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.
Wang
Linda
L
Department of Microbiology, Faculty of Medicine, National University of Singapore, Singapore, Republic of Singapore.
Zhang
Haifeng
H
Zhong
Fei
F
Lu
Jinhua
J
D002460:Cell Line; D019281:Dimerization; D006801:Humans; D015847:Interleukin-4 / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000737:chemistry*; D016328:NF-kappa B / Q000378:metabolism; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000737:chemistry*; D019948:Receptors, Interleukin-4 / Q000737:chemistry*; D051194:Toll-Like Receptor 1; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors; D020798:Two-Hybrid System Techniques
D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011956:Receptors, Cell Surface; D019948:Receptors, Interleukin-4; C495342:TLR2 protein, human; D051194:Toll-Like Receptor 1; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors; D015847:Interleukin-4