Fundamental to eukaryotic cell signaling is the regulation of protein-protein interactions.
We engineered a split enzyme complementation assay to monitor cell fusion, protein translocation, receptor-receptor interactions, and the activation of G-protein coupled receptors in highly sensitive, quantitative assays. Enzyme complementation represents the ability of two inactive enzyme fragments to restore enzymatic activity by interacting with each other. Split β-gal proteins were first described by Jacob and Monod in prokaryotes (Ullman A. et al. (1965), J Mol Biol. 12: 918-23; Ullman A. et al. (1967), J Mol Biol. 24: 339-43). Our lab has been the first to adapt this technology for use in eukaryotic cells.
The generation of low affinity complementing protein fragments represents a major advance that allows monitoring of dynamic protein interactions and their disruptions within the same subcellular compartment, with a major impact for drug discovery. These assays are widely used for drug discovery by pharmaceutical companies and user-friendly kits are now available to academic laboratories from DiscoveRx (contact information)
1) Cell Fusion We have adapted intracistronic complementation of Escherichia Coli lacZ gene for use in mammalian cells. In Escherichia Coli, deletion of the N- and C- terminus of LacZ produces an inactive enzyme that can be complemented by co-expression of a second deletion mutant containing domains lacking in the first. We show that enzymatic activity is produced upon co-expression of two mutant lacZ peptides within single cells or upon fusion of cells expressing the individual mutants. Therefore, complementation by Escherichia Coli lacZ mutants in mammalian cells allows analysis of cell fusion and detection of colocalized interacting proteins.
2) Quantitative Detection of Inducible Nuclear Protein Translocation Using Proximity-Based Enzyme Complementation The Complementation Assay for Protein Translocation (CAPT) is a method that allows quantitative measurement of protein translocation based on proximity. This assay involves the use of two complementing fragments of β-galactosidase, the enzyme fragment ω, which is localized to a particular subcellular region, and a small complementing mutant peptide (α) that is fused to the translocating protein of interest. In response to protein translocation, the increase in local concentration of α in the immediate vicinity of ω leads to complementation and generation of enzyme activity, that is proportional to the concentration of α. Therefore α acts as a genetically encoded biosensor for local protein concentration. The enzymatic amplification of CAPT allows expression of physiological levels of the reporters and the high signal-to-noise ratio facilitates quantitative assessment of protein translocation. Examples include the glucocorticoid receptor, the C1A domain of PKC gamma, and the PH domain of AKT. This system is therefore ideal for studying signaling pathways and for validation of drug screens aimed to identify molecules that perturb protein translocation.
3) Detection of Families of Membrane Receptors: ErbB2 Family of Receptors We used the β-galactosidase system to quantitatively monitor the dynamic interactions of ErbB2 with other members of the EGF family of receptor tyrosine kinases, by measuring the complementation of low affinity mutant subunits of β-galactosidase fused to the receptor proteins. This system allowed us to characterize ErbB2 interactions at the plasma membrane, and to define the mechanism of action of Herceptin (Trastuzumab), an antibody to ErbB2 (HER2/Neu) that reduces recurrence and improves survival in a subset of ErbB2 positive breast cancer patients. The β-galactosidase system is ideal, in that it allows the study of inducible and reversible interactions, the quantitative comparison of receptor homo- and hetero-dimers at the plasma membrane, and exhibits superior signal-to-noise ratio.
4) TrkA and p75 Receptors Nerve growth factor (NGF) binds and activates two structurally distinct transmembrane receptors, TrkA and p75. These receptors have been proposed to cooperate to form the classical “high affinity” NGF binding site through the formation of a heterotrimeric TrkA/NGF/p75 complex. We solved the crystal structure of the extracellular domain of TrkA complexed with NGF but a mechanism for p75 coordination was not obvious. Therefore, we used the β-galactosidase system to investigate the hetero-dimerization of membrane-bound TrkA and p75, on intact mammalian cells. We found that the extracellular domain and the transmembrane region of TrkA are sufficient to mediate homodimerization in the presence of NGF and that p75 exists in the cell in a multimeric form that does not interact with NGF. The use of the β-galactosidase system was key in determining the details of the interaction of NGF with TrkA and p75 receptors.
5) in vivo protein complementation assay in mice (von Degenfeld) We used the β-galactosidase system to quantify activation of G-protein coupled receptors (GPCRs) in vitro and in vivo, by measuring their interaction with β-arrestin. This novel in vitro assay that overcomes the limitations of existing methods, is a universal, robust and specific cell based assay, applicable to high throughput screening. The high sensitivity and specificity of the system allowed real time measurement of GPCR activity in live animals by non-invasive bioluminescence imaging. The in vivo assay constitute a great advance in the field, in that it provides a means to monitor the effect of agonists and inhibitors of GPCR signaling in a physiological context where biodistribution and metabolism are present. PS: The bioluminescence imaging utilizes a luminescent β-galactosidase substrate (Lugal), a caged luciferin molecule that can be cleaved by firefly luciferase only after it has been cleaved by β-galactosidase.