 0.6 to 5.4 micrograms/ml, by the addition of iron in the form of FeCl3, iron-saturated serum, or diferric transferrin, and by the addition of the iron chelator Desferal (desferrioxamine). TfR expression was negatively correlated with exogenous iron content: any treatment that reduced exogenous iron supply by at least 15% resulted in as much as a 1.8-fold increase in external receptors, detected as binding by both transferrin and monoclonal anti-TfR antibodies, and a 1.5-fold increase in the pool of internal receptors, as detected by anti-TfR antibody binding. None of these treatments altered growth rate, total cellular protein content, protein synthetic rate, cell cycle distribution or cell size. The rapid (12 hr) and reversible induction of internal and external receptors by Desferal was inhibited by cycloheximide and therefore may have resulted from de novo synthesis and not just mobilization of internal receptor pool to the cell surface. The correlation between growth rate and TfR expression previously observed in these and other cells must be secondary to cellular mechanisms that maintain intracellular iron pools by regulating synthesis, recycling, and cell surface expression of TfRs.     

Overexpression of hemochromatosis protein, HFE, alters transferrin recycling process in human hepatoma cells

HFE is a MHC class 1-like protein that is mutated in hereditary hemochromatosis. In order to elucidate the role of HFE protein on cellular iron metabolism, functional studies were carried out in human hepatoma cells (HLF) overexpressing a fusion gene of HFE and green fluorescent protein (GFP). The expression of HFE-GFP was found to be localized on cell membrane and perinuclear compartment by fluorescent microscopy. By co-immunoprecipitation and Western blotting, HFE-GFP protein formed a complex with endogenous transferrin receptor and beta(2)-microglobulin, suggesting that this fusion protein has the function of HFE reported previously. We then examined the (59)Fe uptake and release, and internalization and recycling of (125)I-labeled transferrin in order to elucidate the functional roles of HFE in the cell system. In the transfectants, HFE protein decreased the rate of transferrin receptor-dependent iron ((59)Fe) uptake by the cells, but did not change the rate of iron release, indicating that HFE protein decreased the rate of iron influx. Scatchard analysis of transferrin binding to HFE-transfected cells showed an elevation of the dissociation constant from 1.9 to 4. 3 nM transferrin, indicating that HFE protein decreased the affinity of transferrin receptor for transferrin, while the number of transferrin receptors decreased from 1.5x10(5)/cell to 1. 2x10(5)/cell. In addition, the rate of transferrin recycling, especially return from endosome to surface, was decreased in the HFE-transfected cells by pulse-chase study with (125)I-labeled transferrin. Our results strongly suggest an additional role of HFE on transferrin receptor recycling in addition to the decrease of receptor affinity, resulting in the reduced cellular iron.     

Expression of iron transport proteins divalent metal transporter-1, Ferroportin-1, HFE and transferrin receptor-1 in human monocyte-derived dendritic cells

Iron is essential for cell survival and regulates many cell functions. In the context of the immune response, iron-related metabolism is tightly controlled in activated lymphocytes as well as in cells of the innate immunity. More precisely, for dendritic cells (DCs), which are the key cell type in the development of a specific immune response, the importance of iron absorption was recently unravelled by showing that depletion of iron inhibits the maturation of DCs. On this basis, we studied in detail the expression of iron transport proteins and HFE in DCs. We found that iron uptake in this cell type is mediated by divalent-metal transporter 1 (DMT1) and transferrin receptor-1 (TfR) whereas Ferroportin-1 is very weakly expressed. HFE that regulates TfR's activity is also detected at the mRNA level. The expression of DMT1 and HFE barely varies upon endotoxin-induced maturation but TfR is up-regulated and the iron export molecule Ferroportin-1 is down-regulated. As opposed to MHC class II molecules, the intracellular localization of TfR is not changed during maturation. Our results indicate that the uptake of iron during DCs development and maturation is mediated by a strong expression of iron-uptake molecules such as DMT1 and TfR as well as a down-regulation of iron export molecules such as Ferroportin-1.     

Co-localization of the mammalian hemochromatosis gene product (HFE) and a newly identified transferrin receptor (TfR2) in intestinal tissue and cells.

Mutations in the HFE gene and a newly identified second transferrin receptor gene, TfR2, cause hemochromatosis. The cognate proteins, HFE and TfR2, are therefore of key importance in human iron homeostasis. HFE is expressed in small intestinal crypt cells where transferrin-iron entry may determine subsequent iron absorption by mature enterocytes, but the physiological function of TfR2 is unknown. Using specific peptide antisera, we examined the duodenal localization of HFE and TfR2 in humans and mice, with and without HFE deficiency, by confocal microscopy. We also investigated potential interactions of these proteins in human intestinal cells in situ. Duodenal expression of HFE and TfR2 (but not TfR1) in wild-type mice and humans was restricted to crypt cells, in which they co-localized. HFE deficiency disrupted this interaction, altering the cellular distribution of TfR2 in human crypts. In human Caco-2 cells, HFE and TfR2 co-localized to a distinct CD63-negative vesicular compartment showing marked signal enhancement on exposure to iron-saturated transferrin ligand, indicating that HFE preferentially interacts with TfR2 in a specialized early endosomal transport pathway for transferrin-iron. This interaction occurs specifically in small intestinal crypt cells that differentiate to become iron-absorbing enterocytes. Our immunohistochemical findings provide evidence for a novel mechanism for the regulation of iron balance in mammals.     

Titanium preferential binding sites in human serum transferrin at physiological concentrations

Serum transferrin (Tf) is an iron binding glycoprotein that plays a central role in the metabolism of this essential metal but it also binds other metal ions. Four main transferrin forms containing different iron binding states can be distinguished in human serum samples: monoferric (C-site or N-site), holotransferrin (with two Fe atoms) and apotransferrin (with no metal). Recently, it has been reported that Tf binds also Ti even more tightly than does Fe, in artificially Ti(iv) spiked solutions. However, very limited work has been done on the Ti binding to Tf at physiological concentrations in patients carrying intramedullary Ti nails. Here we report the chemical association of Ti to Tf "in vivo" under different chromatographic conditions by elemental mass spectrometry using double focusing inductively coupled plasma (DF-ICP-MS) as detector. For the separation of the Ti/Fe-Tf forms different gradient conditions have been explored. The observed results reveal that human serum Ti (from patients carrying intramedullary Ti nails) is uniquely associated to the N-lobe of Tf. The investigation of the influence of sialic acid in the carbohydrate chain of human serum Tf, studied by incubating the protein with neuraminidase (sialidase) to obtain the monosialilated species, revealed that the binding affinity of Ti was similar for monosialo-Tf and for native-Tf and occurs in the N-lobe. These results suggest that the species Fe(C)Ti(N)-TF might provide a route for Ti entry into cells via the transferrin receptors after the release of the metal from its implants.