The goal of this project is to study how heme trafficking might impact systemic iron homeostasis. As 25 mg of iron is required daily to maintain red cell production, only 1-2 mg/d is absorbed from the GI tract, and 1-2 mg/d is lost through sloughed gastrointestinal, skin and kidney mucosal cells, most iron is recycled. The traditional view is that when macrophages engulf senescent red cells, their hemoglobin is degraded to heme then iron, which is exported via ferroportin to transferrin for delivery to the liver (for storage) or to the marrow (for new red cell production). In previous studies, we determined that the feline leukemia virus-C receptor, FLVCR, specifically exports heme from cells (Cell 118:757-66, 2004). On western analysis, FLVCR is highly expressed in the duodenum, liver and macrophage, tissues critical for iron absorption and trafficking; and Flvcr-deleted mice have excess iron at these sites, in addition to red cell aplasia (Science 319:825-828, 2008). These observations, plus studies of cell lines and primary macrophages in vitro, led us to hypothesize that heme, not only iron, is trafficked. This grant will define FLVCR structure and discern the mechanism by which it exports heme to heme-binding proteins, such as hemopexin and albumin, using mutagenesis analyses, Xenopus oocytes studies, and electron crystallography. We will determine how iron regulates the intercellular localization of FLVCR to aid systemic iron balance. In addition, we will determine the fate of heme from red cells after the cells are ingested by macrophages by studying macrophages from Flvcrflox/flox;LysM-cre mice in vitro and in vivo; determine the fate of heme once delivered to liver by studying Flvcrflox/flox;alb-cre mice; determine if hepatocytes can secrete heme via FLVCR into the bile permitting iron to exit the body; and explore the implications of these findings in models of hemolysis, chronic inflammation, and hemochromatosis. Together these studies should demonstrate that the physiologic regulation of iron is more intricate and complex than previously appreciated. PUBLIC HEALTH RELEVANCE: Systemic iron balance largely depends on macrophage iron recycling (20-25 mg/d in persons) and minimally on the ingestion of iron in the diet (1-2 mg/day). The current concept is that heme from red cells is converted to iron, then recycled to the marrow to make new red cells or transported to the liver for storage. Our data challenge this accepted paradigm; demonstrate that heme, and not only iron, is trafficked; and suggest that this trafficking could help regulate systemic iron balance and/or modify the clinical manifestations of hemolysis, chronic inflammation and hemochromatosis.