Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling.

摘要: TLR4 is the signal-transducing receptor for structurally diverse microbial molecules such as bacterial LPS, respiratory syncytial virus fusion (F) protein, and chlamydial heat shock protein 60. Previous studies associated two polymorphic mutations in extracellular domain of (Asp(299)Gly Thr(399)Ile) with decreased LPS responsiveness. To analyze molecular basis diminished responsiveness, site-specific (singly or coexpressed) were introduced into untagged epitope (Flag)-tagged wild-type (WT) expression vectors to permit a direct comparison WT mutant signal transduction. Coexpression TLR4, CD14, MD-2 HEK293T cells was first optimized achieve optimal LPS-induced NF-kappaB reporter gene expression. Surprisingly, transfection at high input levels often used literature suppressed signaling, whereas supraoptimal CD14 did not. Under conditions where variants comparably expressed, significant differences activation observed response unrelated agonists, 60 RSV F double, cosegregating exhibiting greatest deficiency. Overexpression Flag-tagged resulting agonist-independent signaling led equivalent suggesting that these affect appropriate interaction agonist coreceptor. These data provide new insights importance stoichiometry among components TLR4/MD-2/CD14 complex. A structural model accounts responsiveness polymorphisms agonists proposed.