A systematic scan of interactions with tyrosine motifs in the erythropoietin receptor using a mammalian 2-hybrid approach.
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-gamma (PLC-gamma), 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.
Montoye
Tony
T
Flanders Interiniversity Institute for Biotechnology, Department of Medical Protein Research, Faculty of Medicine and Health Sciences, Ghent University, Belgium.
Lemmens
Irma
I
Catteeuw
Dominiek
D
Eyckerman
Sven
S
Tavernier
Jan
J
D020816:Amino Acid Motifs; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D008894:Milk Proteins / Q000378:metabolism; D010449:Peptide Mapping; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051966:Phospholipase C gamma; D011485:Protein Binding; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine
D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D008894:Milk Proteins; D019869:Phosphatidylinositol 3-Kinases; D051966:Phospholipase C gamma; D017467:Receptors, Erythropoietin; D012097:Repressor Proteins; D050799:STAT5 Transcription Factor; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators; D010738:Type C Phospholipases; D014443:Tyrosine; C106643:SOCS2 protein, human