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Analysis of the Functional Role of Toll-Like Receptor-4 Tyrosine Phosphorylation

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Toll-Like Receptors

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 517))

Summary

Toll-like receptors (TLRs) are principal innate immune sensors critically involved in the recognition of evolutionary conserved microbial and viral structures called “pathogen-associated molecular patterns” (PAMPs). Although recognition patterns of many TLRs have been characterized, molecular mechanisms that initiate TLR signaling are poorly understood. Since posttranslational modifications of many receptor systems are important in initiating signaling, we studied whether tyrosine phosphorylation of TLR4, the principal sensor of Gram-negative bacterial lipopolysaccharide (LPS) plays a role in TLR4 signal-transducing functions. We found that LPS induced TLR4 tyrosine phosphorylation and mutations of tyrosine residues in the Toll-IL-1R signaling domain markedly suppressed TLR4-mediated activation of JNK and p38 MAP kinases and transcription factors NF-κB, RANTES, and IFN-β. This chapter summarizes a combination of methodological approaches that can be used to demonstrate an indispensable role of TLR4 tyrosine phosphorylation in receptor signaling, including transient transfections, site-directed mutagenesis, immunoprecipitation and immunoblot analyses, and analyses of transcription factor activation in reporter assays.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, School of Medicine, University of Maryland, Baltimore, USA

    Andrei E. Medvedev & Wenji Piao

Authors
  1. Andrei E. Medvedev
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  2. Wenji Piao
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Corresponding author

Correspondence to Andrei E. Medvedev .

Editor information

Editors and Affiliations

  1. School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland

    Claire E. McCoy  & Luke A. J. O’Neill  & 

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© 2009 Humana Press, a part of Springer Science + Business Media, LLC

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Medvedev, A.E., Piao, W. (2009). Analysis of the Functional Role of Toll-Like Receptor-4 Tyrosine Phosphorylation. In: McCoy, C.E., O’Neill, L.A.J. (eds) Toll-Like Receptors. Methods in Molecular Biology™, vol 517. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-541-1_10

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  • DOI: https://doi.org/10.1007/978-1-59745-541-1_10

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-934115-72-5

  • Online ISBN: 978-1-59745-541-1

  • eBook Packages: Springer Protocols

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