Bacterial lipopolysaccharide (LPS) is a major factor responsible for toxic manifestations of systemic inflammatory response syndrome (SIRS), reported to cause 100,000 deaths annually in the US. Recently Toll like receptor (TLR) 4 was identified as the murine lps gene product; a missense mutation in its cytoplasmic tail leads to an LSP- hyporesponsive phenotype in C3H/HeJ mice. Critical roles for TLR4 and its adaptor protein MyD88 in LPS signaling are starting to emerge, yet the molecular events involved remain poorly understood. We hypothesize that TLR4 may be activated by a process other than direct binding of LPS; that the response to LPS involves the oligomerization of TLR4 and the association of TLR4 with other proteins in additional to MyD88; and that Hsp90 binds the TLR4 activation complex and participates in its signaling. To test these hypotheses, we have generated a panel of immortalized macrophage cells lines bearing either normal, mutated or deleted TLR4 or MyD88 genes. Using these cells lines, we will generate stable transfectants expressing either intact or altered versions of TLR4 or MyD88. The experimental approaches include (1) comparison of the LPS or Taxol responses between these stable cell lines to define the role of the putative domains of TLR4 and MyD88, (2) detection of TLR4 dimerization by co-IP of differentially tagged-TLR4 molecules after LPS/Taxol treatment, (3) identification of monomeric or dimeric TLR4 interacting proteins by the yeast two- hybrid system and affinity purification/ MALDI-reTOR mass spectrometry, and (4) detection of interactions between Hsp90 and TLR4 complex by co-IP and examination of the effect of geldanamycin on the half-lives or TLR4, MyD88 and IRAK. These studies should enrich and refine our current understanding of how microbial products initiate host responses.
