Please use this identifier to cite or link to this item: https://doi.org/10.1016/j.jim.2004.06.020
Title: A Toll-like receptor-based two-hybrid assay for detecting protein-protein interactions on live eukaryotic cells
Authors: Wang, L. 
Lu, J. 
Zhang, H. 
Zhong, F. 
Keywords: Interaction
Interleukin-4 receptor
Luciferase
NF-κB
Secreted protein
Toll-like receptor
Two-hybrid
Issue Date: 2004
Source: Wang, L., Lu, J., Zhang, H., Zhong, F. (2004). A Toll-like receptor-based two-hybrid assay for detecting protein-protein interactions on live eukaryotic cells. Journal of Immunological Methods 292 (1-2) : 175-186. ScholarBank@NUS Repository. https://doi.org/10.1016/j.jim.2004.06.020
Abstract: Protein-protein interactions underly diverse biological processes. Here, we describe a method for detecting protein interactions on the cell surface. This method is based on the mechanism of TLR2 activation whereby extracellular (EC) domain-mediated heterodimerization of TLR2 and TLR1 activates NF-κB and other signalling processes. Test proteins were expressed as the EC domains of TLR1 and TLR2 in fusion with the transmembrane/cytoplasmic (TM/Cyt) domains, i.e. tmTIR1 and tmTIR2. The feasibility of this TIR1/2-based method was examined by expression of IL-4 and the EC domains of the interleukin-4 receptor α (IL-4Rα) and the cytokine receptor common γ chain (γC) as hybrid receptors with tmTIR1 and tmTIR2. Upon co-expression of IL4Rα-TIR1 and γC-TIR2 in 293T cells, NF-κB activation was found to be inducible by IL-4. Co-expression of IL4-TIR1 with IL4Rα-TIR2, but not γC-TIR2, led to constitutive NF-κB activation. This is consistent with IL-4 primarily binding to IL4Rα but not γC. Co-expression of the IL4Rα-TIR1/2, IL4-TIR1/2 or γC-TIR1/2 hybrid receptor pairs also constitutively activated NF-κB suggesting that IL-4, IL4Rα and γC form homodimers or homotypic interactions. This was confirmed by immunoprecipitation studies. In summary, we report a TIR1/2-based assay for detecting interactions between membrane proteins, receptors/ligands and secreted proteins on live eukaryotic cells. © 2004 Elsevier B.V. All rights reserved.
Source Title: Journal of Immunological Methods
URI: http://scholarbank.nus.edu.sg/handle/10635/31441
ISSN: 00221759
DOI: 10.1016/j.jim.2004.06.020
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