| HFE is a class I MHC-related protein that is mutated in patients with the iron overload disease hereditary hemochromatosis. HFE binds to the transferrin receptor (TfR), reducing its affinity for iron-loaded transferrin (Tf). The prominent role that TfR plays in iron uptake suggests that HFE binding to TfR is critical for the involvement of HFE in the regulation of iron homeostasis. We have characterized the interaction between HFE, TfR and Tf. Using soluble, recombinant forms of HFE and TfR, we demonstrated that HFE binds tightly at the basic pH of the cell surface, but not at the acidic pH of intracellular vesicles. In solution, only one HFE molecule associates with dimeric TfR, as compared with the binding of two Tf molecules by TfR dimers. Further biochemical analyses of the interaction between HFE, TfR and transferrin demonstrated that TfR could bind simultaneously to both HFE and Tf to form a ternary complex, and that HFE inhibits the TfR-Tf interaction by binding at or near the Tf binding site on TfR. Using the 2.6 A crystal structure of HFE, we designed a series of site directed mutants and measured their binding affinities for TfR. These studies allowed localization of the TfR binding site on the HFE structure to the C-terminal portion of the alpha1 domain helix and the loop connecting the first and second beta-strands in alpha1. These results are discussed with reference to the postulated role of HFE in intracellular trafficking, which we have begun to investigate in transfected intestinal cells using an HFE-green fluorescent protein construct. |
