chapter  6
22 Pages

Use of Engineered β-Lactamase Fragment Complementation to Detect the Associations of Toll-Like Receptors and Signaling Adaptors

ByHyun-Ku Lee, Peter S. Tobias

Transfection .......................................................................................99 6.3.2 Complementation Analysis by Fluorescence Microscopy ............... 100 6.3.3 Complementation Analysis by Flow Cytometry .............................. 102

References .............................................................................................................. 105

Toll-like receptors (TLRs) are type I transmembrane proteins and are known as pattern recognition receptors (PRRs) that can recognize conserved pathogen-associated molecular patterns (PAMPs).1-3 As demonstrated by a number of studies, TLRs play an essential role in sensing innate immune signals that result in the production of proinflammatory and anti-inflammatory cytokines including IL-1, IL-6, IL-8, IL-10,

IL-12, TNF-α, and IFNs.4-6 The TLR-mediated innate immune signals also stimulate the host to develop adaptive immunity through induction of major histocompatibility complex molecules as well as co-stimulatory molecules such as CD40, CD80, and CD86.7-9 TLR activation is generally initiated through ligand-associated TLR homodimerization as demonstrated by crystal structure studies of TLR.10,11 Cooperation among TLRs in signal transduction further describes that some TLRs also work as heterodimers.12-16 The TLR dimerizations are sequentially followed by binding their signaling adaptors that further propagate the downstream signaling cascades. The in vivo protein-protein interactions among TLRs as well as between TLRs and their signaling adaptors can be detected by protein complementation assays (PCAs), as previously shown.17 This chapter describes the molecular basis of this technique.