Structural, functional, and tissue distribution analysis of human transferrin receptor-2 by murine monoclonal antibodies and a polyclonal antiserum.

Human transferrin receptor-2 (TFR-2) is a protein highly homologous to TFR-1/CD71 and is endowed with the ability to bind transferrin (TF) with low affinity. High levels of TFR-2 mRNA were found in the liver and in erythroid precursors. Mutations affecting the TFR-2 gene led to hemochromatosis type 3, a form of inherited iron overload. Several issues on distribution and function of the receptor were answered by raising a panel of 9 monoclonal antibodies specific for TFR-2 by immunizing mice with murine fibroblasts transfected with the human TFR-2 cDNA. A polyclonal antiserum was also produced in mice immunized with 3 peptides derived from the TFR-2 sequence, exploiting an innovative technique. The specificity of all the reagents produced was confirmed by reactivity with TFR-2(+) target cells and simultaneous negativity with TFR-1(+) cells. Western blot analyses showed a dominant chain of approximately 90 kDa in TFR-2 transfectants and HepG2 cell line. Analysis of distribution in normal tissues and in representative cell lines revealed that TFR-2 displays a restricted expression pattern--it is present at high levels in hepatocytes and in the epithelial cells of the small intestine, including the duodenal crypts. Exposure of human TFR-2(+) cells to TF-bound iron is followed by a significant up-regulation and relocalization of membrane TFR-2. The tissue distribution pattern, the behavior following exposure to iron-loaded TF, and the features of the disease resulting from TFR-2 inactivation support the hypothesis that TFR-2 contributes to body iron sensing.

Hemochromatosis due to mutations in transferrin receptor 2.

A rare recessive disorder which leads to iron overload and severe clinical complications similar to those reported in HFE-related hemochromatosis has been delineated and sometimes called hemochromatosis type 3. The gene responsible is Transferrin Receptor 2 (TFR2), which maps to chromosome 7q22. The TFR2 gene presents a significative homology to transferrin receptor (TFRC) gene, encodes for a transmembrane protein with a large extracellular domain, is able to bind transferrin, even if with lower affinity than TFRC. The TFR2 function is still unclear. The transcript does not contain IRE elements and is not modified by the cellular iron status. At variance with TFRC, interactions between TFR2 and HFE do not occur, at least in their soluble forms. TFR2 is spliced in two alternative forms, alfa and beta. The alfa form is strongly expressed in the liver. The beta form, codified from a start site in exon 4 of the alpha, has a low and ubiquitous expression. Using anti-TFR2 monoclonal antibodies we have confirmed expression of the protein in the liver but also in duodenal epithelial cells, and studied the protein functional behaviour in cell lines, in response to iron addition, iron deprivation and olo-transferrin exposure. Our results suggest a regulatory role of TFR2 in iron metabolism. Five TFR2 homozygous mutations have been documented in HFE3 patients: a nonsense mutation (Y250X); a C insertion that causes a frameshift and a premature stop codon (E60X); a missense mutation (M172K); a 12 basepair deletion in exon 16, that causes 4 aminoacid loss (AVAQ 594-597del) in the extracellular domain of TFR2; a missense mutation in exon 17 (Q690P). The mutation analysis supports the hypothesis that all are private mutations. The pathogenetic role of TFR2 in hemochromatosis has been recently further demonstrated through the targeted expression of the Y250X human mutation in mice, which develop sings of iron overload identical to the human disease. Although the rarity of TFR2 mutations limits their usefulness in diagnostic/screening programs, their study can contribute to a better understanding of the protein function.