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 Kcell 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 Kcell 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 Kcell 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 Ki is 87 nM.