The Cytoplasmic domain of transferrin receptor 2 dictates its stability and response to holo-transferrin in Hep3B cells

Transferrin receptor 2 (TfR2) is a homolog of transferrin receptor 1 (TfR1), the receptor responsible for the uptake of iron-loaded transferrin (holo-Tf) into cells. Unlike the ubiquitous TfR1, TfR2 is predominantly expressed in the liver. Mutations in TfR2 gene cause a rare autosomal recessive form of the iron overload disease, hereditary hemochromatosis. Previous studies demonstrated that holo-Tf increases TfR2 levels by stabilizing TfR2 at the protein level. In this study we constructed two chimeras, one of which had the cytoplasmic domain of TfR2 and the remaining portion of TfR1 and the other with the cytoplasmic and transmembrane domain of TfR1 joined to the ectodomain of TfR2. Similar to TfR2, the levels of the chimera containing only the cytoplasmic domain of TfR2 increased in a time- and dose-dependent manner after the addition of holo-Tf to the medium. The half-life of the chimera increased 2.7-fold in cells exposed to holo-Tf like the endogenous TfR2 in HepG2 cells. Like TfR2 and unlike TfR1, the levels of the chimera did not respond to intracellular iron content. These results suggest that although holo-Tf binding to the ectodomain is necessary, the cytoplasmic domain of TfR2 is largely responsible for its stabilization by holo-Tf.     

HFE modulates transferrin receptor 2 levels in hepatoma cells via interactions that differ from transferrin receptor 1-HFE interactions

Mutations in the transmembrane glycoproteins transferrin receptor 2 (TfR2) and HFE are associated with hereditary hemochromatosis. Interactions between HFE and transferrin receptor 1 (TfR1) have been mapped to the alpha1- and alpha2-helices in HFE and to the helical domain of TfR1. Recently, TfR2 was also reported to interact with HFE in transfected mammalian cells. To test whether similar HFE residues are important for both TfR1 and TfR2 binding, a mutant form of HFE (W81AHFE) that has an approximately 5,000-fold lower affinity for TfR1 than HFE was employed. As expected, W81AHFE does not interact with TfR1. However, we found that the same mutation in HFE does not affect the TfR2/HFE interaction. This finding indicates that the TfR2/HFE and TfR1/HFE interactions are distinct. We further observed that, unlike TfR1/HFE, Tf does not compete with HFE for binding to TfR2 and that binding is independent of pH (pH 6-7.5). TfR2-TfR1 and HFE-HLA-B7 chimeras were generated to map the domains of the TfR2/HFE interaction. TfR1 and HLA-B7 were chosen because of their similar overall structures with TfR2 and HFE, respectively. We mapped the interacting domains to the putative stalk and protease-like domains of TfR2 located between residues 104 and 250 and to the alpha3 domain of HFE, both of which differ from the TfR1/HFE interacting domains. Furthermore, we found that HFE increases TfR2 levels in hepatic cells independent of holo-Tf.     

The hereditary hemochromatosis protein, HFE, inhibits iron uptake via down-regulation of Zip14 in HepG2 cells

Lack of functional hereditary hemochromatosis protein, HFE, causes iron overload predominantly in hepatocytes, the major site of HFE expression in the liver. In this study, we investigated the role of HFE in the regulation of both transferrin-bound iron (TBI) and non-transferrin-bound iron (NTBI) uptake in HepG2 cells, a human hepatoma cell line. Expression of HFE decreased both TBI and NTBI uptake. It also resulted in a decrease in the protein levels of Zip14 with no evident change in the mRNA level of Zip14. Zip14 (Slc39a14) is a metal transporter that mediates NTBI into cells (Liuzzi, J. P., Aydemir, F., Nam, H., Knutson, M. D., and Cousins, R. J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 13612-13617). Knockdown of Zip14 with siRNA abolished the effect of HFE on NTBI uptake. To determine if HFE had a similar effect on Zip14 in another cell line, HeLa cells expressing HFE under the tetracycline-repressible promoter were transfected with Zip14. As in HepG2 cells, HFE expression inhibited NTBI uptake by approximately 50% and decreased Zip14 protein levels. Further analysis of protein turnover indicated that the half-life of Zip14 is lower in cells that express HFE. These results suggest that HFE decreases the stability of Zip14 and therefore reduces the iron loading in HepG2 cells.     

Iron homeostasis: recently identified proteins provide insight into novel control mechanisms

Iron is an essential nutrient required for a variety of biochemical processes. It is a vital component of the heme in hemoglobin, myoglobin, and cytochromes and is also an essential cofactor for non-heme enzymes such as ribonucleotide reductase, the limiting enzyme for DNA synthesis. When in excess, iron is toxic because it generates superoxide anions and hydroxyl radicals that react readily with biological molecules, including proteins, lipids, and DNA. As a result, humans possess elegant control mechanisms to maintain iron homeostasis by coordinately regulating iron absorption, iron recycling, and mobilization of stored iron. Disruption of these processes causes either iron-deficient anemia or iron overload disorders. In this minireview, we focus on the roles of recently identified proteins in the regulation of iron homeostasis.     

Influence of inter-item symmetry in visual search

Does visual search involve a serial inspection of individual items (Feature Integration Theory) or are items grouped and segregated prior to their consideration as a possible target (Attentional Engagement Theory)? For search items defined by motion and shape there is strong support for prior grouping (Kingstone and Bischof, 1999). The present study tested for grouping based on inter-item shape symmetry. Results showed that target-distractor symmetry strongly influenced search whereas distractor-distractor symmetry influenced search more weakly. This indicates that static shapes are evaluated for similarity to one another prior to their explicit identification as 'target' or 'distractor'. Possible reasons for the unequal contributions of target-distractor and distractor-distractor relations are discussed.     

Cytoskeletal Protein ABP-280 Directs the Intracellular Trafficking of Furin and Modulates Proprotein Processing in the Endocytic Pathway

Furin catalyzes the proteolytic maturation of many proproteins within the trans-Golgi network (TGN)/endosomal system. Furin's cytosolic domain (cd) directs both the compartmentalization to and transit between its manifold processing compartments (i.e., TGN/biosynthetic pathway, cell surface, and endosomes). Here we report the identification of the first furin cd sorting protein, ABP-280 (nonmuscle filamin), an actin gelation protein. The furin cd was used as bait in a yeast two-hybrid screen to identify ABP-280 as a furin-binding protein. Binding analyses in vitro and coimmunoprecipitation studies in vivo showed that furin and ABP-280 interact directly and that ABP-280 tethers furin molecules to the cell surface. Quantitative analysis of both ABP-280-deficient and genetically replete cells showed that ABP-280 modulates the rate of internalization of furin but not of the transferrin receptor, a cycling receptor. However, although ABP-280 directs the rate of furin internalization, the efficiency of sorting of the endoprotease from the cell surface to early endosomes is independent of expression of ABP-280. By contrast, efficient sorting of furin from early endosomes to the TGN requires expression of ABP-280. In addition, ABP-280 is also required for the correct localization of late endosomes (dextran bead uptake) and lysosomes (LAMP-1 staining), demonstrating a pleiotropic role for this actin binding protein in the organization of cellular compartments and directing protein traffic. Finally, and consistent with the trafficking studies on furin, we showed that ABP-280 modulates the processing of furin substrates in the endocytic but not the biosynthetic pathways. The novel roles of ABP-280 and the cytoskeleton in the sorting of furin in the TGN/ endosomal system and the formation of proprotein processing compartments are discussed.