The localization of the MyD88 and TRAM complex in cells.

<p>(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.</p

TRAM Is Involved in IL-18 Signaling and Functions as a Sorting Adaptor for MyD88

<div><p>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 <em>in vitro</em>. 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.</p> </div

The IFN-γ production from IL-18 and/or IL-12 stimulated Th1 cells from TRAM-deficient mice and MyD88 deficient mice.

<p>The IFN-γ production levels were significantly reduced in TRAM deficient mice and MyD88 deficient mice. The black bars show the production levels from IL-18 and IL-12 co-stimulated Th1 cells, grey bars show those from IL-18 solely stimulated Th1 cells, and the white bars show those from no secondary stimulated Th1 cells.</p

The interaction sites of MyD88 with TRAM.

<p>(A) Luciferase reporter gene activities with wild type and mutant types of the MyD88 TIR domain after IL-18 stimulation. The black bars indicate that the residues show significant difference with wild type. (B) The functional assays of IL-18 signaling presented on the 3D structure of the TIR domain of MyD88. Results of the functional assays are mapped onto the molecular surface of the MyD88 TIR domain. The amino acid residues judged to be significant by the luciferase assay are shown in red, while non-significant ones are shown in light brown. The conserved motifs of boxes 1–3 (FDA of box1, VLPG of box2, FW of box3) are shown in blue. (C) Assay to study the binding of the wild type or mutant TRAM TIR domain and MyD88 TIR domain. The representative alanine substitutions at Site II (R196A) or Site III (R288A) in MyD88 caused a reduced interaction with TRAM. The double alanine substituted mutant at Site II and Site III caused the complete abrogation of the interaction with TRAM. (D) Immunoprecipitation assay between MyD88 wild or R196A–R288A mutant, and TRAM.</p

Protein interaction assays.

<p>(A) GST pull-down assay investigating the direct interactions between the GST-MyD88-TIR and the TRAM-TIR or TLR1-TIR. (B) GST pull-down assay between the GST-IL-18 receptor TIRs and TRAM-TIR.</p

Assay of the interaction between MyD88 and TRAM in IL-18 signaling.

<p>(A) MyD88 and TRAM were co-expressed in HEK293T cells along with IL-18Rβ. IL-18 stimulation was carried out as indicated. MyD88 was immunoprecipitated using the Myc-tag antibody; co-immunoprecipitated TRAM was also detected. (B) The opposite direction co-immunoprecipitation assay between MyD88 and TRAM. Myc-MyD88 was detected with immunoprecipitated FLAG-TRAM.</p