The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells

Abstract

Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin-based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, termed mammalian-membrane two-hybrid assay (MaMTH). We show that this technology detects stimulus (hormone or agonist)-dependent and phosphorylation-dependent PPIs. MaMTH can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB receptor variants or by treatment with drugs such as the tyrosine kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFR(L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non-small cell lung cancer cells. MaMTH is a powerful tool for investigating the dynamic interactomes of human integral membrane proteins.

Characterizing Dynamic Protein–Protein Interactions Using the Genetically Encoded Split Biosensor Assay Technique Split TEV

“…Upon ligand binding, RTKs can homodimerize or heterodimerize, leading to a conformational change that transmits the signal into cytosol, where the kinase domains phosphorylate target tyrosine residues in trans [7]. In turn, Src homology 2 (SH2) domain-and phosphotyrosine binding (PTB) domain-containing adapters are recruited to phosphorylated docking sites, a process that can be robustly and precisely monitored by SBA [8][9][10]. An established interaction between an RTK and an adapter protein initiates downstream signaling activities, as these adaptors link RTK activation to downstream signal transduction pathways, such as the MAP kinase or PI3K/AKT signaling cascades.…”

“…To assess dynamic interactions among proteins, various approaches for genetically encoded SBA are available, including, but not limited to, GFP and variants thereof [18][19][20], luciferases (firefly [21,22], Renilla [23,24], Gaussia [25], click beetle luciferases [17,26,27]), β-lactamase [28], β-galactosidase [29,30], ubiquitin [8,31,32], and the tobacco etch virus (TEV) protease [33]. In addition to SBA, proximity assays were developed, such as bioluminescence resonance energy transfer (BRET) assays comprising firefly luciferase and GFP [34,35] and the Tango method that uses a full-length TEV protease fused to an interacting partner like β-Arrestin to monitor GPCR activities [16].…”

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor

“…Another binary interaction assay, luminescencebased mammalian interactome mapping (LUMIER), has been applied to map TGF␤ induced modulation of PPIs with components of the TGF␤ signaling pathway (11). MaMTH, a mammalian version of the split ubiquitin approach, was designed particularly for the analysis of PPIs involving integral membrane proteins, also allowing the detection of functional PPI modulation (12). Efforts to apply purification-based methods for detecting context-dependent PPI modulation recently resulted in the development of AP-SRM (13) and AP-SWATH (14).…”

Application guide for omics approaches to cell signaling

“…The mammalian membrane two--hybrid (MaMTH) 42 is a recently developed PCA--variant based on split--ubiquitin reconstitution and is derived from the original membrane--yeast two--hybrid system (MYTH) 43,44 (Fig. 3c).…”