MAPPIT, a Mammalian Two-Hybrid Method for In-Cell Detection of Protein-Protein Interactions

Abstract

MAPPIT (MAmmalian Protein-Protein Interaction Trap) is a two-hybrid technology that facilitates the detection and analysis of interactions between proteins in living mammalian cells. The system is based on type 1 cytokine receptor signaling. The bait protein of interest is fused to a chimeric signaling-deficient cytokine receptor, the signaling competence of which is restored upon recruitment of a prey protein that is coupled to a functional cytokine receptor domain. MAPPIT exhibits an excellent signal-to-noise ratio, detects a wide variety of protein-protein interactions (PPIs) including transient and indirect interactions, and has been shown to be highly complementary to other two-hybrid methods with respect to the interactions it can detect. Variants of the method were developed to allow large-scale PPI screening, mapping of protein interaction interfaces, PPI inhibitor screening and drug profiling. This chapter describes a basic 4-day MAPPIT protocol for the analysis of interaction between two designated proteins.

A combined binary interaction and phenotypic map of C. elegans cell polarity proteins

“…Finally, we retested 93 randomly selected protein pairs using the MAmmalian Protein-Protein Interaction Trap (MAPPIT), which detects interactions between proteins in living cells based on restoration of the signaling capacity of a signaling-deficient cytokine receptor 16 , 17 ( Supplementary Table 4 ). We examined the fraction that tested positively at a range of assay stringency thresholds, and compared the results with three previously published C. elegans MAPPIT analyses 18 : a positive reference set of 46 low-throughput literature-curated interactions ( Ce PRS), a set of 86 random protein pairs that serves as a negative control ( Ce RRS), and a set of 87 interactions detected in a large scale Y2H screen (WI-2007) 18 .…”

The Zinc-Finger Domain Containing Protein ZC4H2 Interacts with TRPV4, Enhancing Channel Activity and Turnover at the Plasma Membrane

“…We hypothesized that cell-specific regulatory proteins binding to and shaping TRPV4 function could play a role in the pathophysiology of TRPV4-pathies. In order to discover proteins interacting with human TRPV4, we performed a MAPPIT screening [15]. MAPPIT is a two-hybrid technology based on the functional complementation of a human type 1 cytokine receptor.…”

“…It has been suggested that the divergent pathological effects of the disease-causing TRPV4 mutations in various tissues may involve altered interaction with cell-specific regulatory proteins [6]. In this study, we made use of the mammalian protein-protein interaction trap (MAPPIT) [15] to search for proteins that interact with the cytosolic N-terminus of TRPV4. We identified the zinc-finger C4H2-type containing protein (ZC4H2) as a TRPV4 interaction partner, and provide evidence that it augments TRPV4-mediated Ca 2+ signals and enhances channel turnover at the plasma membrane.…”

Targeting Persistent Human Papillomavirus Infection

“…Additional studies illustrate that targeting the interaction between E2 and other identified host cellular proteins could be an efficient therapeutic strategy to eliminate persistent infection [ 47 , 48 , 64 , 65 , 66 ], providing a framework with which to approach developing HPV-specific therapeutics. Highly specific visualization/detection techniques such as bimolecular fluorescence complementation and Mammalian Protein–Protein Interaction Trap (MAPPIT) will likewise prove integral to identifying these synthetic and naturally occurring small molecule inhibitors [ 57 , 90 , 91 , 92 ].…”