HFE and transferrin directly compete for transferrin receptor in solution and at the cell surface

Creators: Giannetti, Anthony M.; Bjorkman, Pamela J.

Abstract

Transferrin receptor (TfR) is a dimeric cell surface protein that binds both the serum iron transport protein transferrin (Fe-Tf) and HFE, the protein mutated in patients with the iron overload disorder hereditary hemochromatosis. HFE and Fe-Tf can bind simultaneously to TfR to form a ternary complex, but HFE binding to TfR lowers the apparent affinity of the Fe-Tf/TfR interaction. This apparent affinity reduction could result from direct competition between HFE and Fe-Tf for their overlapping binding sites on each TfR polypeptide chain, from negative cooperativity, or from a combination of both. To explore the mechanism of the affinity reduction, we constructed a heterodimeric TfR that contains mutations such that one TfR chain binds only HFE and the other binds only Fe-Tf. Binding studies using a heterodimeric form of soluble TfR demonstrate that TfR does not exhibit cooperativity in heterotropic ligand binding, suggesting that some or all of the effects of HFE on iron homeostasis result from competition with Fe-Tf for TfR binding. Experiments using transfected cell lines demonstrate a physiological role for this competition in altering HFE trafficking patterns.

Additional Information

© 2004 The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, February 10, 2004, and in revised form, March 30, 2004. Published, JBC Papers in Press, March 31, 2004, DOI 10.1074/jbc.M401467200. We thank Peter Snow, Inderjit Nangiana, and Cynthia Jones (Caltech Protein Expression Facility) for insect cell expression of TfR constructs; Tim McGraw (Cornell) for providing TRVb and TRVb-1 cells; W. Lance Martin and Jasvinder Nangiana for construction of expression vectors; Rochelle Diamond (Caltech Cell Sorting Facility) for cell sorting; Kirsten Lassila for assistance with CD data collection; the Beckman Imaging Center at Caltech for providing confocal microscopes and support; Andrew Herr, Caroline Enns, Devin Tesar, and Rich Olson for helpful discussions; Dave Myszka for beta versions of Scrubber and Clamp; and members of the Björkman laboratory for critical reading of the manuscript. This work was supported by the Howard Hughes Medical Institute, by National Institutes of Health Grant 1 R01 DK60770 (to P.J.B.), and by National Research Service Award 5T32-GM-7616 (to A.M.G). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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