Stoichiometries of transferrin receptors 1 and 2 in human liver

Abstract

Mutations in either the hereditary hemochromatosis protein, HFE, or transferrin receptor 2, TfR2, result in a similarly severe form of the most common type of iron overload disease called hereditary hemochromatosis. Models of the interactions between HFE, TfR1, and TfR2 imply that these proteins are present in different molar concentrations in the liver, where they control expression of the iron regulatory hormone, hepcidin, in response to body iron loading. The aim of this study was to determine in vivo levels of mRNA by quantitative RT-PCR and concentrations of these proteins by quantitative immunoblotting in human liver tissues. The level of TfR2 mRNA was 21- and 63-fold higher than that of TfR1 and HFE, respectively. Molar concentration of TfR2 protein was the highest and determined to be 1.95 nmol/g protein in whole cell lysates and 10.89 nmol/g protein in microsomal membranes. Molar concentration of TfR1 protein was 4.5- and 6.1-fold lower than that of TfR2 in whole cell lysates and membranes, respectively. The level of HFE protein was below 0.53 nmol/g of total protein. HFE is thus present in substoichiometric concentrations with respect to both TfR1 and TfR2 in human liver tissue. This finding supports a model, in which availability of HFE is limiting for formation of complexes with TfR1 or TfR2.

Introduction

Hereditary hemochromatosis (HH)¹ is an autosomal, inherited disorder of iron homeostasis characterized by hepatocellular iron overload that ranges from mild to severe (reviewed in ref. [1], [2], [3]). HH is associated with mutations in at least five genes. On the basis of the gene involved, HH is classified as type 1 (hereditary hemochromatosis, HFE) [4], type 2A (hemojuvelin, HFE2) [5], type 2B (hepcidin, HAMP) [6], type 3 (transferrin receptor 2, TFR2) [7], and type 4 (ferroportin, FPN) [8]. Type 1 is the most common form of HH [4].

HFE is a type I transmembrane protein that belongs to the MHC-I like family of proteins. Like MHC-I proteins, HFE also forms a heterodimer with β₂-microglobulin (β₂M) [4], [9]. The most common mutation in the HFE protein, C282Y [4], results in destabilization of the α3 domain, which abrogates the interaction between HFE and β₂M [4]. As a result, the mutant C282Y-HFE protein has impaired ability to reach the cell surface [4], [10], [11]. The second most common mutation is H63D [4], but the mechanism by which this mutation causes HH is unknown. Interestingly, there is a considerable variation in iron loading in individuals with these two mutations [1], [2]. Such heterogeneity suggests that HFE function depends on the presence of modifiers, which might be proteins that interact with HFE.

The first identified binding partner of HFE was the transferrin receptor 1 (TfR1) [12], [13], a ubiquitous cell surface receptor that binds and internalizes iron-loaded transferrin (holo-Tf). HFE/TfR1 complex dissociates in the presence of holo-Tf because holo-Tf competes with HFE for binding to TfR1 [14], [15], [16]. The discovery that hepcidin, an iron regulatory hormone predominantly expressed in hepatocytes [17], is decreased in both HH type 1 patients [18] and Hfe^-/- mice [19], [20] and that HFE is also predominantly expressed in hepatocytes [21] indicated that the primary site of HFE effects on iron homeostasis is the liver. These observations lead to a “hepcidin hypothesis,” in which HFE is an upstream regulator of hepcidin expression (reviewed in ref. [22]). Observations that mice lacking Hfe in the crypt- and villi-enterocytes have no detectable iron loading [23] while mice lacking Hfe in hepatocytes manifest iron overload [24] emphasize the importance of HFE expression in hepatocytes.

Recently, transferrin receptor 2 (TfR2), a homolog of TfR1 that is predominantly expressed in hepatocytes [25], was reported to bind to HFE [26]. Interestingly, the interacting domains of HFE and TfR2 [27] are different from those of HFE and TfR1. First, HFE interacts with TfR2 via its α3 domain, versus with TfR1 via its α1 and α2 domains. Second, the Tf binding site of TfR2 does not overlap with the HFE binding site as it does in TfR1. Thus, in contrast to the HFE/TfR1 complex, the HFE/TfR2 complex does not dissociate, even in the presence of high Tf concentrations [27]. This finding suggests a new model of HFE-dependent regulation of hepcidin expression, in which HFE is released from TfR1 and binds to TfR2, with increasing iron-loaded Tf concentrations. Two recent studies expand these findings. The first work analyzes Hfe and Tfr1 interactions in mice models of HH type 1. Expression of mutant forms of mouse Tfr1 that either prevent or stabilize Hfe/Tfr1 interactions results in an Hfe-dependent induction of hepcidin expression, which occurs only when Hfe is dissociated from Tfr1 [28]. The second study demonstrates that in the presence of holo-Tf, human hepatoma cells that express undetectable HFE but readily detectable TfR1 and TfR2 proteins regulate hepcidin expression only when exogenous HFE is expressed [29]. These studies lead to the proposal that TfR1 sequesters HFE from TfR2 under low iron conditions, but under high iron conditions, the increased iron saturation of Tf shifts the balance towards creation of an HFE/TFR2 hepcidin signaling complex [28]. In this process, HFE represents the limiting factor during reorganization of HFE/TfR1 and HFE/TfR2 complexes [29].

In order to better understand the mechanism by which these complexes are formed as well as their response to iron levels, it is important to know the relative amounts of HFE, TfR1, and TfR2 in the liver. Thus, we tested the hypothesis that in human liver, where the HFE-dependent regulation of hepcidin expression occurs, the molar concentration of HFE is similar to or lower than that of TfR1 or TfR2. Both the mRNA and protein levels of HFE, TfR1, and TfR2 in human liver tissues were measured. Our results showed that mRNA and protein levels of TfR2 are significantly higher than TfR1 and HFE levels. The least abundant is the HFE protein, supporting the proposed model of hepcidin regulation in vivo.