A Toll-like receptor-based two-hybrid assay for detecting protein–protein interactions on live eukaryotic cells
Copyright policy )A Toll-like receptor-based two-hybrid assay for detecting protein–protein interactions on live eukaryotic cells
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-kappaB 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 alpha (IL-4Ralpha) and the cytokine receptor common gamma chain (gammaC) as hybrid receptors with tmTIR1 and tmTIR2. Upon co-expression of IL4Ralpha-TIR1 and gammaC-TIR2 in 293T cells, NF-kappaB activation was found to be inducible by IL-4. Co-expression of IL4-TIR1 with IL4Ralpha-TIR2, but not gammaC-TIR2, led to constitutive NF-kappaB activation. This is consistent with IL-4 primarily binding to IL4Ralpha but not gammaC. Co-expression of the IL4Ralpha-TIR1/2, IL4-TIR1/2 or gammaC-TIR1/2 hybrid receptor pairs also constitutively activated NF-kappaB suggesting that IL-4, IL4Ralpha and gammaC 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.
- National University of Singapore Singapore
- National University of Singapore Libraries Singapore
Membrane Glycoproteins, Interaction, Toll-Like Receptors, NF-kappa B, Two-hybrid, 610, Receptors, Cell Surface, 540, Secreted protein, Precipitin Tests, Toll-Like Receptor 1, NF-κB, Toll-Like Receptor 2, Cell Line, Receptors, Interleukin-4, Toll-like receptor, Two-Hybrid System Techniques, Humans, Interleukin-4, Interleukin-4 receptor, Luciferase, Dimerization
Membrane Glycoproteins, Interaction, Toll-Like Receptors, NF-kappa B, Two-hybrid, 610, Receptors, Cell Surface, 540, Secreted protein, Precipitin Tests, Toll-Like Receptor 1, NF-κB, Toll-Like Receptor 2, Cell Line, Receptors, Interleukin-4, Toll-like receptor, Two-Hybrid System Techniques, Humans, Interleukin-4, Interleukin-4 receptor, Luciferase, Dimerization
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