FLVCRb: a Mitochondrial FLVCR Isoform Important for Erythropoiesis

Abstract 4243

Feline Leukemia Virus subgroup C Receptor (FLVCR) was originally identified and cloned as a cell-surface protein receptor for feline leukemia virus subgroup C, causing pure red blood cell aplasia in cats. Recent studies have demonstrated that FLVCR is a heme exporter which is essential for erythropoiesis. The heme efflux via FLVCR was shown to be essential for erythroid differentiation in K562 cells as well as in CD34+ precursors cells¹. Moreover, Keel and co-authors have reported that Flvcr-null mice die in utero due to the failure of fetal erythropoiesis; also post-natal mice lacking FLVCR showed severe anemia. In addition to the erythroid defect, Flvcr-null embryos display defective growth and developmental anomalies².

We have identified an alternative transcription start site giving rise to a novel FLVCR isoform (FLVCRb). Flvcr-b transcript completely lacks the first exon of the canonical isoform (FLVCRa) and code for a putative 6 transmembrane domain containing protein ubiquitously expressed. In vitro over-expression of FLVCRa and FLVCRb showed that the two proteins display different subcellular localization. As expected, FLVCRa is localized at the cell membrane while FLVCRb is in the mitochondrial compartment. The mitochondrial localization of this novel isoform is further confirmed by the identification of a N-terminal mitochondrial sorting presequence.

The mitochondrion is the site in which heme biosynthesis occurs. Although all the enzymatic reactions involved in heme synthesis are well characterized, how heme is exported to the cytosol is largely unknown. Because of FLVCRa is a heme exporter at the cell membrane, we hypothesized that FLVCRb could be the mitochondrial heme exporter. According to this hypothesis, FLVCRb expression increased following the stimulation of heme biosynthesis in vitro, in correlation with the increase in hemoglobin production. The ability of FLVCRb to bind and export heme out of the mitochondria is still under investigation.

To gain insights into the specific roles of the two isoforms, we have generated Flvcr mutant mice different from those previously reported². Keel and co-author generated a mouse model in which both FLVCRa and FLVCRb have been deleted. In our mouse model, FLVCRa has been specifically deleted while FLVCRb is still expressed (FLVCRa-null mice).

Flvcr-a +/− mice were grossly normal, fertile and indistinguishable from their wild-type littermates. When Flvcr-a +/− mice were intercrossed, no Flvcr-a homozygous knock-out newborns were obtained. The analysis of the embryos from timed Flvcr-a +/− intercrosses showed that the Flvcr-a homozygous knock-out genotype was lethal between E14.5 and the birth.

E13.5 Flvcr-a-null embryos showed multifocal and extended hemorrhages, visible in the limbs, head and throughout the body wall, as well as subcutaneous edema. Imcomplete vasculogenesis in the Flvcr-a-null embryos was observed at E11.5, a developmental stage in which hemorrhages were not still evident. This suggests that hemorrhages arise from a defect in the development of embryonic vasculature. Moreover, FLVCRa-null embryos showed skeletal abnormalities as demonstrated by Alcian blue-alizarin red staining. Skeletal malformations were evident in the limb where digits did not form properly and in the head where Meckel's cartilage was incomplete. It is interesting to note that this kind of malformations also occurs in Diamond Blackfan Anemia (DBA) patients. Surprisingly, flow cytometric analyses of E14.5 fetal liver cells double-stained for Ter119 (erythroid-specific antigen) and CD71 (transferrin receptor) showed normal erythropoiesis in Flvcr-a-null embryos, in opposition to what occurs in the previously reported Flvcr-null mice².

Taken together, these data demonstrated that FLVCRb is sufficient to support fetal erythropoiesis when the expression of FLVCRa is loss, likely exporting heme out of the mithocondrion for hemoglobin synthesis. Moreover, the loss of FLVCRa leads to incomplete vasculogenesis, hemorrhages and skeletal malformations highlighting new roles of FLVCRa in these processes.

1. Quigley JG et al. Identification of a human heme exporter that is essential for erythropoiesis. Cell 2004

2. Keel SB et al. A heme export protein is required for red blood cell differentiation and iron homeostasis. Science 2008.