EDITORIAL FOCUS
From genetics to cellular physiology.
Focus on "Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells"

Curtis T. Okamoto

Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089-9121


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HEREDITARY HEMOCHROMATOSIS (HH) is a common inherited disorder of people of Northern European descent, affecting some 1 in 400 people. HH is a disorder of iron metabolism characterized by iron overload in many organs, particularly in the liver, pancreas, heart, and pituitary, leading to multiorgan dysfunction and premature death. Positional cloning of the gene for HH resulted in the identification of a relatively widely expressed gene, named HFE, that was curiously homologous in its predicted amino acid sequence and structure to major histocompatibility complex (MHC) class I molecules (1). Thus, from the genetic data, the role of HFE in iron metabolism could not be immediately deduced.

However, since the initial cloning of HFE, progress in the characterization of its role in iron metabolism at the cellular and molecular level has been impressively rapid. By using physiological systems and heterologous expression systems, several groups have begun to characterize the role of HFE in cellular iron uptake (2-9), with the latest contribution by Schwake et al., the current article in focus (Ref. 10, see p. C973 in this issue). The collective data from these groups have dramatically changed the way in which we think about cellular iron uptake mediated by the transferrin receptor (TfR), the key cell surface receptor that mediates cellular iron uptake by binding to extracellular iron-loaded transferrin and endocytosing it. Such data include showing in immunoprecipitation studies that wild-type HFE is physically associated with the TfR (2-4, 6, 9). Wild-type HFE was also found to colocalize with the TfR at the plasma membrane and in intracellular compartments, presumably endocytic organelles (3, 7). An apparent functional consequence of the association of wild-type HFE with TfR is that the affinity of cell surface TfR for transferrin is lowered (2, 4, 5, 9). All of these data suggest that wild-type HFE cotraffics with the TfR, and this association may regulate cellular iron uptake into cells by altering the affinity of the TfR for transferrin either at the cell surface or in endocytic compartments. Further supportive evidence for a role of HFE in the regulation of TfR-mediated iron uptake is that the most common mutation found in HFE, the missense C282Y mutation occurring in over 80% of HH patients, nearly eliminates the capacity of HFE to interact with the TfR (2), and the mutated HFE protein is retained in the endoplasmic reticulum/Golgi apparatus (7). In addition, expression of the mutated C282Y HFE did not alter the affinity of cell surface TfR for transferrin (2). Thus, in cells expressing mutated C282Y HFE, transferrin (iron) uptake would be predicted to be higher, which would provide a plausible mechanism for iron overload in cells and organs of HH patients.

In the study in focus here, Schwake et al. (10) have applied a powerful biophysical approach, cell-attached and whole cell capacitance measurements, to characterize the role of HFE in transferrin internalization. They were able to measure transferrin-dependent endocytotic events in a heterologous cell system in which the expression level of HFE and the mutated C282Y HFE could be regulated by a tetracycline-sensitive promoter. Their data suggest an alternate, although not necessarily mutually exclusive, function for HFE: a negative regulator of TfR internalization from the cell surface. This study makes several important, novel contributions, not only with respect to characterization of HFE-regulated cellular iron uptake but also to cell physiology in general. First, this study provides surprising evidence suggesting that transferrin stimulates endocytotic events, presumably by stimulation of the internalization of the TfR, a receptor traditionally considered to be a constitutively endocytosing receptor (that is, one whose endocytosis is not dependent on the presence of ligand). Thus the TfR, perhaps by virtue of its interaction with HFE, may actually be a tightly regulated endocytotic receptor for iron uptake. Second, as the role of HFE in the regulation of TfR internalization has been controversial, the characterization by capacitance measurements of the role of HFE as a negative regulator of TfR internalization is significant, particularly with respect to understanding the overall mechanism by which HFE regulates iron metabolism. However, because there are conflicting data in this area (4, 8, 9), it will be an important one to resolve in the future. Third, Schwake et al. have been able to show that the C282Y mutation significantly attenuates HFE's negative regulatory function in transferrin-stimulated endocytosis, providing an alternative or additional mechanism to account for increased iron uptake in cells and organs of HH patients. Fourth, they demonstrate that capacitance measurements are sensitive enough to characterize the kinetics of ligand-receptor endocytotic events and may therefore be applicable to the study of a wide variety of endocytosing receptors and be an approach of significant interest to cell biologists studying receptor-mediated endocytosis.

Intriguing questions remain with respect to HFE and its role in iron metabolism. By virtue of its homology to MHC class I molecules, which bind to beta 2-microglobulin, HFE predictably has also been shown to bind to beta 2-microglobulin (2, 4, 6, 9). Thus the TfR may exist in a ternary complex with beta 2-microglobulin and HFE. Although the contribution of beta 2-microglobulin in iron metabolism has yet to be fully characterized, it plays at least an indirect role because the binding of beta 2-microglobulin to HFE regulates HFE trafficking along the biosynthetic pathway (7). In addition, the HFE C282Y mutation abrogates this interaction, resulting in the retention of mutated HFE in the early biosynthetic pathway (7). Clearly, the regulation of the dynamic interactions among HFE, beta 2-microglobulin, and the TfR represents an important future area of investigation in the cellular physiology of iron metabolism, perhaps employing additional novel approaches. More importantly, hints of bench-to-bedside benefits of the research into HFE come from the report that soluble truncated fragments of HFE complexed to beta 2-microglobulin inhibit transferrin binding to the TfR (2); perhaps such soluble fragments or derivatives thereof could be used therapeutically to alleviate iron overload in HH patients. In conclusion, there remains continued promise for success in an area that has progressed rapidly from genetics to cellular and molecular physiology, using novel approaches such as that used in the article in focus.


    ACKNOWLEDGEMENTS

This essay was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-51588.


    FOOTNOTES

Address for reprint requests and other correspondence: C. T. Okamoto, Dept. of Pharmaceutical Sciences, School of Pharmacy, Univ. of Southern California, 1985 Zonal Ave., Los Angeles, CA 90089-9121 (E-mail: cokamoto{at}hsc.usc.edu).

10.1152/ajpcell.00009.2002


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1.   Feder, JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, Domingo R, Jr, Ellis MC, Fullan A, Hinton LM, Jones NL, Kimmel BE, Kronmal GS, Lauer P, Lee VK, Loeb DB, Mapa FA, McClelland E, Meyer NC, Mintier GA, Moeller N, Moore T, Morikang E, Prass CE, Quintana L, Starnes SM, Schatzman RC, Brunke KJ, Drayna DT, Risch NJ, Bacon BR, and Wolff RK. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 13: 399-408, 1996[ISI][Medline].

2.   Feder, JN, Penny DM, Irrinki A, Lee VK, Lebrón JA, Watson N, Tsuchihashi Z, Sigal E, Bjorkman PJ, and Schatzman RC. The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding. Proc Natl Acad Sci USA 95: 1472-1477, 1998[Abstract/Free Full Text].

3.   Gross, CN, Irrinki A, Feder JN, and Enns CA. Co-trafficking of HFE, a nonclassical major histocompatibility complex class I protein, with the transferrin receptor implies a role in intracellular iron regulation. J Biol Chem 273: 22068-22074, 1998[Abstract/Free Full Text].

4.   Ikuta, K, Fujimoto Y, Suzuki Y, Tanaka K, Saito H, Ohhira M, Sasaki K, and Kohgo Y. Overexpression of hemochromatosis protein, HFE, alters transferrin recycling process in human hepatoma cells. Biochim Biophys Acta 1496: 221-231, 2000[ISI][Medline].

5.   Lebrón, JA, West AP, Jr, and Bjorkman PJ. The hemochromatosis protein HFE competes with transferrin for binding to the transferrin receptor. J Mol Biol 294: 239-245, 1999[ISI][Medline].

6.   Parkkila, S, Waheed A, Britton RS, Bacon BR, Zhou XY, Tomatsu S, Fleming RE, and Sly WS. Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis. Proc Natl Acad Sci USA 94: 13198-13202, 1997[Abstract/Free Full Text].

7.   Ramalingam, TS, West AP, Jr, Lebrón JA, Nangiana JS, Hogan TH, Enns CA, and Bjorkman PJ. Binding to the transferrin receptor is required for endocytosis of HFE and regulation of iron homeostasis. Nat Cell Biol 2: 953-957, 2000[ISI][Medline].

8.   Roy, CN, Penny DM, Feder JN, and Enns CA. The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells. J Biol Chem 274: 9022-9028, 1999[Abstract/Free Full Text].

9.   Salter-Cid, L, Brunmark A, Li Y, Leturcq D, Peterson PA, Jackson MR, and Yang Y. Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis. Proc Natl Acad Sci USA 96: 5434-5439, 1999[Abstract/Free Full Text].

10.   Schwake, L, Henkel AW, Riedel HD, Schlenker T, Both M, Migala A, Hadaschik B, Henfling N, and Stremmel W. Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells. Am J Physiol Cell Physiol 282: C973-C979, 2002[Abstract/Free Full Text].


Am J Physiol Cell Physiol 282(5):C971-C972
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society