Kinetics and Specificity of Feline Leukemia Virus Subgroup C Receptor (FLVCR) Export Function and Its Dependence on Hemopexin

Abstract

The feline leukemia virus subgroup C receptor (FLVCR) is a heme export protein that is required for proerythroblast survival and facilitates macrophage heme iron recycling. However, its mechanism of heme export and substrate specificity are uncharacterized. Using [55 Fe]heme and the fluorescent heme analog zinc mesoporphyrin, we investigated whether export by FLVCR depends on the availability and avidity of extracellular heme-binding proteins. Export was 100-fold more efficient when the medium contained hemopexin (K d < 1 pM) compared with albumin (K d ‫؍‬ 5 nM) at the same concentration and was not detectable when the medium lacked heme-binding proteins. Besides heme, FLVCR could export other cyclic planar porphyrins, such as protoporphyrin IX and coproporphyrin. However, FLVCR has a narrow substrate range because unconjugated bilirubin, the primary breakdown product of heme, was not transported. As neither protoporphyrin IX nor coproporphyrin export improved with extracellular hemopexin (versus albumin), our observations further suggest that hemopexin, an abundant protein with a serum concentration (6.7-25 M) equivalent to that of the iron transport protein transferrin (22-31 M), by accepting heme from FLVCR and targeting it to the liver, might regulate macrophage heme export and heme iron recycling in vivo. Final studies show that hemopexin directly interacts with FLVCR, which also helps explain why FLVCR, in contrast to some major facilitator superfamily members, does not function as a bidirectional gradient-dependent transporter. Together, these data argue that hemopexin has a role in assuring systemic iron balance during homeostasis in addition to its established role as a scavenger during internal bleeding or hemolysis.The biological roles of heme are diverse and encompass most areas of cell metabolism and gene regulation. Heme serves as a prosthetic group in the hemeproteins, which are involved in crucial biological functions, including oxygen binding (hemoglobin and myoglobin), oxygen metabolism (oxidases, peroxidases, catalases, and hydroxylases), electron transfer (cytochromes) (4), and signal transduction (nitric-oxide synthases) (5). In addition, heme serves as a signaling molecule in erythropoiesis and other physiological systems. Because heme generally signals by binding and inactivating a transcriptional repressor (e.g. Bach1) or translational inhibitor (e.g. heme-regulated inhibitor and thus eIF␣2 kinase), its impact is immediate (6 -8). Perhaps for this reason, heme is utilized in time-sensitive processes, such as the regulation of circadian rhythm (9), N-end rule pathway/protein ubiquitination (10), and globin synthesis. Heme is also required for microRNA processing (11). However, excess free or non-protein-bound heme damages lipid, protein, and DNA through the generation of reactive oxygen species, resulting in cellular injury and death (12). Therefore, free cellular heme levels must be tightly regulated to provide an adequate supply yet avoid heme toxicities (4,(13)(14)(15).Most studies of h...

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

“…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. Hemopexin facilitates heme export by FLVCR1 by binding to a 69-amino acid peptide (residues 132-201), which contains two exoplasmic loops and two TMDs (44). In silico examination of the membrane topology of FLVCR2 predicts a 12-TMD protein with several strategically placed, evolutionarily conserved, heme-binding ligands in the exoplasmic and cytoplasmic loops and within the TMDs.…”

Wearing Red for Signaling: The Heme-Bach Axis in Heme Metabolism, Oxidative Stress Response and Iron Immunology

“…Hemopexin, an abundant heme carrier protein in the serum, accepts heme by directly interacting with FLVCR ( Fig. 1) (Yang et al 2010). FLVCR achieves a balance between heme and globin by exporting excess heme, preventing the toxicity of free heme.…”

Heme Utilization in the Caenorhabditis elegans Hypodermal Cells Is Facilitated by Heme-responsive Gene-2

“…The feline leukemia virus subgroup C cellular receptor (FLVCR), a major facilitator superfamily protein, is involved in heme export in red blood cells and macrophages (8,9). Additionally, a number of proteins such as hemopexin (10,11), p22 HBP (12), HBP23 (13), and certain classes of glutathione S-transferases (GSTs) (14 -16) have been shown to associate with heme, and correspondingly, these proteins have been implicated in heme homeostasis.…”