Characterization of the Hepcidin-Ferroportin Complex: Co-localization, Internalization and Degradation
Johnson, Marianna B.
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Iron metabolism is essential for life, but its imbalance can lead to disorders such as anemia (iron deficiency) or hemochromatosis (iron excess). Membrane-bound ferroportin (Fpn), the only known iron export protein, is regulated by hepcidin (Hepc), which binds to Fpn and triggers internalization and subsequent cellular iron sequestration.1 Recent reports suggest that Janus kinase 2 (Jak2) is required for endocytosis and degradation of the Fpn/Hepc complex through the phosphorylation of Fpn on tyrosines 302 and 303 and subsequent recruitment of Jak2.2,3 The published data were evaluated through fluorescence microscopy. First, the Fpn/Hepc co-localization and endocytosis were characterized in Fpn transduced HEK293 cells. Afterward, the presence of Fpn phosphorylation and Jak2 recruitment in endocytosis were assessed with four cell lines. To evaluate the phosphorylation of Fpn, two mutant Fpn (Y2F and Y4F) HEK293 cell lines were used in which either two (302 and 303) or all four (20, 302, 303 and 538) of the proposed internal tyrosines were mutated to phenylalanine. To evaluate the recruitment of Jak2, a Jak2 deficient and reconstituted gamma-2A (γ-2A) cell line was used. The results of this study demonstrate that neither these tyrosines nor Jak2 are essential for internalization. Both Fpn containing either two or four tyrosine mutations and Fpn in Jak2 deficient cell lines are internalized with similar trends to that of wild type Fpn.