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

Juxing Chen,Caroline A Enns

doi:10.1074/jbc.m114.632778

Abstract

Mutations in transferrin receptor 2 (TfR2) cause a rare form of the hereditary hemochromatosis, resulting in iron overload predominantly in the liver. TfR2 is primarily expressed in hepatocytes and is hypothesized to sense iron levels in the blood to positively regulate the expression of hepcidin through activation of the BMP signaling pathway. Hepcidin is a peptide hormone that negatively regulates iron egress from cells and thus limits intestinal iron uptake. In this study, a yeast two-hybrid approach using the cytoplasmic domain of TfR2 identified CD81 as an interacting protein. CD81 is an abundant tetraspanin in the liver. Co-precipitations of CD81 with different TfR2 constructs demonstrated that both the cytoplasmic and ecto-transmembrane domains of TfR2 interact with CD81. Knockdown of CD81 using siRNA significantly increased TfR2 levels by increasing the half-life of TfR2, indicating that CD81 promotes degradation of TfR2. Previous studies showed that CD81 is targeted for degradation by GRAIL, an ubiquitin E3 ligase. Knockdown of GRAIL in Hep3B-TfR2 cells increased TfR2 levels, consistent with inhibition of CD81 ubiquitination. These results suggest that down-regulation of CD81 by GRAIL targets TfR2 for degradation. Surprisingly, knockdown of CD81 decreased hepcidin expression, implying that the TfR2/CD81 complex is involved in the maintenance of hepcidin mRNA. Moreover, knockdown of CD81 did not affect the stimulation of hepcidin expression by BMP6 but increased both the expression of ID1 and SMAD7, direct targets of BMP signaling pathway, and the phosphorylation of ERK1/2, indicating that the CD81 regulates hepcidin expression differently from the BMP and ERK1/2 signaling pathways.

Highlights

Loss of function mutations in transferrin receptor 2 (TfR2) cause iron overload in the body
Recent studies suggest that the bone morphogenetic protein (BMP) receptor/ SMAD1,5,8 (BMPR/SMAD) and ERK1/2 signaling pathways are involved in the TfR2-mediated regulation of hepcidin expression, but these results are somewhat controversial (16 – 18)
In the present study we find that CD81, a tetraspanin scaffold protein, serves to maintain TfR2-sensitive hepcidin expression through BMPR/SMAD- and ERK1/2-independent mechanisms

Summary

Background

Loss of function mutations in TfR2 cause iron overload in the body. Results: CD81, a scaffold protein, controls the level of TfR2 and links TfR2 to E3 ligase, GRAIL. Mutations in transferrin receptor 2 (TfR2) cause a rare form of the hereditary hemochromatosis, resulting in iron overload predominantly in the liver. Knockdown of GRAIL in Hep3B-TfR2 cells increased TfR2 levels, consistent with inhibition of CD81 ubiquitination These results suggest that down-regulation of CD81 by GRAIL targets TfR2 for degradation. Hepcidin (Hamp1) expression increases in response to iron loading, preventing further iron uptake and decreases with iron deficiency to promote increased iron absorption [12, 13] In the liver, both HAMP1 and TfR2 are primarily expressed in hepatocytes. Recent studies suggest that the BMP receptor/ SMAD1,5,8 (BMPR/SMAD) and ERK1/2 signaling pathways are involved in the TfR2-mediated regulation of hepcidin expression, but these results are somewhat controversial (16 – 18). In the present study we find that CD81, a tetraspanin scaffold protein, serves to maintain TfR2-sensitive hepcidin expression through BMPR/SMAD- and ERK1/2-independent mechanisms

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS