Analysis of the functional role of Toll-like receptor-4 tyrosine phosphorylation

Toll-like receptors (TLRs) are principal innate immune sensors critically involved in the recognition of evolutionary conserved microbial and viral structures called "pathogen-associated molecular patterns" (PAMPs). Although recognition patterns of many TLRs have been characterized, molecular mechanisms that initiate TLR signaling are poorly understood. Since posttranslational modifications of many receptor systems are important in initiating signaling, we studied whether tyrosine phosphorylation of TLR4, the principal sensor of Gram-negative bacterial lipopolysaccharide (LPS) plays a role in TLR4 signal-transducing functions. We found that LPS induced TLR4 tyrosine phosphorylation and mutations of tyrosine residues in the Toll-IL-1R signaling domain markedly suppressed TLR4-mediated activation of JNK and p38 MAP kinases and transcription factors NF-kappaB, RANTES, and IFN-beta. This chapter summarizes a combination of methodological approaches that can be used to demonstrate an indispensable role of TLR4 tyrosine phosphorylation in receptor signaling, including transient transfections, site-directed mutagenesis, immunoprecipitation and immunoblot analyses, and analyses of transcription factor activation in reporter assays.