IL-14 MAPPIT: a versatile METHOD to study protein-protein interactions in mammalian cells.

Protein interactions underlie the structural and functional integrity of many subcellular processes. The notion that most of the proteins within a cell are part of higher order complexes regulating signal transduction, gene expression, apoptosis and other crucial events, becomes generally accepted. Existing methodologies for large-scale protein interaction mapping include yeast two-hybrid, phage display and mass spectrometric analysis. Although these approaches have found wide application, each of them suffers from intrinsic limitations, not at least from the fact that interactions are analyzed in a non-physiological context. We have developed a cytokine receptor-based two-hybrid method in mammalian cells. Incorporation of an interaction trap in a signalling-deficient receptor allows identification of protein-protein interactions using a STAT-dependent complementation assay. Interaction between ‘bait’ and ‘prey’ leads to ligand-dependent STAT activation, which is detected by the use of STAT-dependent reporter/selector genes. Using this Mammalian Protein-Protein Interaction Trap (MAPPIT) we were able to demonstrate both modification-independent and phosphorylation-dependent interactions. MAPPIT can be used as screening tool, either to detect novel protein-protein interactions, or to search for inhibitors of known interactions. This MAPPIT procedure places protein-protein interactions in their normal physiological context and may be particularly instrumental for the in situ analysis of signal transduction pathways. [ABSTRACT FROM AUTHOR]