Giannetti, Anthony Michael (2004) Biochemical, biophysical, and cellular investigations of the interactions of transferrin receptor with transferrin and the hereditary hemochromatosis protein, HFE. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-05262004-173612
Hereditary hemochromatosis (HH) is a prevalent genetic disorder that results in the daily excess absorption of dietary iron. If untreated this disease leads to systemic organ failure and death. HH is caused by mutations to the gene coding for a protein called HFE, a type I transmembrane glycoprotein with a demonstrated role in regulating cellular iron homeostasis. HFE binds to the cell-surface receptor transferrin receptor (TfR), a dimeric type II transmembrane glycoprotein responsible for iron uptake into most mammalian cell types. TfR binds iron-loaded transferrin (Fe-Tf) from the blood and transports it to acidic recycling endosomes where iron is released from Fe-Tf in a TfR-facilitated process. Iron-free transferrin (apo-Tf) remains bound to TfR and is recycled to the cell surface, where apo-Tf rapidly dissociates from TfR upon exposure to the basic pH of blood. 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 reduction could result from direct competition between HFE and Fe-Tf for receptor binding sites, from negative cooperativity, or both. We sought to understand the mechanism of HFE, Fe-Tf, and apo-Tf binding by TfR to help define HFE's role in iron homeostasis. We determined the binding constants for HFE, Fe-Tf, and apo-Tf to an extensive set of site-directed TfR mutants and discovered that HFE and Tf bind to an overlapping site on TfR, indicating the two proteins compete with each other for receptor binding. The mutagenesis results also identified differences in the contact points between TfR and the two forms of Tf, Fe-Tf and apo-Tf. By combining the mutations that are required for apo-Tf, but not Fe-Tf, binding we find that a highly conserved hydrophobic patch on the TfR surface is required for the receptor-mediated stimulation of iron release from Fe-Tf. From these data we propose a structure-based model for the mechanism of TfR-assisted iron release.
To explore the mechanism of the HFE-induced affinity reduction for Fe-Tf binding by TfR, we engineered a heterodimeric TfR (hdTfR) that contains mutations such that one TfR chain binds only HFE and the other binds only Fe-Tf. Competition binding experiments using hdTfR demonstrate that TfR does not exhibit cooperativity in heterotropic ligand binding, suggesting that some or all of HFE's effects on iron homeostasis result from competition with Fe-Tf for TfR binding. Using transfected cell lines we show that HFE is dependent on its interactions with TfR for transport to endosomal compartments and that competition with extracellular Fe-Tf can alter HFE trafficking patterns. These data suggest that HFE's role in iron homeostasis is as a sensor of body iron status.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||analytical ultracentrifugation; apo-Tf; Biacore; bivalent ligand; chinese hamster ovary; confocal microscopy; Fe-Tf; functional epitope; green fluorescent protein; hereditary hemochromatosis; heterodimer; HFE; iron; iron overload; protein engineering; protein-protein binding; sedimentation velocity; site directed mutagenesis; stoichiometry; structural epitope; surface plasmon resonance; TfR; tissue culture; transfection; transferrin receptor; TRVb|
|Degree Grantor:||California Institute of Technology|
|Major Option:||Biochemistry and Molecular Biophysics|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||18 May 2004|
|Author Email:||tonyg (AT) caltech.edu|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||27 May 2004|
|Last Modified:||26 Dec 2012 02:46|
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