The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding

Abstract

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis called HFE. The gene product, a member of the major histocompatibility complex class I-like family, was found to have a mutation, Cys-282 --> Tyr (C282Y), in 85% of patient chromosomes. This mutation eliminates the ability of HFE to associate with beta2-microglobulin (beta2m) and prevents cell-surface expression. A second mutation that has no effect on beta2m association, H63D, was found in eight out of nine patients heterozygous for the C282Y mutant. In this report, we demonstrate in cultured 293 cells overexpressing wild-type or mutant HFE proteins that both the wild-type and H63D HFE proteins form stable complexes with the transferrin receptor (TfR). The C282Y mutation nearly completely prevents the association of the mutant HFE protein with the TfR. Studies on cell-associated transferrin at 37 degrees C suggest that the overexpressed wild-type HFE protein decreases the affinity of the TfR for transferrin. The overexpressed H63D protein does not have this effect, providing the first direct evidence for a functional consequence of the H63D mutation. Addition of soluble wild-type HFE/beta2m heterodimers to cultured cells also decreased the apparent affinity of the TfR for its ligand under steady-state conditions, both in 293 cells and in HeLa cells. Furthermore, at 4 degrees C, the added soluble complex of HFE/beta2m inhibited binding of transferrin to HeLa cell TfR in a concentration-dependent manner. Scatchard plots of these data indicate that the added heterodimer substantially reduced the affinity of TfR for transferrin. These results establish a molecular link between HFE and a key protein involved in iron transport, the TfR, and raise the possibility that alterations in this regulatory mechanism may play a role in the pathogenesis of hereditary hemochromatosis.

Figure 1

Cell-surface labeling of HFE and association with TfR. (A) HFE antibodies immunoprecipitate 12, 49, 100, and 200 kDa surface-labeled proteins from wild-type HFE expressing cells. The coimmunoprecipitating proteins are greatly reduced or absent in immune complexes from parental 293 or C282Y HFE mutant expressing cells. (B) FLAG epitope antibodies also immunoprecipitate 12, 49, 100, and 200 kDa surface-labeled proteins in wild-type HFE expressing cells. As in A, the coimmunoprecipitating proteins are greatly reduced or absent in immune complexes from parental 293 or C282Y HFE mutant expressing cells. (C) TfR antibodies immunoprecipitate 100- and 200-kDa surface-labeled proteins from parental 293, wild-type, and C282Y HFE expressing cells and in addition, immunoprecipitate 12- and 49-kDa proteins from wild-type HFE expressing cells. (D) HLA-ABC antibodies fail to immunoprecipitate 100- and 200-kDa proteins from parental 293 cells.

Figure 2

Direct association of TfR with HFE. (A) HFE antibodies coimmunoprecipitate TfR from wild-type and H63D HFE expressing cells but very little from parental 293 or C282Y HFE mutant expressing cells. (B) HFE antibodies immunoprecipitate substantial amounts of HFE protein from wild-type, C282Y and H63D HFE expressing cells. (C) TfR antibodies coimmunoprecipitate HFE from wild-type and H63D HFE expressor cells but not parental 293 and only a trace from C282Y mutant expressing cells. (D) TfR antibodies immunoprecipitate TfR protein from parental 293, and wild-type, C282Y and H63D HFE expressing cells. (E) FLAG epitope (M2) antibodies coimmunoprecipitate TfR from wild-type and H63D HFE expressing cells but not parental 293 or C282Y HFE mutant expressing cells.

Figure 3

Effect of HFE on cell-association of [¹²⁵I]-transferrin. (A) Cell association of [¹²⁵I]-transferrin in cells that overexpress the C282Y (intracellular) mutant form of HFE. Approximately 9 × 10⁵ cells (clone 10, □; and clone 12, ▪) were incubated with various concentrations of transferrin at 37°C for 20 min (Inset). The data represent the mean of duplicate determinations corrected for nonspecific binding. Scatchard analysis revealed an K_{cell association} of ≈12 and 14 nM, respectively. (B) Cell-association of [¹²⁵I]-transferrin in cells overexpressing the wild-type (surface) form of HFE (clone 7, ○; clone 3, •). Scatchard analysis revealed that the K_{cell association} for transferrin was 180 and 40 nM, respectively. (C) Cell-association of [¹²⁵I]-transferrin in cells overexpressing the H63D mutant form of HFE (clone 3, ▵; clone 8, ▴). Scatchard analysis revealed a K_{cell association} for transferrin to be 12 and 20 nM, respectively. (D) Inhibition of cell-associated [¹²⁵I]-transferrin to the TfR on parental 293 cells by addition of soluble HFE/β₂m heterodimers. Parental 293 cells were preincubated with various amounts of soluble HFE/β₂m heterodimers followed by addition of 10 nM [¹²⁵I]-transferrin. The amount of specific cell-associated transferrin was determined and plotted as a percent of that observed in cells with no added HFE. The apparent K_i is 87 nM.

Figure 4

Soluble HFE/β₂m heterodimers reduce the affinity of the TfR for transferrin. (A) Effect of HFE/β₂m heterodimers on the cell-association of [¹²⁵I]-transferrin to HeLa cells at 37°C. Cell-associated [¹²⁵I]-transferrin in HeLa cells in the presence of 0, 150, or 1,000 nM HFE or 100 nM soluble HLA-B27/β₂m heterodimers (Inset). Scatchard analysis of cell associated [¹²⁵I]-transferrin in the presence of 0 nM (□), 150 nM (▴), or 1,000 nM HFE (▵) revealed K_{cell association} values of 4.7 nM, 11 nM, and 50 nM, respectively and ≈1.0 × 10⁶ transferrin binding sites per cell. (Note: the addition of 1,000 nM soluble HFE/β₂m heterodimers dramatically reduces cell-associated transferrin such that an accurate x-intercept is difficult to obtain; the K_{cell association} 50 nM is estimated utilizing 1.0 × 10⁶ binding sites per cell.) Addition of 100 nM soluble HLA-B27/β₂m heterodimers (▪) to the assay had no effect. (B) Determination of the apparent K_i of soluble HFE/β₂m heterodimers for cell-associated transferrin inhibition in HeLa cells. Cells were preincubated at 37°C in various amounts of soluble HFE/β₂m heterodimers and then [¹²⁵I]-transferrin was added to 5 nM. The amount of specific cell-associated transferrin was determined and plotted as a percent of that observed in cells with no added HFE. The apparent K_i for HeLa is 39 nM. (C) Effect of HFE/β₂m heterodimers on the binding of [¹²⁵I]-transferrin to HeLa cells at 4°C. Binding of [¹²⁵I]-transferrin to HeLa cells in the absence or presence of 0, 35, or 1,000 nM HFE (inset). Scatchard analysis of [¹²⁵I]-transferrin binding in the presence of 0 (□), 35 (▴), or 1,000 nM (▵) HFE revealed apparent K_D values of 5 nM, 12 nM, and 75 nM, respectively and ≈1.0 × 10⁶ transferrin binding sites per cell. (Note: the addition of 1,000 nM soluble HFE/β₂m heterodimers dramatically reduces transferrin binding such that an accurate x intercept is difficult to obtain; the apparent K_D of 75 nM is estimated utilizing 1.0 × 10⁶ binding sites per cell.)