The hereditary hemochromatosis protein, HFE, lowers intracellular iron levels independently of transferrin receptor 1 in TRVb cells

Abstract

Hereditary hemochromatosis (HH) is an autosomal recessive disease that leads to parenchymal iron accumulation. The most common form of HH is caused by a single amino acid substitution in the HH protein, HFE, but the mechanism by which HFE regulates iron homeostasis is not known. In the absence of transferrin (Tf), HFE interacts with transferrin receptor 1 (TfR1) and the 2 proteins co-internalize, and in vitro studies have shown that HFE and Tf compete for TfR1 binding. IntroductionHereditary hemochromatosis (HH) is a common autosomal recessive disease of iron metabolism characterized by gradual accumulation of excess iron in organs such as the liver, heart, pancreas, and thyroid, resulting in symptoms, including hepatic cirrhosis and hepatocellular carcinoma, cardiomyopathy and arrhythmias, diabetes, and hypogonadotropic hypogonadism. 1,2 The most common form of HH is caused by a single base pair mutation in the HH gene that results in the substitution of tyrosine for cysteine at amino acid position 260 (C260Y; The numbering system for both HFE and low-density lipoprotein receptor [LDLR] is based on the mature protein and does not include the signal sequence) in the mature protein, HFE. 3 HFE is an atypical major histocompatibility complex (MHC) class 1-related protein. The C260Y mutation disrupts a disulfide bond in its ␣3 domain, which abrogates its association with the ␤2-microglobulin (␤2M) light chain and subsequent trafficking to the cell surface. 4 Although the importance of functional HFE-␤2M heterodimers in the maintenance of iron homeostasis has been further documented in studies using HFE knock-out mice, 5,6 the mechanism by which HFE regulates iron metabolism in cells is still unknown.In vitro and in vivo studies show that HFE associates with transferrin receptor 1 (TfR1) at neutral pH [7][8][9][10] and that the binding sites for HFE and Tf overlap in TfR. [11][12][13] In vitro experiments demonstrate that HFE competes with Tf for binding to TfR1 at concentrations of diferric Tf lower than 100 nM. 11 These experiments explain the early observation that HFE appears to lower the binding affinity of TfR to diferric Tf. 7,14 Indeed, in HeLa, HEK293, and H1299 cells, expression of exogenous HFE results in the lowering of intracellular iron levels. However, HeLa and H1299 cells expressing HFE have about 30% lower rates of Tf-mediated iron uptake than parental controls, even in the presence of sufficiently high concentrations of diferric Tf that HFE does not affect the uptake of Tf. 15,16 In addition, the W81A HFE mutant regulates Tf-mediated iron uptake to the same extent as wild-type HFE in HeLa cells, 17 despite showing a 5000-fold lower affinity for TfR1 binding. 18 These studies show that the effect of HFE on iron homeostasis does not depend solely on the interaction with TfR1 and is more complex than simple competition between HFE and Tf for binding to TfR1.In this study we took advantage of a Chinese hamster ovary cell line, TRVb cells, which lacks endogenous transferrin receptors. 19 These cells w...

The Hereditary Hemochromatosis Protein, HFE, Inhibits Iron Uptake via Down-regulation of Zip14 in HepG2 Cells

“…HFE reduces NTBI uptake in Chinese hamster ovary cells lacking endogenous TfR1 and TBI uptake in Chinese hamster ovary cells transfected with TfR1 (18). A single divalent iron transporter could explain the shared pathway for TBI and NTBI uptake, which functions in the endosome for TBI uptake and on cell membrane for NTBI uptake.…”

“…Hepcidin is a hormone secreted by the liver that negatively regulates dietary iron uptake by the intestine. HFE can also lower NTBI uptake in isolated primary mouse hepatocytes (9) and in Chinese hamster ovary cells lacking endogenous TfR1 (18). Thus, the mechanism by which HFE regulates iron metabolism still remains elusive.…”

“…Iron Treatment of Cells-Fe-NTA was freshly prepared by mixing 500 mM FeCl 3 and 530 mM NTA at a volume ratio of 1:3.77 (18). HepG2 cells and HepG2/HFE cells were seeded in a 6-well plate at a concentration of 2 ϫ 10 5 cells/well.…”

Src Regulates Tyr20 Phosphorylation of Transferrin Receptor-1 and Potentiates Breast Cancer Cell Survival

“…TfR1 is a type II transmembrane glycoprotein, which contains a large extracellular C-terminal domain (671 amino acids), a single-pass transmembrane domain (28 amino acids), and a short intracellular N-terminal domain (61 amino acids) (3). The tetrapeptide 20 YTRF 23 motif within the cytoplasmic region of TfR1 has been shown as the structural recognition motif to TfR1 endocytosis (4).…”

Ubiquitination of lysine-331 by Kaposi's sarcoma-associated herpesvirus protein K5 targets HFE for lysosomal degradation

“…At physiological concentrations of iron-bound transferrin (10 μM holo-Tf), it was shown that HFE could not compete for binding to TfR1 because of its lower affinity, but that cellular iron levels were reduced by HFE expression. HFE therefore appeared to be regulating some aspect of iron import in the endosome, independent of TfR1 binding (8)(9)(10). HFE may also regulate iron export in some cell types (11,12).…”