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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 interaction sites of MyD88 with TRAM.(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.
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pone-0038423-g006: The interaction sites of MyD88 with TRAM.(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.

Mentions: Having established that MyD88 and TRAM directly interact and that the interaction is critical in IL-18 signaling, we next carried out experiments to identify the amino acid residues of MyD88 that are important in this interaction. A cell-based reporter assay system was utilized to examine various mutations in the MyD88-TIR as previously reported [12]. The results are shown in Figure 6A. An alanine substitution of any one of eight residues (Arg196, Asp197, Lys214, Arg217, Lys238, Arg269, Lys282 or Arg288) caused significantly reduced dominant negative inhibitory effects on IL-18 signaling indicating that these residues are involved in the signal transduction. These eight residues are mapped on the protein structure of the MyD88-TIR (PDB code: 2z5v) (Figure 6B). In our previous experiments for LPS/TLR4 signaling using the same luciferase reporter system, three discrete functional sites were found on the surface of the MyD88-TIR, which we designated Site I, Site II, and Site III (Figure 6B) [12]. Five out of the eight residues, Arg196 (Site II), Asp197 (Site II), Arg217 (Site I), Lys282 (Site III) or Arg288 (Site III) were previously found to be important in the LPS/TLR4 signaling. We then examined the direct binding of the representative mutants of each functional sites of MyD88-TIR to the TRAM-TIR using GST pull-down assays (Figure 6C). The results indicate that the binding between the MyD88-TIR and TRAM-TIR is dependent on Sites II and III because the alanine substitution of either Arg196 or Arg288 resulted in decreased binding. Furthermore, the interaction between the MyD88-TIR and TRAM-TIR was completely abrogated when both Arg196 and Arg288 were mutated. Additionally, co-immunoprecipitation assay also showed the reduction of interaction between MyD88 R196A-R288A mutant and TRAM (Figure 6D). In contrast, a Site I mutant, R217A, did not show a significant decrease in binding affinity. Overall, these interactions are very similar to those observed between the MyD88-TIR and Mal-TIR [12], indicating that TRAM and Mal share the same binding sites on MyD88-TIR.


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 interaction sites of MyD88 with TRAM.(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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369926&req=5

pone-0038423-g006: The interaction sites of MyD88 with TRAM.(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.
Mentions: Having established that MyD88 and TRAM directly interact and that the interaction is critical in IL-18 signaling, we next carried out experiments to identify the amino acid residues of MyD88 that are important in this interaction. A cell-based reporter assay system was utilized to examine various mutations in the MyD88-TIR as previously reported [12]. The results are shown in Figure 6A. An alanine substitution of any one of eight residues (Arg196, Asp197, Lys214, Arg217, Lys238, Arg269, Lys282 or Arg288) caused significantly reduced dominant negative inhibitory effects on IL-18 signaling indicating that these residues are involved in the signal transduction. These eight residues are mapped on the protein structure of the MyD88-TIR (PDB code: 2z5v) (Figure 6B). In our previous experiments for LPS/TLR4 signaling using the same luciferase reporter system, three discrete functional sites were found on the surface of the MyD88-TIR, which we designated Site I, Site II, and Site III (Figure 6B) [12]. Five out of the eight residues, Arg196 (Site II), Asp197 (Site II), Arg217 (Site I), Lys282 (Site III) or Arg288 (Site III) were previously found to be important in the LPS/TLR4 signaling. We then examined the direct binding of the representative mutants of each functional sites of MyD88-TIR to the TRAM-TIR using GST pull-down assays (Figure 6C). The results indicate that the binding between the MyD88-TIR and TRAM-TIR is dependent on Sites II and III because the alanine substitution of either Arg196 or Arg288 resulted in decreased binding. Furthermore, the interaction between the MyD88-TIR and TRAM-TIR was completely abrogated when both Arg196 and Arg288 were mutated. Additionally, co-immunoprecipitation assay also showed the reduction of interaction between MyD88 R196A-R288A mutant and TRAM (Figure 6D). In contrast, a Site I mutant, R217A, did not show a significant decrease in binding affinity. Overall, these interactions are very similar to those observed between the MyD88-TIR and Mal-TIR [12], indicating that TRAM and Mal share the same binding sites on MyD88-TIR.

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