Development of a novel Toll-like receptor-based two-hybrid assay for detecting protein-protein interactions and its application in the study of CD14 dimerization and FcyRIIA activation

Abstract

Protein-protein interactions form the basis of diverse biological processes. We describe here a method that allows us to detect protein-protein interactions on the surface of live mammalian cells. It is based on the activation of TLR1/TLR2-mediated NF-N:B signaling. This signal is transduced through the dimerization of cytoplasmic TIR domains of TLR1 and TLR2 (i.e. TIR1 and TIR2). In this assay, the extracellular (EC) domains of TLR2 and TLR1 are deleted to express secreted proteins or the EC domains of other receptors as hybrids with the transmembrane/cytoplasmic (TM/Cyt) domains of TLR1 and TLR2, i.e.TIR1 and TIR2. Dimerization of test proteins is detected using NF-N:B luciferase reporter plasmids. This method was tested using the IL-4/receptor system. IL-4 and the EC domains of the interleukin-4 receptor N1 (IL-4RN1o )) and the common N3 (N3C) subunits were expressed as TIR1 and TIR2 chimeras. This TIR1/TIR2-based two-hybrid assay has been used to investigate CD14-CD14 interactions and FcN3RIIA activation.