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. 2010 Nov;30(22):5318-24.
doi: 10.1128/MCB.00690-10. Epub 2010 Sep 7.

The Fowler syndrome-associated protein FLVCR2 is an importer of heme

Simon P Duffy  1 , Jennifer Shing, Punit Saraon, Lloyd C Berger, Maribeth V Eiden, Andrew Wilde, Chetankumar S Tailor
Affiliations

The Fowler syndrome-associated protein FLVCR2 is an importer of heme

Simon P Duffy et al. Mol Cell Biol. 2010 Nov.

Abstract

Mutations in FLVCR2, a cell surface protein related by homology and membrane topology to the heme exporter/retroviral receptor FLVCR1, have recently been associated with Fowler syndrome, a vascular disorder of the brain VSports手机版. We previously identified FLVCR2 to function as a receptor for FY981 feline leukemia virus (FeLV). However, the cellular function of FLVCR2 remains unresolved. Here, we report the cellular function of FLVCR2 as an importer of heme, based on the following observations. First, FLVCR2 binds to hemin-conjugated agarose, and binding is competed by free hemin. Second, mammalian cells and Xenopus laevis oocytes expressing FLVCR2 display enhanced heme uptake. Third, heme import is reduced after the expression of FLVCR2-specific small interfering RNA (siRNA) or after the binding of the FY981 FeLV envelope protein to the FLVCR2 receptor. Finally, cells overexpressing FLVCR2 are more sensitive to heme toxicity, a finding most likely attributable to enhanced heme uptake. Tissue expression analysis indicates that FLVCR2 is expressed in a broad range of human tissues, including liver, placenta, brain, and kidney. The identification of a cellular function for FLVCR2 will have important implications in elucidating the pathogenic mechanisms of Fowler syndrome and of phenotypically associated disorders. .

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Figures (V体育安卓版)

FIG. 1.
FIG. 1.
Hemin binding by FLVCR1 and FLVCR2. (A) Immunoblots of cell lysate samples from CHO cells and CHO cells expressing the HA-tagged huFLVCR1, huFLVCR2, and feTHTR1 proteins incubated with and without hemin-agarose. Proteins were detected by using anti-HA-horseradish peroxidase (HRP) antibody. (B) Hemin-agarose binding by HA-tagged huFLVCR2 after preincubation with 25 μM and 50 μM free hemin. The percent bound huFLVCR2 relative to the control (0 μM) is shown below the blot.
FIG. 2.
FIG. 2.
Heme import by human FLVCR2. (A) Inhibition of ZnMP uptake by expression of the FY981 FeLV envelope (Env) protein in TELCeB6 (T6) cells. T6 cells (control) and T6 cells expressing the FeLV-C (T6/C Env) or FY981 (T6/FY Env) Env protein were incubated with ZnMP and analyzed by flow cytometry to determine the mean fluorescence intensity (MFI). The percent MFI value of ZnMP uptake was determined relative to the uptake by control T6 cells. MFI values are averages from three uptake experiments. Standard deviation (SD) bars are shown. (B) CHO cells (control) and CHO cells expressing huFLVCR1 or huFLVCR2 were incubated with ZnMP, and the MFI was determined by flow cytometry. ZnMP uptake is relative to that of control CHO cells and is the average of data from three uptake experiments. (C) [55Fe]hemin uptake in Xenopus oocytes. Oocytes injected with huFLVCR2 cRNA or water were incubated with [55Fe]hemin at room temperature. [55Fe]hemin uptake is an average of 10 oocytes used per group for each experiment. Results were repeated in three separate uptake experiments. The P value was calculated by using a Student's t test.
FIG. 3.
FIG. 3.
Downregulation of human FLVCR2 disrupts heme uptake. (A) Validation of FLVCR2 knockdown by FLVCR2 siRNA. CHO/huFLVCR2 or CHO/huFLVCR1 cells were transiently transfected with huFLVCR2-specific siRNA oligonucleotide S1 or S2 or with scrambled (Scr) siRNA, and the expression of the HA-tagged huFLVCR2 or huFLVCR1 (control) protein was analyzed by Western blotting. (B) ZnMP uptake in human TE671 cells transiently transfected with FLVCR2-specific siRNA oligonucleotide S1 or S2 or scrambled (Scr) siRNA. The MFI value of ZnMP uptake was determined by flow cytometry, and the percent MFI was calculated relative to ZnMP uptake by TE671 cells expressing the Scr siRNA oligonucleotide. The MFI is the average of data from three uptake experiments. SD bars are shown. The P value was calculated by using a Student's t test.
FIG. 4.
FIG. 4.
FLVCR2-expressing CHO cells are more sensitive to heme toxicity. CHO cells (control) and CHO/huFLVCR1 and CHO/huFLVCR2 cells were plated in a 24-well plate and exposed to increasing doses of hemin for 24 hours. Cell survival was analyzed by staining with trypan blue (which stains dead cells), and the percentage of unstained viable cells compared to stained dead cells was calculated. Results are averages of data from three experiments. SD bars and P values are shown. The P value was calculated by using a Student's t test.
FIG. 5.
FIG. 5.
FLVCR2 transcript expression in human tissues. The tissue distribution of FLVCR2 mRNA was determined by quantitative real-time PCR using cDNA from various hematopoietic and nonhematopoietic human tissues and using FLVCR2-specific primers. The FLVCR2 mRNA expression level is relative to that of β-actin mRNA expression. Data presented are averages of data from three independent real-time PCR experiments. SD are shown.
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