The Fowler Syndrome-Associated Protein FLVCR2 Is an Importer of Heme

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. 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 heminconjugated 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.Membrane transporters play essential roles in cellular homeostasis by importing substrates critical for cell growth and differentiation or by exporting substrates that cause toxicity. There are five major categories of membrane transporters consisting of over 550 transporter superfamilies (41). The major facilitator superfamily (MFS) is the largest and most diverse superfamily, consisting of over 10,000 members (31, 41). Transporters in this superfamily consist of 12 to 14 transmembrane (TM)-spanning segments and transport substrates as diverse as sugars, polyols, drugs, neurotransmitters, amino acids, organic/inorganic ions, and peptides (31). Recently, a disruption of MFS transporters that is associated with human diseases has been described, further confirming their role in the maintenance of normal cell homeostasis. The DIRC2 MFS transporter (substrate transported unknown) is disrupted in renal cell carcinoma cosegregating with a t(2;3)(q35;q21) chromosomal translocation (4). Mutations in the thiamine transporter THTR1 have been shown to be responsible for Rogers syndrome (14, 21), a thiamine-responsive megaloblastic anemia. We have recently reported that a disruption in the heme exporter FLVCR1 (MFSD7B) plays a role in Diamond Blackfan anemia (DBA) (40), a fatal infant anemia characterized by a block in erythroid progenitor cell development (3, 12, 13). The abrogation of FLVCR1 function in primary human hematopoietic stem cells (40) or in a human erythroid cell line (37) specifically disrupts erythropoiesis, mimicking the hematological features observed for patients with DBA. We have reported previously that FLVCR1 is disrupted not as a consequence of mutations in the FLVCR1 coding region but due to the aberrant...

Topologically Conserved Residues Direct Heme Transport in HRG-1-related Proteins

“…FLVCR2, a member of the major facilitator superfamily, was recently reported to import heme in mammalian cells (28). Although we did not observe heme import in our assay conditions, given the high degree of homology between FLVCR2 and the heme effluxer FLVCR1 (41)(42)(43), it is possible that FLVCR2 may export heme.…”

“…We next determined whether the assays employed to determine the heme import function of hHRG-1 can be extended to FLVCR2, another transporter recently postulated to be a heme importer in humans cells (28). hFLVCR2 was abundantly expressed in hem1⌬ yeast and localized primarily to the plasma membrane.…”

A Novel Population of α-Smooth Muscle Actin-Positive Cells Activated in a Rat Model of Stroke: An Analysis of the Spatio-Temporal Distribution in Response to Ischemia

“…However, transforming growth factorb has been reported to induce aSMA in the brain (Verbeek et al, 1994) and retinal (Sieczkiewicz and Herman, 2003) pericytes, perhaps suggesting that aSMA-expression (aSMA + ) can be associated with a more mature phenotype. The possibility that pericytes might express the feline leukemia virus C receptor 2 (FLVCR2) transporter (Brasier et al, 2004) of heme (Duffy et al, 2010) as described during angiogenesis in fetal development of the brain (Thomas et al, 2010) warranted an investigation of its expression in the adult brain by the aSMA + cells described in this study.…”

Non-Canonical Heme-Binding Proteins

“…A simple approach is the capture of apo-proteins by means of resins, usually agarose crosslinked with hemin. 39 This method has facilitated the isolation of plasma, 40 intracellular 41-43 and membrane-linked 44,45 HBPs. Cleared cell lysates are incubated with immobilized hemin, then washed with buffer and the proteins are eluted by changing the ionic strength.…”