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Essential roles of CD14 and lipopolysaccharideâbinding protein for activation of tollâlike receptor (TLR)2 as well as TLR4: Reconstitution of TLR2â and TLR4âactivation by distinguishable ligands in LPS preparations
- Muta, Tatsushi, Takeshige, Koichiro
- European journal of biochemistry 2001 v.268 no.16 pp. 4580-4589
- Toll-like receptor 2, Toll-like receptor 4, blood serum, humans, kidneys, lipid A, mutants, point mutation, polymyxin B, transcription factor NF-kappa B, transfection
- Although genetic studies have revealed a critical role for the tollâlike receptor (TLR) 4 in the biological response to lipopolysaccharide (LPS), the activities of ectopically expressed TLR4 and TLR2 are controversial. We have found that under appropriate transfection conditions, both TLR2 and TLR4 mediate LPSâinduced NFâÎºB activation in human embryonic kidney 293 cells. The reconstitution systems we established here allow direct biochemical characterization and comparison of activation of each receptor. TLR4 is ââ100âfold more sensitive to LPS than TLR2. In contrast to the response to commercial LPS preparations, TLR2 is unresponsive to repurified LPS or synthetic lipidâA, indicating the requirement for an additional molecule(s). On the other hand, a lipidâAâneutralizing reagent, polymyxin B, blocks the ability of the LPS preparation to stimulate both receptors, suggesting that lipidâA is also involved in the activation of TLR2. Mutant TLRs harboring a point mutation in the cytoplasmic domain is inactive in transducing the signal upon stimulation, and act as dominantânegative mutants specifically inhibiting the activation of corresponding type of the receptor but not the other type. Thus, the two receptors are independently activated by distinguishable ligands. Nevertheless, the responses of both TLRs to the LPS preparation are strongly dependent on serum and CD14 and LPSâbinding protein are essential for the activation of both of the two receptors. Supporting its functional significance, both receptors are found to associate with CD14.