Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis

Abstract

Hereditary hemochromatosis is a common autosomal recessive disorder of iron metabolism. Recent demonstration of an association between transferrin receptor (TfR) and HFE, a major histocompatibility complex class I-like molecule that has been implicated to play a role in hereditary hemochromatosis, further strengthens the notion that HFE is involved in iron metabolism. Herein we show that TfR is required for and controls the assembly and the intracellular transport and surface expression of HFE. Because surface-expressed HFE and TfR remain firmly associated physically, only the fraction of TfR that is associated with HFE during biosynthesis is affected functionally. Moreover, we show that HFE binding reduces the number of functional transferrin binding sites and impairs TfR internalization, thus reducing the uptake of transferrin-bound iron. Thus, iron homeostasis is indirectly regulated by HFE, a negative modulator of TfR.

Figure 1

Biosynthesis, assembly, and intracellular transport of HFE and TfR. (A) Association of β₂m and TfR with HFE. LS10 cells grown for 48 h in the absence of tetracycline were labeled with [³⁵S]methionine for 1 h (lanes 1–4) followed by a 4-h chase (lanes 5–8). Nonidet P-40 lysates were first incubated with the monoclonal antibody 10G4 (lanes 1 and 5). Immunoprecipitates were then boiled in the presence of 0.1% SDS and reimmunoprecipitated with 10G4 and anti-β₂m K355 (lanes 2 and 6), HFE-C (lanes 3 and 7), and anti-TfR (lanes 4 and 8) antibodies. Molecular mass markers are indicated in kDa on the left. (B and C) Pulse–chase analysis of the expression and intracellular transport of HFE in LS10 cells. Expression of HFE in LS10 cells was induced for 48 h. Cells were labeled for 30 min and then chased for various times: B, 0 min (lanes 1 and 6), 30 min (lanes 2 and 7), 1 h (lanes 3 and 8), 3 h (lanes 4 and 9), and 6 h (lanes 5 and 10); C, 0 min (lanes 1 and 7), 30 min (lanes 2 and 8), 1 h (lanes 3 and 9), 2 h (lanes 4 and 10), 4 h (lanes 5 and 11), and 6 h (lanes 6 and 12). The asterisk indicates nonspecific bands. Immunoprecipitated materials with 10G4 (B) or HFE-C (C) were divided into two portions and incubated at 37°C for 16 h with or without endo H. (D) Recognition of the C-terminal epitope of HFE by HFE-C. LS10 cells were labeled for 30 min and chased for 0 min (lanes 1 and 4), 2 h (lanes 2 and 5), and 4 h (lanes 3 and 6). Nonidet P-40 lysates were divided into two aliquots, one of which was boiled in the presence of 0.1% SDS. Samples were immunoprecipitated with HFE-C.

Figure 2

Effect of TfR on surface expression of HFE. (A) Effect of TfR supertransfection on the biosynthesis and intracellular transport of HFE. LS10 cells without (lanes 1–3) or with (lanes 4–6) expression of additional TfR were labeled for 30 min and chased for 0 min (lanes 1 and 4), 2 h (lanes 2 and 5), or 4 h (lanes 3 and 6). Immunoprecipitates with 10G4 were analyzed by SDS/PAGE and fluorography. (B) Densitometric analysis of intracellular transport of HFE^H and TfR. Values were expressed relative to the level of HFE^H or TfR in LS10 cells at zero chase time point, which was arbitrarily set at 1.0 arbitrary units (A.U.). Four experiments were performed.

Figure 3

Effect of HFE on Tf uptake. HeLa cells transiently expressing HFE were incubated with FITC-Tf for 30 min at 37°C and examined by confocal fluorescence microscopy. (A) Cells expressing HFE (e.g., the cell on the upper left corner) were identified by incubation with the antiserum HFE-C and Texas red-conjugated goat anti rabbit IgG. (B) Differences in intensities of intracellular FITC-Tf between cells with (e.g., the cell on the upper left corner) and without (e.g., the cell on the lower right corner) HFE expression was apparent. The presence of surface-expressed HFE was also detected in an aliquot of the same cells with 10G4 (data not shown).

Figure 4

Effect of HFE on number of cell-surface Tf-binding sites. LS10 cells were grown for 48 h in medium containing tetracycline at concentrations of 1.0 μg/ml (A) or 0 μg/ml (B). Saturation binding curves were determined after incubating cells for 90 min at 4°C in the presence of various concentrations of ¹²⁵I-labeled Tf. Results are expressed as means ± SEM of triplicate measurements. Scatchard analyses were performed with the prizm software program (GraphPad, San Diego). It was calculated that in the absence of HFE the apparent K_D for TfR-bound Tf was 4.9 ± 1.0 nM, whereas in the presence of HFE the K_D was 4.9 ± 1.1 nM. Four experiments were performed.

Figure 5

Effect of HFE on TfR internalization. LS10 cells were grown for 48 h in the presence of tetracycline at 1.0 μg/ml (No HFE), 0.01 μg/ml (Lo HFE), or 0 μg/ml (Hi HFE). (A) The S/I ratio represents the ratio of the endocytic externalization to internalization rate constants. (B) Determination of the internalization constants. Cells were incubated for 2, 4, 6, or 8 min at 37°C with ¹²⁵I-labeled Tf, and an acid wash was used to distinguish between membrane-bound and internalized Tf. A plot of the ratio of internalized to steady-state surface Tf binding versus time yields a straight line whose slope is the internalization rate constant. All measured k_int values are presented in Table 1. (C) Determination of the externalization constants. After cell surface-bound ¹²⁵I-labeled Tf was removed by the deferoxamine wash, cells were then incubated at 37°C for 2, 4, 8, or 16 min, and the radioactivity that remained associated with the cells as well as that was released into the media was determined. Values were corrected for nonspecific binding and the values for k_ext are presented in Table 1. The results presented are the means ± SEM from triplicate determinations. Three experiments were performed.