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TAG, a splice variant of the adaptor TRAM, negatively regulates the adaptor MyD88–independent TLR4 pathway

Nature Immunology volume 10, pages 579–586 (2009)Cite this article

Abstract

Toll-like receptor 4 (TLR4) signals the induction of transcription factor IRF3–dependent genes from the early endosome via the adaptor TRAM. Here we report a splice variant of TRAM, TAG ('TRAM adaptor with GOLD domain'), which has a Golgi dynamics domain coupled to TRAM's Toll–interleukin 1 receptor domain. After stimulation with lipopolysaccharide, TRAM and TAG localized to late endosomes positive for the GTPase Rab7a. TAG inhibited activation of IRF3 by lipopolysaccharide. Knockdown of TAG with small interfering RNA enhanced induction of the chemokine CCL5 (RANTES), but not of interleukin 8, by lipopolysaccharide in human peripheral blood mononuclear cells. TAG displaced the adaptor TRIF from TRAM. TAG is therefore an example of a specific inhibitor of the adaptor MyD88–independent pathway activated by TLR4. Targeting TAG could be useful in the effort to boost the immunostimulatory effect of TLR4 without causing unwanted inflammation.

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Figure 1: TRAM and TAG are generated by alternative splicing.
Figure 2: Expression and localization of TAG and TRAM.
Figure 3: Live-cell imaging of the cellular localization of TRAM and TAG.
Figure 4: Overexpression of TAG inhibits TLR4 signaling pathways.
Figure 5: Negative regulatory function of TAG in LPS signaling.

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Acknowledgements

We thank K. Fitzgerald (University of Massachusetts) for TRAM-GFP and TRAM-CFP; M. Zerial (Max Planck Institute of Molecular Cell Biology and Genetics) for Rab7a-CFP and Rab7a-GFP; H. Stanmark (The Norweigen Radium Hospital) for EEA1-GFP; C. Basler (Mount Sinai School of Medicine) for hemagglutinin-tagged-TRIF; R.Y. Tsien (University of California Davis) for pRSET-B-mCherry; and E. Latz (University of Massachusetts) and E. Samstad (University of Massachusetts) for mCherry-tagged TLR4-HEK293 cells. M.H. and H.H. contributed equally to this work. Supported by the Health Research Board Ireland and Science Foundation Ireland.

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Author notes

  1. Eva M Palsson-McDermott, Sarah L Doyle and Anne F McGettrick: These authors contributed equally to this work.

Authors and Affiliations

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

    Eva M Palsson-McDermott, Sarah L Doyle, Anne F McGettrick, Kathy Banahan & Luke A J O'Neill

  2. CSL, Parkville, Victoria, Australia

    Matthew Hardy

  3. Norwegian University of Science and Technology, Institute of Cancer Research and Molecular Medicine, Trondheim, Norway

    Harald Husebye & Terje Espevik

  4. University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Mei Gong & Douglas Golenbock

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Contributions

E.M.P.-M. did the experiments in Figures 3g, 4d,e and 5a,b,d,e; S.L.D. did the experiments in Figures 2, 3, 4a–c and 5c; A.F.M. cloned TAG and did 5′ and 3′ RACE and initial transfection, siRNA and confocal experiments; M.H. found TAG by genomic analysis; K.B. provided technical assistance; H.H. and T.E. helped with the confocal experiments; M.G. and D.G. made the immortalized TRAM-deficient bone marrow–derived macrophages; and L.A.J.O. directed the program and wrote the manuscript with E.M.P.-M., S.L.D. and A.F.M.

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Correspondence to Luke A J O'Neill.

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Palsson-McDermott, E., Doyle, S., McGettrick, A. et al. TAG, a splice variant of the adaptor TRAM, negatively regulates the adaptor MyD88–independent TLR4 pathway. Nat Immunol 10, 579–586 (2009). https://doi.org/10.1038/ni.1727

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