Iron is efficiently recycled after erythrocytes are turned over by the reticuloendothelial system. Most retrieved iron is complexed to the iron- transport/-scavenging proteins transferrin, hemopexin (heme), haptoglobin (hemoglobin), serum ferritin, and lactoferrin (Lf) and delivered to the liver where it is endocytosed by hepatocytes. In hepatocytes, iron is stored indefinitely or packaged with transferrin and exported. We have found that hepatocytes endocytose Lf and its bound iron by specific Ca2+- dependent recycling binding sites. The high number and endocytic vigor of these sites suggests that Lf-mediated iron delivery may constitute the dominant iron scavenging pathway in liver. Hepatocyte clearance of plasma Lf also regulates (i) its inhibitory effects on myelopoiesis and chylomicron remnant uptake, and (ii) its association with glycated serum proteins. The role of Lf plays in normal hepatic iron metabolism and aberrant hepatic iron overload, however, is poorly understood, and the hepatic Lf receptor has not been identified. Our broad long-term objectives are to understand the molecular basis of Lf-cell interactions, Lf's iron transport activity, and the biological effects of Lf on cells that bind it. Studies outlined in this proposal focus on two critical issues: (i) explaining Lf delivery of iron to hepatocytes, and (ii) identification and molecular analysis of the hepatic Lf receptor. We propose three specific aims to accomplish this. 1. Analyze hepatocyte uptake and processing of diferric Lf. We will extend our previous studies by examining the cellular itinerary followed by endocytosed Lf and its bound iron and determining the intracellular site of Lf-iron dissociation. We will use vesicular and cytoplasmic fractions isolated from hepatocytes to reconstitute iron transport from the endocytic compartment to the cytoplasm. Because of the high endocytic capacity of hepatocytes for iron-loaded Lf, these studies will allow us to analyze iron translocation in vitro. 2. Confirm the 120 kDa Lf-binding protein as the hepatic Lf Receptor. We have identified a 120 kDa hepatocyte protein (p120) that possesses Lf receptor activity. Anti-p120 immunoglobulins will be affinity-purified from anti-p120 sera on hand and assayed for their ability to block endocytosis of Lf by hepatocytes. Chemical and photoactivatable crosslinkers will be used to determine if Lf binds p120 on intact cells. These approaches will (i) confirm whether p120 is the bona fide hepatocyte Lf receptor and (ii) identify other cellular Lf- binding proteins. 3. Cloning and sequencing the hepatocyte Lf receptor. Lf receptor peptides will be sequenced. This information will be used to develop oligonucleotide probes for the isolation and cloning of hepatocyte Lf receptor gene. Lf receptor cDNAs will be isolated from a rat liver cDNA library, cloned, and sequenced. The deduced amino acid sequence will provide information about the receptor's important structural motifs which will be used to determine experimentally the receptor's functional properties and expression during development.
