Wild-type and mutant ferroportins do not form oligomers in transfected cells

Gonçalves, Ana Sofia; Muzeau, Françoise; Blaybel, Rand; Hetet, Gilles; Driss, Fathi; Delaby, Constance; Canonne‐Hergaux, François; Beaumont, Carole

doi:10.1042/bj20051682

Cited by 42 publications

(49 citation statements)

References 38 publications

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“…Presumably FPN1 dimers persist in the second dimension because of insufficient equilibration of reducing agent and SDS during preparation of the first-dimension gels for the second-dimension electrophoresis. The apparent molecular mass of ϳ70 kDa for the FPN1 detected in the more slowly migrating complex also confirms that FPN1 can exist as monomers (15). Although the functional activity of the FPN1-Heph complexes cannot be determined from these studies, the presence of a dimeric form of FPN1 offers a possible explanation as to how heterozygotes for FPN1 mutations can result in iron overload (8,23).…”

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confidence: 83%

Iron feeding induces ferroportin 1 and hephaestin migration and interaction in rat duodenal epithelium

Yeh

Yeh²,

Mims³

et al. 2009

American Journal of Physiology-Gastrointestinal and Liver Physi

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Yeh KY, Yeh M, Mims L, Glass J. Iron feeding induces ferroportin 1 and hephaestin migration and interaction in rat duodenal epithelium. Am J Physiol Gastrointest Liver Physiol 296: G55-G65, 2009. First published October 30, 2008 doi:10.1152/ajpgi.90298.2008.-Intestinal iron absorption involves proteins located in the brush border membrane (BBM), cytoplasm, and basolateral membrane (BLM) of duodenal enterocytes. Ferroportin 1 (FPN1) and hephaestin (Heph) are necessary for transport of iron out of enterocytes, but it is not known whether these two proteins interact during iron absorption. We first examined colocalization of the proteins by cotransfection of HEK293 cells with pDsRed-FPN1 with pEmGFP-Heph or with the COOH-terminal truncated pEmGFP-Heph⌬43 or -Heph⌬685 and found that FPN1 and Heph with or without the COOH terminus colocalized. In rat duodenal enterocytes, within 1 h of iron feeding prominent migration of FPN1 from the apical subterminal zone to the basal subnuclear zone of the BLM occurred and increased to at least 4 h after feeding. Heph exhibited a similar though less prominent migration after iron ingestion. Analysis using rat duodenal epithelial cell sheets demonstrated that 1) by velocity sedimentation ultracentrifugation, FPN1 and Heph occupied vesicles of different sizes prior to iron feeding and migrated to similar fractions 1 h after iron feeding; 2) by blue native/SDS-PAGE, FPN1, and Heph interacted to form two complexes, one containing dimeric FPN1 and intact Heph and the other consisting of monomeric FPN1 and a Heph fragment; and 3) by immunoprecipitation, anti-Heph or anti-FPN1 antiserum coimmunoprecipitated FPN1 and Heph. Thus the data indicate that FPN1 and Heph migrate and interact during iron feeding and suggest that dimeric FPN1 is associated with intact Heph.DsRed-FPN1; EmGFP-Heph; FPN1 and Heph migration; blue native/ SDS-PAGE; coimmunoprecipitation IRON IS AN ESSENTIAL NUTRIENT absorbed through the duodenal epithelium, and iron homeostasis is achieved by regulation of the absorptive process (20). In the current concept of intestinal iron absorption, dietary iron as Fe 3ϩ is reduced to Fe 2ϩ by the duodenal cytochrome b reductase (Dcytb) (17, 27). The reduced iron is then absorbed through the divalent metal transporter (DMT1) on the brush border membrane (BBM) of duodenal enterocytes (17,27). The Fe 2ϩ then traverses the enterocyte cytoplasm to the basolateral membrane (BLM) where Fe 2ϩ is transported by ferroportin (FPN1) (1, 9, 27) and converted to Fe 3ϩ by the multicopper ferroxidase hephaestin (Heph) (38) before release to transferrin in the systemic circulation. Since Fe 2ϩ is highly reactive and potentially toxic, the molecular mechanisms for handling the absorbed iron as it transverses the enterocyte from BBM to the BLM are important to elucidate. The detailed molecular relationships of the two membrane transporters, DMT1 and FPN1, and the accessory proteins necessary for iron transport remain largely unknown. The Fe 2ϩ transported into the enterocyte may bind to cytosolic...

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confidence: 83%

Iron feeding induces ferroportin 1 and hephaestin migration and interaction in rat duodenal epithelium

Yeh

Yeh²,

Mims³

et al. 2009

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…The most common phenotype characterized by iron loading of macrophages and normal transferrin saturation results from mutations causing mislocalization of Fpn (eg V162del) or the loss of iron export function (eg, N174I). [7][8][9][10]22 The less common phenotype with parenchymal iron loading similar to classical hemochromatosis results from mutations that cause a gain of function, because they interfere with hepcidin-mediated internalization and degradation of Fpn (N144D/ T/H, C326S/Y, Y64N). 8,[11][12][13][14][15][16][17] Some mutations (eg, A77D) appear to cause a mixed phenotype, with both parenchymal and macrophage iron deposition, and normal to high transferrin saturation.…”

Section: Discussionmentioning

confidence: 99%

“…The loss-of-function mutations cause decreased cellular iron export because the mutant protein is retained inside the cell or has impaired iron-exporting function. [7][8][9][10] As a result, iron accumulates in macrophages, leading to very high ferritin levels, but the patients have normal-to-low transferrin saturation, and a tendency to develop anemia particularly when phlebotomized. 6 It is not yet clear whether this condition impacts health or requires treatment.…”

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confidence: 99%

The molecular basis of hepcidin-resistant hereditary hemochromatosis

Fernandes

Preza

Phung

et al. 2009

Blood

151

139

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The interaction between the hormone hepcidin and the iron exporter ferroportin (Fpn) regulates plasma iron concentrations. Hepcidin binds to Fpn and induces its internalization and degradation, resulting in decreased iron efflux from cells into plasma. Fpn mutations in N144, Y64N, and C326 residue cause autosomal dominant disease with parenchymal iron overload, apparently due to the resistance of mutant Fpn to hepcidin-mediated internalization. To define the mechanism of resistance, we generated human Fpn constructs bearing the pathogenic mutations. The mutants localized to the cell surface and exported iron normally, but were partially or completely resistant to hepcidinmediated internalization and continued to export iron despite the presence of hepcidin. The primary defect with exofacial C326 substitutions was the loss of hepcidin binding, which resulted in the most severe phenotype. The thiol form of C326 was essential for interaction with hepcidin, suggesting that C326-SH homology is located in or near the binding site of hepcidin. In contrast, N144 and Y64 residues were not required for hepcidin binding, but their mutations impaired the subsequent internalization of the ligandreceptor complex. Our observations explain why the mutations in C326 Fpn residue produce a severe form of hemochromatosis with iron overload at an early age. (Blood. 2009;114:437-443) IntroductionHereditary hemochromatosis is a disease characterized by high transferrin saturation and parenchymal iron overload. Most forms are caused by autosomal-recessive mutations in HFE, transferrin receptor 2, hemojuvelin, or hepcidin. These mutations lead to deficiency of the iron-regulatory hormone hepcidin. Rarely, hereditary hemochromatosis is caused by autosomal-dominant mutations in the hepcidin receptor ferroportin (Fpn). 1 Fpn is the only known cellular iron exporter in vertebrates and is expressed on all tissues that handle major iron flows, including absorptive enterocytes, iron-recycling macrophages, and iron-storing hepatocytes. 2 The rate of iron efflux from these cells is a major determinant of iron concentration in plasma and is directly proportional to the number of Fpn molecules on the cell surface. Furthermore, by controlling the absorption of dietary iron, Fpn expression on the basolateral membranes of enterocytes ultimately determines total body iron. The concentration of cell surface Fpn is regulated mainly by the interaction with its ligand hepcidin. Hepcidin binding to Fpn results in internalization and degradation of the ligand-receptor complex and leads to decreased iron efflux from cells into plasma. 3 The hepcidin-Fpn interaction is critical for normal iron homeostasis and underlies the pathogenesis of iron disorders, including not only hereditary hemochromatosis, but also anemia of inflammation and iron-loading anemias. 4 Although the complete loss of Fpn is embryonic lethal in zebrafish and mice, 2,5 some heterozygous missense mutations in the Fpn gene result in an autosomal-dominant form of iron overload disease called ...

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“…It has been suggested that mutations in Fpn might result in cellular iron overload due to haploinsufficiency 10,12 ; however, mice that are heterozygous for a targeted gene deletion of Fpn do not show evidence of iron-overload disease. 14 To further explore the possibility that haploinsufficiency could explain decreased iron export, we used RNAi to silence mouse Fpn in macrophages.…”

Section: Mutant Fpn Ffe Acts As a Dominant Negative In Macrophages Ismentioning

confidence: 99%

“…[9][10][11] The mechanism by which the disease mutations exert a dominant effect is unclear. Some groups that study the disease suggest that it results from haploinsufficiency, 10,12 whereas others suggest that the disorder results from a dominantnegative effect of the mutant allele. 9,13 Importantly, all human mutations are missense mutations and mice that are heterozygous for a targeted deletion of Fpn do not show the disease.…”

Section: Introductionmentioning

confidence: 99%

The flatiron mutation in mouse ferroportin acts as a dominant negative to cause ferroportin disease

Zohn

Domenico

Pollock

et al. 2007

Blood

105

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IntroductionHereditary hemochromatosis is a common disorder in humans, characterized by iron overload resulting in tissue injury and ultimately organ failure. Typically, hemochromatosis exhibits an autosomal-recessive pattern of inheritance and is associated with mutations in HFE, hemojuvelin, hepcidin, or transferrin receptor 2. 1,2 Targeted deletion of these genes in the mouse results in hemochromatosis, providing mouse models for most forms of the disease. Hemochromatosis type IV, also referred to as ferroportin (Fpn) disease, results from mutations in the iron transporter ferroportin. Fpn is the only known iron exporter in mammalian cells and is present on the surface of macrophages, intestinal enterocytes, hepatocytes, and placental cells. [3][4][5] The level of cell surface Fpn is regulated by its interaction with hepcidin, a peptide secreted by the liver in response to iron stores and inflammation. Hepcidin binds to Fpn, inducing its internalization and degradation, thus regulating the export of iron from cells to plasma. 6 Mutations in Fpn lead to iron-overload disease but, in contrast to other forms of hemochromatasis, ferroportin disease exhibits an autosomal-dominant pattern of inheritance. 7 The disorder has different presentations depending on the Fpn mutation. Mutations leading to Fpn that is not internalized by hepcidin result in iron accumulation in hepatocytes and high transferrin saturation. 8,9 Mutations leading to Fpn that is not appropriately targeted to the cell surface result in iron accumulation in Kupffer cells and low transferrin saturation. [9][10][11] The mechanism by which the disease mutations exert a dominant effect is unclear. Some groups that study the disease suggest that it results from haploinsufficiency, 10,12 whereas others suggest that the disorder results from a dominantnegative effect of the mutant allele. 9,13 Importantly, all human mutations are missense mutations and mice that are heterozygous for a targeted deletion of Fpn do not show the disease. 14 Treatment for hemochromatosis aims to decrease iron load by repeated phlebotomy and this treatment works well for most patients. Many patients with ferroportin disease, however, become anemic with phlebotomy, highlighting the need for a mouse model to develop better treatments.We report here on a missense mutation in mouse Fpn that results in a disorder that is identical to classic human ferroportin disease. We show that macrophages isolated from mutant mice have no Fpn on their cell surface and that expression of Fpn constructs containing the missense mutation (H32R) affects the behavior of wild-type Fpn. These results show that Fpn disease is due to a dominant-negative effect of the mutant allele and provide the first mouse model for this disorder. Materials and methods Generation of mutant mice and identification of Fpn mutationThe ffe mouse line was identified in a screen for recessive ethylnitrosourea (ENU)-induced mutations that cause morphologic abnormalities at embryonic day (E) 12.5. [15][16][17] The ffe mutation...

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Wild-type and mutant ferroportins do not form oligomers in transfected cells

Cited by 42 publications

References 38 publications

Iron feeding induces ferroportin 1 and hephaestin migration and interaction in rat duodenal epithelium

Iron feeding induces ferroportin 1 and hephaestin migration and interaction in rat duodenal epithelium

The molecular basis of hepcidin-resistant hereditary hemochromatosis

The flatiron mutation in mouse ferroportin acts as a dominant negative to cause ferroportin disease

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