THE HEMOCHROMATOSIS GENE-PRODUCT COMPLEXES WITH THE TRANSFERRIN RECEPTOR AND LOWERS ITS AFFINITY FOR LIGAND-BINDING

We recently reported the positional cloning of a candidate gene for he reditary hemochromatosis called HFE, The gene product, a member of the major histocompatibility complex class I-like family, was found to ha ve a mutation, Cys-282 --> Tyr (C282Y), in 85% of patient chromosomes, This mutation eliminates the ability of HFE to associate with beta(2) -microglobulin (beta(2)m) and prevents cell-surface expression, A seco nd mutation that has no effect on beta(2)m association, H63D, was foun d in eight out of nine patients heterozygous for the C282Y mutant, In this report, we demonstrate in cultured 293 cells overexpressing mild- type or mutant HFE proteins that both the wild-type and H63D HFE prote ins form stable complexes with the transferrin receptor (TfR). The C28 2Y mutation nearly completely prevents the association of the mutant H FE protein with the TfR. Studies on cell-associated transferrin at 37 degrees C suggest that the overexpressed wild-type HFE protein decreas es the affinity of the TfR for transferrin, The overexpressed H63D pro tein does not have this effect, providing the first direct evidence fo r a functional consequence of the H63D mutation, Addition of soluble w ild-type HFE/beta(2)m heterodimers to cultured cells also decreased th e apparent affinity of the TfR for its ligand under steady-state condi tions, both in 293 cells and in HeLa cells, Furthermore, at 4 degrees C, the added soluble complex of HFE/beta(2)m inhibited binding of tran sferrin to HeLa cell TfR in a concentration-dependent manner. Scatchar d plots of these data indicate that the added heterodimer substantiall y reduced the affinity of TfR for transferrin. These results establish a molecular link between HFE and a key protein involved in iron trans port, the TfR, and raise the possibility that alterations in this regu latory mechanism may play a role in the pathogenesis of hereditary hem ochromatosis.