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.     

Hereditary hemochromatosis protein, HFE, interaction with transferrin receptor 2 suggests a molecular mechanism for mammalian iron sensing

HFE and transferrin receptor 2 (TFR2) are membrane proteins integral to mammalian iron homeostasis and associated with human hereditary hemochromatosis. Here we demonstrate that HFE and TFR2 interact in cells, that this interaction is not abrogated by disease-associated mutations of HFE and TFR2, and that TFR2 competes with TFR1 for binding to HFE. We propose a new model for the mechanism of iron status sensing that results in the regulation of iron homeostasis.     

Hereditary hemochromatosis

The advent of the genetics era has profoundly changed the way we look at iron related diseases, particularly hemochromatosis. New discoveries have challenged historical concepts about the disease, such as its monogenic nature, intestinal origin or complete phenotypic penetrance. This review presents a new concept of hemochromatosis which stems from the idea that, beyond their genetic diversities, all known hemochromatoses have in common the same metabolic abnormality: the genetically determined failure to prevent unneeded iron from entering the circulatory pool. Inappropriate levels of hepcidin, the iron hormone, appear now as the central pathogenic event in all forms of hemochromatosis: depending on the protein involved, and its effect on hepatic production of hepcidin, the phenotype varies, ranging from massive early-onset iron loading with severe organ disease (e.g., associated with homozygous mutations of hemojuvelin or hepcidin itself) to the milder late-onset phenotype characterizing the classic and highly prevalent HFE-related form or the rare transferrin receptor 2-related form. In vitro and in vivo studies will be needed to dissect the consequences of each hereditary hemochromatosis allele and increase our understanding of the precise contribution of each gene to the hereditary hemochromatosis phenotype.     

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.     

Hereditary hemochromatosis in Spain

The C282Y mutation of the HFE gene has been reported as the main cause of hereditary hemochromatosis (HH). Another missense mutation (H63D) has also been detected at an increased frequency in a compound heterozygote state with the C282Y mutation in HH patients. However, these two mutations are not present in all of the HH patients, indicating that other mutations in the HFE gene, or in other loci, should exist. The present study reports the frequencies of the C282Y and H63D mutations in 74 Spanish HH patients and the results of the sequencing analysis of the HFE exons, intron-exon boundaries, and 588 bp of the 5' region in 5 patients negative for the C282Y mutation. We have detected a high frequency of the C282Y mutation (85.1%) in Spanish HH patients, indicating that this mutation is the most common defect associated with the disease in Spain. The screening of the HFE regions in our patients without the C282Y mutation has revealed the presence of five polymorphisms. However, no other pathological mutations have been found. Therefore, further efforts to characterize the unscreened part of the HFE gene or other loci should be taken to identify the potential genetic factors causing HH in the C282Y-negative patients.     

Defective trafficking and localization of mutated transferrin receptor 2: implications for type 3 hereditary hemochromatosis

Transferrin receptor 2 (TfR2), a homologue of transferrin receptor 1 (TfR1), is a key molecule involved in the regulation of iron homeostasis. Mutations in TfR2 result in iron overload with similar features to HFE-associated hereditary hemochromatosis. The precise role of TfR2 in iron metabolism and the functional consequences of disease-causing mutations have not been fully determined. We have expressed wild-type and various mutant forms of TfR2 that are associated with human disease in a mouse liver cell line. Intracellular and surface analysis shows that all the TfR2 mutations analyzed cause the intracellular retention of the protein in the endoplasmic reticulum, whereas the wild-type protein is expressed in endocytic structures and at the cell surface. Our results indicate that the majority of mutations that cause type 3 hereditary hemochromatosis are a consequence of the defective localization of the protein.     

Molecular and cellular characterization of transferrin receptor 2

Iron is an essential component of many biological processes. However, an excess of iron in the body is also toxic; thus, the levels of this element are tightly regulated. Our knowledge of the mechanism by which iron levels are maintained has been bolstered by the dramatic increase in the discovery of novel molecules implicated in iron homeostasis. The transferrin receptor-transferrin pathway is the main mechanism by which cells take up iron. The recently identified homolog of transferrin receptor, its characterization and its role in iron metabolism is the subject of this review.     

Hereditary hemochromatosis: an opportunity for gene therapy

Levels of body iron should be tightly controlled to prevent the formation of oxygen radicals, lipoperoxidation, genotoxicity, and the production of cytotoxic cytokines, which result in damage to a number of organs. Enterocytes in the intestinal villae are involved in the apical uptake of iron from the intestinal lumen: iron is further exported from the cells into the circulation. The apical divalent metal transporter-1 (DMT1) transports ferrous iron from the lumen into the cells, while the basolateral transporter ferroportin extrudes iron from the enterocytes into the circulation. Patients with hereditary hemochromatosis display an accelerated transepithelial uptake of iron, which leads to body iron accumulation that results in cirrhosis, hepatocellular carcinoma, pancreatitis, and cardiomyopathy. Hereditary hemochromatosis, a recessive genetic condition, is the most prevalent genetic disease in Caucasians, with a prevalence of one in 300 subjects. The majority of patients with hereditary hemochromatosis display mutations in the gene coding for HFE, a protein that normally acts as an inhibitor of transepithelial iron transport. We discuss the different control points in the homeostasis of iron and the different mutations that exist in patients with hereditary hemochromatosis. These control sites may be influenced by gene therapeutic approaches; one general therapy for hemochromatosis of different etiologies is the inhibition of DMT1 synthesis by antisense-generating genes, which has been shown to markedly inhibit apical iron uptake by intestinal epithelial cells. We further discuss the most promising strategies to develop gene vectors and deliver them into enterocytes.     

The transferrin receptor

The isolation and analysis of the transferrin receptor has been greatly aided by the use of monoclonal antibodies. The receptor is a disulphide-linked homo-dimer which spans the membrane and binds two molecules of transferrin. Controlling genes for this receptor in humans have been mapped to chromosome 3 using cell hybrids. The expression of transferrin receptors is related to the obligatory and ubiquitous iron requirements associated with cell proliferation or the special iron demand of haemoglobin synthesizing cells and trophoblasts. However, transferrin receptors may also be involved in cell interactions regulating cell growth.     

脑内的铁,转铁蛋白及转铁蛋白受体

脑铁异常增高可能参与脑神经变性疾病的发生发展。这一发现使得脑铁代谢成为近年广为关注和研究较为广泛的领域。本文综述了这一领域某些方面的目前认识。包括：（１）脑铁分布及功能;（２）铁转铁蛋白及转铁蛋白受体在脑内的合成与分布;（３）脑铁摄取和运输。此外,对铁与某些金属离子,转的蛋白和转铁蛋白受体与脑神经变性疾病的关系,以及转铁蛋白受体内吞在生物大分子跨血脑屏障运输中的作用也作了简要讨论。     

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.     

Hereditary transferrin system in healthy subjects and schizophrenic patients]

Hereditary polymorphism of transferrin is studied by means of starch gel electrophoresis in a group of healthy inhabitants of Minsk (250 persons) and in a group of schizophrenic patients (128 persons). It is concluded that the inhabitants of Minsk do not differ considerably from the population, living in the European territory by distributing the frequency of transferrin alleles. No considerable differences are found between the group of healthy people and schizophrenic patients.     

Hereditary hemochromatosis: An update vision of the laboratory diagnosis

Haemochromatosis (HC) is an inherited disorder of iron metabolism. The 85–90% of Hereditary hemochromatosis cases are caused by mutations in HFE gene (HC type 1). The remaining 10–15% of HC cases are caused by mutations in other non-HFE genes (HJV, HAMP, TRF2, SLC40A1, BMP6). The study of patients for the diagnosis of HC has an important laboratory approached: analysis of biochemical parameters and genetic studies. To confirm a case, it is necessary to carry out a genetic study of the C282Y and H63D mutations. The presence of C282Y mutation in homozygosis is compatible with the diagnosis of HC type 1. Due to the incomplete penetrance of this mutation and the variable phenotypic expression, the severe forms of the disease are relatively rare. The study of variants in non-HFE genes allows more detailed study of both non-classic HC cases and those with more severe clinical expression. The genotype characterization of a patient not always justified the phenotype expression of the symptoms in this disease. All laboratory clinicians must consider recommendation provide by the experts in the Materia.     

Iron absorption and hepatic iron uptake are increased in a transferrin receptor 2 (Y245X) mutant mouse model of hemochromatosis type 3

Hereditary hemochromatosis type 3 is an iron (Fe)-overload disorder caused by mutations in transferrin receptor 2 (TfR2). TfR2 is expressed highly in the liver and regulates Fe metabolism. The aim of this study was to investigate duodenal Fe absorption and hepatic Fe uptake in a TfR2 (Y245X) mutant mouse model of hereditary hemochromatosis type 3. Duodenal Fe absorption and hepatic Fe uptake were measured in vivo by 59Fe-labeled ascorbate in TfR2 mutant mice, wild-type mice, and Fe-loaded wild-type mice (2% dietary carbonyl Fe). Gene expression was measured by real-time RT-PCR. Liver nonheme Fe concentration increased progressively with age in TfR2 mutant mice compared with wild-type mice. Fe absorption (both duodenal Fe uptake and transfer) was increased in TfR2 mutant mice compared with wild-type mice. Likewise, expression of genes participating in duodenal Fe uptake (Dcytb, DMT1) and transfer (ferroportin) were increased in TfR2 mutant mice. Nearly all of the absorbed Fe was taken up rapidly by the liver. Despite hepatic Fe loading, hepcidin expression was decreased in TfR2 mutant mice compared with wild-type mice. Even when compared with Fe-loaded wild-type mice, TfR2 mutant mice had increased Fe absorption, increased duodenal Fe transport gene expression, increased liver Fe uptake, and decreased liver hepcidin expression. In conclusion, despite systemic Fe loading, Fe absorption and liver Fe uptake were increased in TfR2 mutant mice in association with decreased expression of hepcidin. These findings support a model in which TfR2 is a sensor of Fe status and regulates duodenal Fe absorption and liver Fe uptake.     

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.     

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.     

Segregation of genetic hemochromatosis indexed by latent capacity of transferrin.

A genetic analysis of the segregation of hereditary hemochromatosis, indexed by the measurement of latent capacity of transferrin (LCAP), was undertaken in an ascertained sample of 147 pedigrees from Brittany, France. There were no mean differences by sex in the distribution of LCAP in the control sample, although in the family data there was a higher representation of males with low values than of females with low values, consistent with the higher proportion of affected males. The results of bivariate segregation analysis revealed no systematic evidence for heterozygous expression either in the biochemical domain of LCAP abnormalities or in increased liability to overt symptomatic disease. Joint consideration of the quantitative variable with hemochromatosis affection status allowed clear resolution of a recessive single-gene inheritance pattern in these families.     

Oxidative stress and transferrin receptor recycling

Perturbation of the oxidative balance in biological systems plays an important role in numerous pathological states as well as in many physiological processes such as receptor activity. In order to evaluate if oxidative stress induced by menadione influences membrane receptor processes, a study was conducted on the transferrin receptor. Consequently, biochemical, biophysical and ultrastructural studies were carried out on different cell lines. The results obtained seem to indicate that oxidative stress is able of inducing a rapid and specific down-modulation of membrane transferrin receptor due to a block of receptor recycling on the cell surface without affecting binding affinity.