Abstract

[Introduction and aim] Transferrin receptor 2 (TfR2) is a homologue of the classical transferrin receptor 1 (TfR1), and TfR2 has two isoforms, α and β. Like TfR1, TfR2α is a type II membrane protein expressing mainly in the hepatocytes 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 diferric transferrin (Tf), and is therefore thought likely to be involved in cellular iron metabolism. In fact, mutations of TfR2 cause hemochromatosis, implying that the function of TfR2 might be a regulation of iron metabolism. The aim of the present study was to investigate the interaction of TfR2α with Tf by functional assays and atomic force microscopy (AFM), which would be a powerful tool for investigation of the interaction between ligand and receptor on living cell surface.

[Methods] To investigate the functional properties of TfR2α, we established the stable clone that expresses TfR2α protein with FLAG-tagging from transferrin receptor-deficient Chinese hamster ovary (CHO) cells (TRVb). That clone was applied for the 125I –labeled Tf (125I –Tf) binding study at 4 oC. The cells were applied for Tf and iron uptake study using 125I –Tf and 59Fe –loaded Tf at 37 oC. AFM is the method that can investigate the unbinding force between receptor and ligand at single molecule level when their binding is physically detached, and we applied this method for determine the interaction between Tf and TfR2α using the transiently transfected human hepatome-derived HLF cells with TfR2α-expression vector.

[Results] The association constant for binding of 125I-Tf to TfR2α was calculated to be 5.6 x 106 M−1 from non-linear least squares curve fitting to a saturable binding isotherm, which is much lower than that of TfR1. Although CHO cells showed a receptor-independent non-specific association with Tf at 37 oC, we observed cell-associated Tf persisting after acid-washing in TfR2α overexpressing cells, We also confirmied the existence of internalized Tf into cells via TfR2α. Overexpressed TfR2α protein was also shown to mediate iron uptake although its rate of iron donation is slower than TfR1. The interaction between Tf and TfR2α was also confirmed by AFM, but the unbinding force was different from that between Tf and TfR1. This report might be the first that showed the binding and unbinding properties between Tf and TfR2α by AFM.

[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 functional assays. AFM study also showed the binding between Tf and TfR2α on cell surface, but the properties of unbinding between them were different from those between Tf and TfR1. In conclusion, TfRα binds to Tf at cell surface and functions as iron donator to the cells like TfR1, but there should be the difference in the properties of binding with Tf between TfR1 and TfR2α, implying that the difference makes them having different physiological role in living organisms.

Disclosures: No relevant conflicts of interest to declare.

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