title: The Asp299Gly polymorphism alters TLR4 signaling by interfering with recruitment of MyD88 and TRIF.
abstract: Asp(299)Gly (D299G) and, to a lesser extent, Thr(399)Ile (T399I) TLR4 polymorphisms have been associated with gram-negative sepsis and other infectious diseases, but the mechanisms by which they affect TLR4 signaling are unclear. In this study, we determined the impact of the D299G and T399I polymorphisms on TLR4 expression, interactions with myeloid differentiation factor 2 (MD2), LPS binding, and LPS-mediated activation of the MyD88- and Toll/IL-1R resistance domain-containing adapter inducing IFN-β (TRIF) signaling pathways. Complementation of human embryonic kidney 293/CD14/MD2 transfectants with wild-type (WT) or mutant yellow fluorescent protein-tagged TLR4 variants revealed comparable total TLR4 expression, TLR4-MD2 interactions, and LPS binding. FACS analyses with anti-TLR4 Ab showed only minimal changes in the cell-surface levels of the D299G TLR4. Cells transfected with D299G TLR4 exhibited impaired LPS-induced phosphorylation of p38 and TANK-binding kinase 1, activation of NF-κB and IFN regulatory factor 3, and induction of IL-8 and IFN-β mRNA, whereas T399I TLR4 did not cause statistically significant inhibition. In contrast to WT TLR4, expression of the D299G mutants in TLR4(-/-) mouse macrophages failed to elicit LPS-mediated induction of TNF-α and IFN-β mRNA. Coimmunoprecipitation revealed diminished LPS-driven interaction of MyD88 and TRIF with the D299G TLR4 species, in contrast to robust adapter recruitment exhibited by WT TLR4. Thus, the D299G polymorphism compromises recruitment of MyD88 and TRIF to TLR4 without affecting TLR4 expression, TLR4-MD2 interaction, or LPS binding, suggesting that it interferes with TLR4 dimerization and assembly of intracellular docking platforms for adapter recruitment.
authors.affiliation: Department of Medical and Research Technology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors.forename: Leandra
authors.initials: L
authors.lastname: Figueroa
authors.forename: Yanbao
authors.initials: Y
authors.lastname: Xiong
authors.forename: Chang
authors.initials: C
authors.lastname: Song
authors.forename: Wenji
authors.initials: W
authors.lastname: Piao
authors.forename: Stefanie N
authors.initials: SN
authors.lastname: Vogel
authors.forename: Andrei E
authors.initials: AE
authors.lastname: Medvedev
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D011110:Polymorphism, Genetic / Q000276:immunology*; D011494:Protein Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D055503:Protein Multimerization / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D050838:Interferon Regulatory Factor-3; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D011494:Protein Kinases; D051197:Toll-Like Receptor 4; C495377:LY96 protein, human; C411728:Ly96 protein, mouse; C507779:MYD88 protein, human; C507780:Myd88 protein, mouse; C476286:Ticam1 protein, mouse; C470409:TICAM1 protein, human; C495345:TLR4 protein, human; C493487:Tlr4 protein, mouse