Intracellular Recycling and Iron Donating Ability of Transferrin Receptor 2α Protein Expressed in Transferrin Receptor-Deficient Chinese Hamster Ovary Cells.

[Introduction] Transferrin receptor 2 (TfR2), a homologue of the transferrin receptor 1 (TfR1), is found in two isoforms, α and β. Like the classical TfR1, TfR2α is a type II membrane protein expressing mainly in the hepatocytes as homodimer on their cell surface, but the β form lacks intracellular and transmembrane portions and therefore is likely to be an intracellular protein. Although the main physiological functions of these two isoforms are still not fully understood, TfR2α binds transferrin (Tf), and is therefore thought likely to be involved in cellular iron metabolism. Disruption of TfR2 leads to hemochromatosis, implying that the function of TfR2 might be a regulation of iron metabolism. However, TfR1 have remarkably high binding affinity to diferric Tf and only little expression on the cell surface of hepatocytes so that TfR1 must be fully saturated under physiological conditions. Besides, TfR2 is upregulated in some conditions in that iron accumulation was observed, for instance in the liver of the patients of hepatitis C, indicating that TfR2 might also function as the iron donator for the hepatocytes. The aim of the present study was to investigate the interactions of TfR2α with Tf, and to elucidate whether TfR2α have the ability of iron donation to the cells.

[Methods] To investigate the functional properties of TfR2α, we expressed TfR2α protein with FLAG-tagging in transferrin receptor-deficient Chinese hamster ovary (CHO) cells (TRVb). The stably transfected cells expressing TfR2α-FLAG were applied for the ¹²⁵I -labeled Tf (¹²⁵I -Tf) binding study at 4 °C. The cells were applied for Tf and iron uptake study using ¹²⁵I -Tf and ⁵⁹Fe -loaded at 37 °C. To determine the fate of Tf internalized into the cells, the release of internalized Tf were then investigated. Degradation of released Tf was also investigated by precipitation using trichloroacetic acid/phosphotungustic acid.

[Results] The association constant for binding of ¹²⁵I-Tf to TfR2α was calculated to be 5.6 × 10⁶ M⁻¹ from non-linear least squares curve fitting to a saturable binding isotherm, which is about 50 times lower than that of TfR1. Although CHO cells showed a receptor-independent non-specific association with Tf at 37 °C, we observed cell-associated Tf persisting after acid-washing in TfR2α overexpressing cells, confirming that the existence of Tf internalization via TfR2α. After internalization, efflux of Tf, without substantial degradation, is also confirmed. Thus, Tf internalized by TfR2α, and presumably recycles, like TfR1 pathway. Overexpressed TfR2α protein was also shown to mediate iron uptake although its rate of iron donation is slower than TfR1.

[Discussion and Conclusions] TfR2α binds to Tf although its affinity for Tf is low, and TfR2α possesses iron donating ability to the cells when TfR2α is over-expressed in our study. Although TfR2α has been reported to be involved in iron sensing and regulation of hepcidin expression, the involvement of the Tf binding and iron donating properties of TfR2α should be considered when the pathophysiology of iron metabolism is investigated, especially in the conditions that TfR2α is upregulated, for instance in hepatitis C.