Diferric transferrin regulates transferrin receptor 2 protein stability

Johnson, Martha B.; Enns, Caroline A.

doi:10.1182/blood-2004-06-2477

Cited by 207 publications

(230 citation statements)

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“…In cells treated with Fe 2 Tf, an increased level of TfR2 protein correlated with an increased half-life of the protein ( Figure 4B). The magnitude of stabilization in Hep3B/TfR2WT cells, from 10 to 28 h, matched that in HepG2 cells, from 4 to 14 h (Johnson and Enns, 2004). Due to the low level of endogenous TfR2 expression in HepG2 cells, assessment of the effect of Fe 2 Tf on TfR2 biosynthetic rate was not feasible.…”

Section: Characterization Of Wild-type Tfr2 In Transfected Hep3b Cellsmentioning

confidence: 54%

“…Fe 2 Tf increases the half-life of TfR2 from 4 to 14 h in HepG2 cells (Johnson and Enns, 2004), indicating that TfR2 degradation is a regulated process. To further understand this process, we set out to determine whether TfR2 degradation occurs in the lysosome.…”

Section: Tfr2 Undergoes Lysosomal Degradationmentioning

confidence: 96%

“…Whereas ligand-receptor interactions frequently result in receptor down-regulation, addition of Fe 2 Tf to the medium of HepG2 cells increases TfR2 by extending the half-life of TfR2 from 4 to 14 h (Johnson and Enns, 2004). Interestingly, regulation of TfR2 by its ligand has only been observed in hepatoma cell lines (Johnson and Enns, 2004;, suggesting the mechanism involves proteins, compartments, or pathways specific to the hepatocyte. Moreover, TfR2 regulation observed in HepG2 cells seems to recapitulate physiological regulation.…”

Section: Introductionmentioning

confidence: 99%

“…Because Trfr2 mutation or knockout results in iron overload, TfR2 appears to function, not principally in cellular iron uptake and delivery, but rather in systemic iron homeostasis. The exact function of TfR2, however, is not known.To TfR2 from 4 to 14 h (Johnson and Enns, 2004 …”

mentioning

confidence: 99%

“…Total protein concentration was measured by bicinchoninic acid assay (Pierce Chemical, Rockford, IL). Samples containing 10 -20 g of total protein were diluted into 4X Laemmli buffer, heated to 95°C for 5 min, loaded on 10% denaturing gels, and analyzed by SDS-PAGE, followed by Western blot with HRP-conjugated or fluorescence-conjugated secondary antibodies as described previously (Johnson and Enns, 2004). …”

mentioning

confidence: 99%

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Transferrin Receptor 2: Evidence for Ligand-induced Stabilization and Redirection to a Recycling Pathway

Johnson

Chen

Murchison

et al. 2007

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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. INTRODUCTIONA truncation mutant of transferrin receptor 2 (TfR2), TfR2/ Y250X, causes a rare form of hereditary hemochromatosis (type 3, HFE3), an iron overload disorder characterized by excess absorption of dietary iron and consequent deposition of iron in liver and other parenchymal tissues (Camaschella et al., 1999(Camaschella et al., , 2000. The analogous mutation or knockout of Trfr2 in mice reproduces the disease phenotype (Fleming et al., 2002;Wallace et al., 2005). Thus, TfR2 is required for normal iron homeostasis.TfR2, cloned in 1999, is a homologue of transferrin receptor 1 (TfR1; Kawabata et al., 1999). The extracellular domains of the two receptors are 45% identical and 67% similar. TfR1 functions to deliver iron to cells through receptor-mediated endocytosis of its ligand transferrin (Tf), a serum protein that transports iron (Dautry-Varsat et al., 1983;Klausner et al., 1983). On the cell surface, TfR1 binds iron-saturated transferrin (Fe 2 Tf) at slightly basic pH. Fe 2 Tf-TfR1 then internalizes in clathrin-coated vesicles to early endosomes.In the acidic pH of early endosomes, iron releases from Tf, whereas Tf remains bound to TfR1. The complex then recycles, from either early or recycling endosomes, to the cell surface. At the slightly basic pH of the cell surface, TfR1 releases unsaturated Tf (apoTf) and again binds Fe 2 Tf. TfR1 expression is ubiquitous, consistent with its role in cellular iron delivery. The stability of TfR1 mRNA is negatively regulated by intracellular iron levels through iron-responsive elements (IREs) in the 3Ј untranslated region (Mattia et al., 1984;Ward et al., 1984;Rao et al., 1985;Sciot et al., 1987;Owen and Kuhn, 19...

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Section: Characterization Of Wild-type Tfr2 In Transfected Hep3b Cellsmentioning

confidence: 54%

Section: Tfr2 Undergoes Lysosomal Degradationmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Transferrin Receptor 2: Evidence for Ligand-induced Stabilization and Redirection to a Recycling Pathway

Johnson

Chen

Murchison

et al. 2007

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Iron: Molecular Basis of Hemochromatosis

Schmidt

2004

Encyclopedia of Inorganic and Bioinorganic Chemistry

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Iron can be deleterious in excess because of its ability to generate reactive oxygen species. Consequently, the appropriate maintenance of organismal iron balance is crucial. Disturbances in iron metabolism are the root cause of many prevalent human diseases. Hereditary hemochromatosis is a genetic disorder resulting in a chronic increase of iron uptake from the diet leading to disease. Although first mentioned in the medical literature by Trousseau in 1865, until recently, the genetic and the pathophysiological bases of this disorder were poorly understood. The vast majority of cases are caused by mutations in HFE , the classic hemochromatosis gene. More recently, genetic studies have implicated hemochromatosis disease‐causing mutations in hemojuvelin ( HJV ), hepcidin ( HAMP ), transferrin receptor 2 ( TFR2 ), and ferroportin ( SLC40A1 ). Research involving these five genetically distinct causes of hemochromatosis has increased our knowledge of the underlying pathogenic mechanisms of the disease and has led to a better general understanding of iron metabolism. Current thought places hepcidin, a soluble 25‐amino‐acid peptide, as the master regulator of iron homeostasis. Hepcidin acts as a ligand for the iron transporter ferroportin, negatively regulating iron release from red blood cell‐recycling macrophages or dietary iron uptake in the duodenum. Perturbance of the hepcidin–ferroportin regulatory apparatus in hemochromatosis leads to dysregulated dietary iron uptake eventuating in disease. Recent work has augmented our understanding of how iron regulates hepcidin expression. Hemojuvelin, a bone morphogenetic protein ( BMP ) coreceptor, plays a central role in the regulation of hepcidin expression through a SMAD ‐mediated signaling pathway. Both HFE and TFR2 are thought to play essential roles in the sensing of total body iron needs, but the mechanism by which these molecules affect HJV / BMP / SMAD ‐mediated hepcidin control is not completely elucidated. Recent insights into the molecular basis of hereditary hemochromatosis and iron metabolism will be reviewed in this article.

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mRNA expression of iron regulatory genes in β-thalassemia intermedia and β-thalassemia major mouse models

et al. 2006

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b-Thalassemia is an inherited anemia in which synthesis of the hemoglobin b-chain is decreased. The excess unmatched a-globin chains accumulate in the growing erythroid precursors, causing their premature death (ineffective erythropoiesis). Clinical features of b-thalassemia include variably severe anemia and iron accumulation due to increased intestinal iron absorption. The most anemic patients require regular blood transfusions, which exacerbate their iron overload and result in damage to vital organs. The hepatic peptide hepcidin, a key regulator of iron metabolism in mammals, was recently found to be low in the urine of b-thalassemia patients, compared with healthy controls, despite their iron overload. In our work, we measured by RQ-PCR the liver mRNA expression of hepcidin and other iron regulatory genes in b-thalassemia major mouse model (C57Bl/6 Hbb th3/th3 ), and compared it with b-thalassemia intermedia mouse model (C57Bl/6 Hbb th3/þ ) and control mice. We found decreased expression of hepcidin and TfR2 and increased expression of TfR1 and NGAL in the b-thalassemia mouse models, compared with the control mice. Significant down-regulation of hepcidin expression in b-thalassemia major, despite iron overload, might explain the increased iron absorption typically observed in thalassemia. Am. J. Hematol. 81:479-483, 2006. V V C 2006 Wiley-Liss, Inc.

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Diferric transferrin regulates transferrin receptor 2 protein stability

Cited by 207 publications

References 52 publications

Transferrin Receptor 2: Evidence for Ligand-induced Stabilization and Redirection to a Recycling Pathway

Transferrin Receptor 2: Evidence for Ligand-induced Stabilization and Redirection to a Recycling Pathway

Iron: Molecular Basis of Hemochromatosis

mRNA expression of iron regulatory genes in β-thalassemia intermedia and β-thalassemia major mouse models

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