Effect of a mutation in transferrin receptor 2 on the uptake of iron

Hereditary haemochromatosis is an iron overload disorder associated with misregulation of iron metabolism. Subtype III arises from mutations in the transferrin receptor 2 {TfR2) gene. TfR2 is thought to mediate transferrin bound iron (TBI) uptake by a lowaffinity receptor-mediated endocytic process. The V22F mutation in TfR2 is situated adjacent to the putative TfR2 endocytosis signal and results in a valine to phenylalanine substitution and has been shown to cause haemochromatosis. The aims of this study were to investigate the functional effects of the V22F mutation on the uptake of iron. To study the functional effects of TfR2V22F on cellular iron uptake, the V22F mutation (TfR2inF) was generated by site-directed mutagenesis and transfected into the Chinese Hamster Ovary (CHO-TRVb) cell line that is deficient in endogenous transferrin receptors. To confirm the subcellular localisation of TfR2, cells containing each construct were subjected to immunocytochemistry using a specific antibody, before being visualised by fluorescence microscopy. The distribution of TfR2 V22F and TfR2WT was predominantly on the cellular surface, consistent with being a surface receptor. Cells were incubated with l25I-Tf-59Fe and treated with Pronase to separate into membrane-bound and internalised fractions. Higher levels of surface and internalised transferrin and iron were observed in cells containing TfR2WT compared with TfR2VEC, signifying that TfR2 mediates TBI uptake. Mutant cells exhibited increased TBI uptake. suggesting that the mutation causes an up-regulation in TBI uptake compared with TfR2WT. To investigate the effects of the mutation on TfR2 cycling, the endocytic and exocytic rates were determined. Cells were incubated with different concentrations of transferrin (0.1 to 10|iM) to measure endocytosis. On increasing concentrations, transferrin internalisation in mutants and wild-type was saturable, consistent with a receptor-mediated process. Similar Kms were obtained for CHO-TRVbV22F. and CHO-TRVwt , however, the mutants exhibited a 1.7-fold higher Kmax, suggestive of an increased rate of endocytosis. Similarly, after loading cells with 125l-Tf-59Fe, followed by incubation in efflux medium, CHO-TRVbV22F demonstrated a 1.6-fold higher rate of transferrin release when compared to CHO-TRYbVVT. Rates of iron release were similar between cells, showing the mutation does not affect iron release but increases the exocytic rate of transferrin When cells were incubated with non-transferrin bound iron (NTBI) in the form of ^Feaccumulated low levels of NTBI in contrast to cells expressing and TfR2WT, suggesting that TfR2 mediates NTBI uptake. The rate of uptake was markedly higher in the mutants than wild-type, which implies that the V22F citrate, CHO-TRVbVEC TfR2V22F mutation induces an increased rate of uptake of NTBI over and above that apparent in CHO-TRVbWT. The results suggest that cells containing TfR2V22F, in comparison to cells containing ^ WT TfR2 , have an enhanced ability to internalise TBI and NTBI in vitro. These findings are consistent with the notion that the V22F mutation affects TfR2-mediated endocytosis, leading to type III haemochromatosis in vivo