MAPPIT: a versatile tool to study cytokine receptor signalling

Abstract

MAPPIT (mammalian protein-protein interaction trap) is a cytokine receptor-based two-hybrid method that operates in intact mammalian cells. A bait is fused C-terminally to a STAT (signal transducer and activator of transcription) recruitment-deficient receptor, whereas the prey is linked to functional STATbinding sites. When bait and prey interact a ligand-dependent complementation of the STAT recruitment deficiency occurs, leading to activation of a STAT-responsive reporter. MAPPIT is very well suited to study protein interactions involving activated cytokine receptors as the technique allows modification of the bait protein in a physiologically optimal environment. Cytokines and their receptorsCytokines are small messenger molecules that are involved in cell-cell communication and are critical to many vital processes such as embryonic development, growth, haemopoiesis and immune responses. They cannot cross the cellular membrane but make use of transmembrane receptors that capture cytokines and transmit their signal inside the cell. In this mini-review, we will mainly focus on homomeric type I cytokine receptors such as the EpoR (erythropoietin receptor), GHR (growth hormone receptor), LR (leptin receptor) and GCSFR (granulocyte colony-stimulating factor receptor). These cytokine receptors have no intrinsic kinase activity but make use of associated JAKs (Janus kinases). Upon ligand binding the JAKs are brought into close proximity enabling them to cross-phosphorylate each other. Subsequently the JAKs phosphorylate tyrosine residues in the cytoplasmic receptor tail, thereby creating docking sites for signalling molecules such as STAT (signal transducer and activator of transcription) proteins. After binding of these STATs they are phosphorylated in turn by the JAKs and translocate as dimers to the nucleus where they initiate transcription of target genes ( Figure 1A). Several mechanisms exist to modulate and terminate cytokine signalling. Among these, the SOCS (suppressor of cytokine signalling) protein Key words: cytokine receptor, mammalian protein-protein interaction trap (MAPPIT), signal transducer and activator of transcription (STAT), signal transduction, suppressor of cytokine signalling (SOCS), Toll-like receptor (TLR). Abbreviations used: βc, beta common; Epo, erythropoietin; EpoR, Epo receptor; GCSFR, granulocyte colony-stimulating factor receptor; GHR, growth hormone receptor; GM-CSF, granulocyte/macrophage colony-stimulating factor; gp130, glycoprotein 130; HEK-293T cells, human embryonic kidney cells expressing the large T-antigen of SV40 (simian virus 40); IRAK, interleukin-1-receptor-associated kinase; IRS4, insulin receptor substrate 4; JAK, Janus kinase; LR, leptin receptor; MAPPIT, mammalian protein-protein interaction trap; MyD88, myeloid differentiation primary response protein 88; Mal, MyD88 adaptor-like protein; PLCγ , phospholipase Cγ ; SH2, Src homology 2; CIS, cytokine-inducible SH2 domain-containing protein; SOCS, suppressor of cytokine signalling; STAT, signal transducer and ...

Using proteomics to identify the HBx interactome in hepatitis B virus: how can this inform the clinic?

“…In addition, several new approaches have emerged as powerful tools to map PPIs, including MAPPIT, luminescencebased mammalian interactome and protein-fragment complementation assay. These technologies allow discovering true interactions with true biological meaning in true physiological context, such as modification-independent and phosphorylation-dependent interactions, induced and constitutive PPIs in mammalian cells [127]. These emerging technologies combined with modern bioinformatics will be utilized to establish HBx-interactome and search therapeutic blockers of abnormal PPIs.…”