The Cytoplasmic Domain of Transferrin Receptor 2 Dictates Its Stability and Response to Holo-transferrin in Hep3B Cells

Abstract

Transferrin receptor 2 (TfR2) is a homolog of transferrin receptor 1 (TfR1), the receptor responsible for the uptake of iron-loaded transferrin (holo-Tf) into cells. Unlike the ubiquitous TfR1, TfR2 is predominantly expressed in the liver. Mutations in TfR2 gene cause a rare autosomal recessive form of the iron overload disease, hereditary hemochromatosis. Previous studies demonstrated that holo-Tf increases TfR2 levels by stabilizing TfR2 at the protein level. In this study we constructed two chimeras, one of which had the cytoplasmic domain of TfR2 and the remaining portion of TfR1 and the other with the cytoplasmic and transmembrane domain of TfR1 joined to the ectodomain of TfR2. Similar to TfR2, the levels of the chimera containing only the cytoplasmic domain of TfR2 increased in a time-and dose-dependent manner after the addition of holo-Tf to the medium. The half-life of the chimera increased 2.7-fold in cells exposed to holo-Tf like the endogenous TfR2 in HepG2 cells. Like TfR2 and unlike TfR1, the levels of the chimera did not respond to intracellular iron content. These results suggest that although holo-Tf binding to the ectodomain is necessary, the cytoplasmic domain of TfR2 is largely responsible for its stabilization by holo-Tf.

Defective trafficking and localization of mutated transferrin receptor 2: implications for type 3 hereditary hemochromatosis

“…Two studies have shown that the TfR2 protein is stabilized by diferric transferrin (17,28). The cytoplasmic domain of TfR2 appears to be responsible for this stabilization, because a chimeric receptor containing the cytoplasmic domain of TfR2 fused to the transmembrane and ectodomain of TfR1 retained the ability to be stabilized by diferric transferrin (4). Other experiments have shown that diferric transferrin increases the fraction of TfR2 being targeted to the recycling pathway and decreases the amount being targeted to the late endosomes and lysosomes for degradation (16).…”

CD81 Promotes Both the Degradation of Transferrin Receptor 2 (TfR2) and the Tfr2-mediated Maintenance of Hepcidin Expression

“…Down-regulation of GRAIL did not abolish the increase in stability of TfR2 in the presence of Tf. 3 Previous studies indicate that Tf increases the recycling of TfR2 from endosomal compartments back to the plasma membrane (21,23,32). These results suggest that recycling occurs from an earlier compartment than the CD81/GRAIL-mediated degradation signal occurs.…”

“…The pcDNA3-TfR2⌬CD encoding TfR2 lacking its cytoplasmic domain was generated from pcDNA3-TfR2 by PCR. The chimera, TfR2CD/TfR1-f, containing the cytoplasmic domain of TfR2 and the transmembrane and ecto-domains of TfR1 followed by a Flag epitope was described previously (21). GRAIL cDNA was amplified from the RNA isolated from Hep3B cells, cloned into the pcDNA3 vector, and verified by sequencing.…”

“…The generation of Hep3B stable cell lines expressing TfR2 (Hep3B-TfR2), Hep3B-TfR2CD/TfR1-f, or Hep3B-TfR2⌬CD cell lines were described previously (21). The stable cell lines were maintained in the minimum essential medium with addition of 400 g/ml Geneticin (G418; Calbiochem).…”

“…Proteins were transferred to nylon-supported nitrocellulose (Maine Manufacturing, Sanford, ME). Immunoblot analysis was carried out using primary antibodies followed by fluorescently labeled secondary antibodies and quantified using a LI-COR infrared fluorescence detector as described previously (21).…”