Analysis of Tlr4-mediated LPS signal transduction in macrophages by mutational modification of the receptor.
In mouse macrophages (RAW 264.7 cells), toll-like receptor 4 (Tlr4) is a limiting factor in lipopolysaccharide (LPS) signal transduction. The expression of only 1-2 x 10(4) copies of recombinant Tlr4 per cell enhances sensitivity to LPS, shifting the EC50 by 30-fold to the left. Expression of the Tlr4(Lps-d) isoform of Tlr4 (found in C3H/HeJ mice) shifts the EC50 2600-fold to the right, essentially abolishing LPS responses. A truncated form of Tlr4, lacking a cytoplasmic domain, exerts only a weak inhibitory effect on signal transduction. Similarly, the normal or Tlr4(Lps-d) forms of protein lacking an ectodomain [corrected], cause modest inhibition of LPS signaling. Manipulations of Tlr4 structure and expression cause changes in LPS sensitivity that range over 3 to 4 orders of magnitude. These findings support the view that Tlr4 is an integral component of a solitary pathway for LPS signal transduction in macrophages and permit inferences related to the mechanism of signaling and its blockade.
Du
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Howard Hughes Medical Institute, Dallas, TX 75235-9050, USA.
Poltorak
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A
Silva
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Beutler
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B
D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins; D007739:L Cells; D008070:Lipopolysaccharides / Q000378:metabolism*; D008264:Macrophages / Q000737:chemistry* / Q000502:physiology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000494:pharmacology* / Q000502:physiology*; D051379:Mice; D009154:Mutation; D020836:Protein Structure, Quaternary; D011956:Receptors, Cell Surface / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000187:drug effects
D029721:Drosophila Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha