PTPN3, a Tyrosine Phosphatase, Regulates Expression of the Erythroid Heme Transporter, FLVCR.

Abstract 779

The Feline Leukemia Virus Subgroup CReceptor, FLVCR, a member of the Major Facilitator Superfamily (MFS) of secondary permeases, plays a critical role in erythropoiesis through regulation of cellular heme export. In addition, the receptor is highly expressed at sites of heme trafficking, such as the small intestine, liver, and macrophages. Accordingly, neonatal conditional knockdown of FLVCR function in a mouse model results in arrested erythroid differentiation and the rapid development of systemic iron overload (Keel SB, et al. 2008 Science 319:825–28). As there are wide disparities between FLVCR protein and gene expression, we hypothesized that, similar to other MFS transporters such as the iron exporter ferroportin, FLVCR expression is likely regulated in a post-translational manner through control of its intracellular trafficking. Thus, we performed yeast-two-hybrid screens of a human liver cDNA library using the intracytosolic C-terminal potential PDZ domain–binding motif of FLVCR. This screen identified a candidate binding protein, PTPN3, a tyrosine phosphatase containing both PDZ and FERM (cytoskeletal attachment) domains. Using in vitro and in vivo co-immunoprecipitation assays, we demonstrate that PTPN3 interacts with and regulates FLVCR expression. Notably, in co-transfection assays, we demonstrate marked positive regulation of FLVCR expression by PTPN3 (∼4-fold). Furthermore, a pool of PTPN3 that associates with the cell membrane specifically immunoprecipitates FLVCR, suggesting their existence in a protein complex at the cell surface. As predicted, deletion of the PDZ domain–binding motif of FLVCR or the PTPN3 PDZ domain, or both, abrogates their interaction in vivo. In addition, a single amino acid mutation of PTPN3 that results in a catalytically inactive phosphatase significantly diminishes its interaction with the receptor, indicating the potential importance of phosphorylation/dephosphorylation of FLVCR for trafficking. By analogy to the ferroportin trafficking pathway (De Domenico I, et al. 2009 Proc. Natl. Acad. Sci. 106:3800–805), we surmise that tyrosine residues located within the intracellular loop of FLVCR (Y319, Y321) are the likely regulatory targets of phosphorylation/dephosphorylation reactions. Thus, both the scaffolding and the phosphatase functions of PTPN3 are important for the regulation of FLVCR expression. Finally, our preliminary analysis of mutation of the dileucine motif located within the intracytosolic N-terminus of FLVCR suggests the motif also contributes to correct cell membrane targeting in transfected cells. In addition to analyzing endogenous interactions between FLVCR and PTPN3, we are currently investigating the importance of both FLVCR trafficking in polarized and erythroid cell lines, utilizing defined deletion constructs, and FLVCR function using heme transport assays. Notwithstanding its importance in erythropoiesis and systemic iron homeostasis, functional insights into FLVCR regulation will increase our understanding of its role in cellular protection from heme toxicity, an issue of considerable relevance for hemolytic anemias, thalassemia, and malaria.