Transferrin receptor 2 (TfR2) is a homologue of transferrin receptor 1 (TfR1), the protein that delivers iron to cells through receptor-mediated endocytosis of diferric transferrin (Fe 2 Tf). TfR2 also binds Fe 2 Tf, but it seems to function primarily in the regulation of systemic iron homeostasis. In contrast to TfR1, the trafficking of TfR2 within the cell has not been extensively characterized. Previously, we showed that Fe 2 Tf increases TfR2 stability, suggesting that trafficking of TfR2 may be regulated by interaction with its ligand. In the present study, therefore, we sought to identify the mode of TfR2 degradation, to characterize TfR2 trafficking, and to determine how Fe 2 Tf stabilizes TfR2. Stabilization of TfR2 by bafilomycin implies that TfR2 traffics to the lysosome for degradation. Confocal microscopy reveals that treatment of cells with Fe 2 Tf increases the fraction of TfR2 localizing to recycling endosomes and decreases the fraction of TfR2 localizing to late endosomes. Mutational analysis of TfR2 shows that the mutation G679A, which blocks TfR2 binding to Fe 2 Tf, increases the rate of receptor turnover and prevents stabilization by Fe 2 Tf, indicating a direct role of Fe 2 Tf in TfR2 stabilization. The mutation Y23A in the cytoplasmic domain of TfR2 inhibits its internalization and degradation, implicating YQRV as an endocytic motif. INTRODUCTIONA truncation mutant of transferrin receptor 2 (TfR2), TfR2/ Y250X, causes a rare form of hereditary hemochromatosis (type 3, HFE3), an iron overload disorder characterized by excess absorption of dietary iron and consequent deposition of iron in liver and other parenchymal tissues (Camaschella et al., 1999(Camaschella et al., , 2000. The analogous mutation or knockout of Trfr2 in mice reproduces the disease phenotype (Fleming et al., 2002;Wallace et al., 2005). Thus, TfR2 is required for normal iron homeostasis.TfR2, cloned in 1999, is a homologue of transferrin receptor 1 (TfR1; Kawabata et al., 1999). The extracellular domains of the two receptors are 45% identical and 67% similar. TfR1 functions to deliver iron to cells through receptor-mediated endocytosis of its ligand transferrin (Tf), a serum protein that transports iron (Dautry-Varsat et al., 1983;Klausner et al., 1983). On the cell surface, TfR1 binds iron-saturated transferrin (Fe 2 Tf) at slightly basic pH. Fe 2 Tf-TfR1 then internalizes in clathrin-coated vesicles to early endosomes.In the acidic pH of early endosomes, iron releases from Tf, whereas Tf remains bound to TfR1. The complex then recycles, from either early or recycling endosomes, to the cell surface. At the slightly basic pH of the cell surface, TfR1 releases unsaturated Tf (apoTf) and again binds Fe 2 Tf. TfR1 expression is ubiquitous, consistent with its role in cellular iron delivery. The stability of TfR1 mRNA is negatively regulated by intracellular iron levels through iron-responsive elements (IREs) in the 3Ј untranslated region (Mattia et al., 1984;Ward et al., 1984;Rao et al., 1985;Sciot et al., 1987;Owen and Kuhn, 19...