The mechanisms by which the hereditary hemochromatosis protein, HFE, decreases 
transferrin-mediated iron uptake were examined. Coimmunoprecipitation studies 
using solubilized cell extracts demonstrated that transferrin (Tf) competed with 
HFE for binding to the transferrin receptor (TfR) similar to previous in vitro 
studies using soluble truncated forms of HFE and the TfR. At concentrations of 
Tf approaching those found in the blood, no differences in Tf binding to cells 
were detected, which is consistent with the lower binding constant of HFE for 
TfR versus Tf. However, cells expressing HFE still showed a decrease in 
Tf-mediated iron uptake at concentrations of Tf sufficient to dissociate HFE 
from the TfR. These results indicate that the association of HFE with TfR is not 
essential for its ability to lower intracellular iron stores. To test the effect 
of HFE on lowering intracellular iron levels independently of its association 
with TfR, a mutated HFE (fW81AHFE) that shows greatly reduced affinity for the 
TfR was transfected into tetracycline-controlled transactivator HeLa cells. HeLa 
cells expressing fW81AHFE behaved in a similar manner to cells expressing 
wild-type HFE with respect to decreased intracellular iron levels measured by 
iron regulatory protein gel-shift assays and ferritin levels. The results 
indicate that HFE can lower intracellular iron levels independently of its 
interaction with the TfR.