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Blood, 1 June 2005, Vol. 105, No. 11, pp. 4264-4271.
Prepublished online as a Blood First Edition Paper on January 11, 2005; DOI 10.1182/blood-2004-07-2733.
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HEMATOPOIESIS
A systematic scan of interactions with tyrosine motifs in the erythropoietin receptor using a mammalian 2-hybrid approach
Tony Montoye,
Irma Lemmens,
Dominiek Catteeuw,
Sven Eyckerman, and
Jan Tavernier
From the Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium.
Signaling via the erythropoietin receptor (EpoR) depends on the interaction of several proteins with phosphorylated tyrosine-containing motifs in its cytosolic domain. Detailed mapping of these interactions is required for an accurate insight into Epo signaling. We recently developed a mammalian protein-protein interaction trap (MAPPIT), a cytokine receptor-based 2-hybrid method that operates in intact Hek293-T mammalian cells. As baits, we used intracellular segments of the EpoR containing 1 or 2 tyrosines. Several known signaling molecules, including cytokine-inducible SH2-containing protein (CIS), suppressor of cytokine signaling-2 (SOCS2), phosphatidylinositol 3'-kinase (PI3-K), phospholipase C- (PLC-), and signal transducer and activator of transcription 5 (STAT5) were used as prey. We also extended the MAPPIT method to enable interaction analysis with wild-type EpoR. In this relay MAPPIT approach, instead of using isolated EpoR fragments as bait, we used the full-length EpoR itself as a "receptor bait." Finally, we introduced MAPPIT in the erythroleukemic TF-1 cell line, which is a more natural setting of the EpoR. With these strategies several known interactions with the EpoR were analyzed and evidence for new interactions was obtained.
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