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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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Assay of the interaction between MyD88 and TRAM in IL-18 signaling.(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.
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pone-0038423-g002: Assay of the interaction between MyD88 and TRAM in IL-18 signaling.(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.

Mentions: Five TIR containing adaptor proteins have been previously identified: MyD88, Mal, TRIF, TRAM and SARM. Mal and TRAM were reported to be the sorting adaptors for MyD88 and TRIF, respectively [13]. Because a model structure of the TIR domain of TRAM represents a substantially large negatively charged surface area (Figure S1), while the TIR domain of MyD88 is covered by positive charge, we expected some interaction between these two adaptor molecules. We thus further hypothesized that TRAM also functions as the sorting adaptor for MyD88 in the IL-18 signaling pathway. To test these ideas, we first examined the direct interaction between MyD88 and TRAM. As both of the adaptors contain the TIR domain, which mediates the protein-protein interaction generally via homomeric or heteromeric TIR-TIR interactions, the direct interaction between the MyD88-TIR and the TRAM-TIR was examined with a GST-pull down assay. The results indicated that the wild-type MyD88-TIR directly bound the TRAM-TIR with a higher affinity than Mal, while the interaction between MyD88-TIR and TLR1-TIR, which had been shown not to bind the MyD88-TIR [14], was not detected in this method (Figure 1A). We then examined the interaction between MyD88 and TRAM in cells using a co-immunoprecipitation analysis. When Myc-MyD88 and FLAG-TRAM were co-expressed in HEK293 cells, the Myc-MyD88 constitutively associated with the FLAG-TRAM (Figure 2), which is consistent with our GST-pull down assay. Strikingly, upon stimulation of the cells with IL-18, the MyD88-TRAM complex gradually dissociated over a 30- to 120-minute time course. The HEK293 cells inherently express IL-18Rα (formerly called IL-1Rrp), which is a necessary component for IL-18 signaling, but lack IL-18Rβ (formerly called IL-1AcPL). Thus, we additionally co-expressed IL-18Rβ in the cells for this experiment.


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)

Assay of the interaction between MyD88 and TRAM in IL-18 signaling.(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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369926&req=5

pone-0038423-g002: Assay of the interaction between MyD88 and TRAM in IL-18 signaling.(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.
Mentions: Five TIR containing adaptor proteins have been previously identified: MyD88, Mal, TRIF, TRAM and SARM. Mal and TRAM were reported to be the sorting adaptors for MyD88 and TRIF, respectively [13]. Because a model structure of the TIR domain of TRAM represents a substantially large negatively charged surface area (Figure S1), while the TIR domain of MyD88 is covered by positive charge, we expected some interaction between these two adaptor molecules. We thus further hypothesized that TRAM also functions as the sorting adaptor for MyD88 in the IL-18 signaling pathway. To test these ideas, we first examined the direct interaction between MyD88 and TRAM. As both of the adaptors contain the TIR domain, which mediates the protein-protein interaction generally via homomeric or heteromeric TIR-TIR interactions, the direct interaction between the MyD88-TIR and the TRAM-TIR was examined with a GST-pull down assay. The results indicated that the wild-type MyD88-TIR directly bound the TRAM-TIR with a higher affinity than Mal, while the interaction between MyD88-TIR and TLR1-TIR, which had been shown not to bind the MyD88-TIR [14], was not detected in this method (Figure 1A). We then examined the interaction between MyD88 and TRAM in cells using a co-immunoprecipitation analysis. When Myc-MyD88 and FLAG-TRAM were co-expressed in HEK293 cells, the Myc-MyD88 constitutively associated with the FLAG-TRAM (Figure 2), which is consistent with our GST-pull down assay. Strikingly, upon stimulation of the cells with IL-18, the MyD88-TRAM complex gradually dissociated over a 30- to 120-minute time course. The HEK293 cells inherently express IL-18Rα (formerly called IL-1Rrp), which is a necessary component for IL-18 signaling, but lack IL-18Rβ (formerly called IL-1AcPL). Thus, we additionally co-expressed IL-18Rβ in the cells for this experiment.

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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