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TRAM is involved in IL-18 signaling and functions as a sorting adaptor for MyD88.

Ohnishi H, Tochio H, Kato Z, Kawamoto N, Kimura T, Kubota K, Yamamoto T, Funasaka T, Nakano H, Wong RW, Shirakawa M, Kondo N - PLoS ONE (2012)

Bottom Line: These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction.The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal.MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan. ohnishih@gifu-u.ac.jp

ABSTRACT
MyD88, a Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR) or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

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The localization of the MyD88 and TRAM complex in cells.(A–C) The localizations of the DsRed-TRAM (Red) and/or GFP-MyD88 (Green) in HEK293T cells. DAPI stained nuclei of HEK293T cells are shown in blue. Complexes of the DsRed fusion protein and GFP fusion protein are shown in yellow.
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pone-0038423-g005: The localization of the MyD88 and TRAM complex in cells.(A–C) The localizations of the DsRed-TRAM (Red) and/or GFP-MyD88 (Green) in HEK293T cells. DAPI stained nuclei of HEK293T cells are shown in blue. Complexes of the DsRed fusion protein and GFP fusion protein are shown in yellow.

Mentions: Next, we further investigated whether MyD88 actually interacted with TRAM in human HEK293T cells by live cell fluoroimaging. When we transiently transfected a DsRed-TRAM construct into HEK293T cells, the protein localized to the plasma membrane region, which was consistent with a previous report [10] (Figure 5A). In contrast, GFP-MyD88 was dominantly found as foci in the cytosol in the cells expressing the protein; this has also been shown by others [17] (Figure 5B). Strikingly, when HEK293T cells were co-transfected with expression plasmids for GFP-MyD88 and DsRed-TRAM, MyD88 proteins moderately co-localized with TRAM in the membrane regions (Figure 5C). These data strongly suggest that this transient interaction between the two proteins dramatically altered the localization of GFP-MyD88 from the cytosol to the membranes. Thus, TRAM both bound and endowed MyD88 with membrane targeting properties as has been previously demonstrated for Mal [5].


TRAM is involved in IL-18 signaling and functions as a sorting adaptor for MyD88.

Ohnishi H, Tochio H, Kato Z, Kawamoto N, Kimura T, Kubota K, Yamamoto T, Funasaka T, Nakano H, Wong RW, Shirakawa M, Kondo N - PLoS ONE (2012)

The localization of the MyD88 and TRAM complex in cells.(A–C) The localizations of the DsRed-TRAM (Red) and/or GFP-MyD88 (Green) in HEK293T cells. DAPI stained nuclei of HEK293T cells are shown in blue. Complexes of the DsRed fusion protein and GFP fusion protein are shown in yellow.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369926&req=5

pone-0038423-g005: The localization of the MyD88 and TRAM complex in cells.(A–C) The localizations of the DsRed-TRAM (Red) and/or GFP-MyD88 (Green) in HEK293T cells. DAPI stained nuclei of HEK293T cells are shown in blue. Complexes of the DsRed fusion protein and GFP fusion protein are shown in yellow.
Mentions: Next, we further investigated whether MyD88 actually interacted with TRAM in human HEK293T cells by live cell fluoroimaging. When we transiently transfected a DsRed-TRAM construct into HEK293T cells, the protein localized to the plasma membrane region, which was consistent with a previous report [10] (Figure 5A). In contrast, GFP-MyD88 was dominantly found as foci in the cytosol in the cells expressing the protein; this has also been shown by others [17] (Figure 5B). Strikingly, when HEK293T cells were co-transfected with expression plasmids for GFP-MyD88 and DsRed-TRAM, MyD88 proteins moderately co-localized with TRAM in the membrane regions (Figure 5C). These data strongly suggest that this transient interaction between the two proteins dramatically altered the localization of GFP-MyD88 from the cytosol to the membranes. Thus, TRAM both bound and endowed MyD88 with membrane targeting properties as has been previously demonstrated for Mal [5].

Bottom Line: These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction.The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal.MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan. ohnishih@gifu-u.ac.jp

ABSTRACT
MyD88, a Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR) or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

Show MeSH