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. 2001 Oct 23;98(22):12654-8.
doi: 10.1073/pnas.231471798. Epub 2001 Oct 16.

Drosophila MyD88 is an adapter in the Toll signaling pathway

T Horng  1 , R Medzhitov
Affiliations

Drosophila MyD88 is an adapter in the Toll signaling pathway

T Horng et al. Proc Natl Acad Sci U S A. .

Abstract

Toll-like receptors comprise a family of cell surface receptors that play a crucial role in the innate immune recognition of both Drosophila and mammals. Previous studies have shown that Drosophila Toll-1 mediates the induction of antifungal peptides during fungal infection of adult flies. Through genetic studies, Tube, Pelle, Cactus, and Dif have been identified as downstream components of the Toll-1 signaling pathway. Here we report characterization of a Drosophila homologue of human MyD88, dMyD88. We show that dMyD88 is an adapter in the Toll signaling pathway that associates with both the Toll receptor and the downstream kinase Pelle. Expression of dMyD88 in S2 cells strongly induced activity of a Drosomycin reporter gene, whereas a dominant-negative version of dMyD88 potently inhibited Toll-mediated signaling. We also show that dMyD88 associates with the death domain-containing adapter Drosophila Fas-associated death domain-containing protein (dFADD), which in turn interacts with the apical caspase Dredd. This pathway links a cell surface receptor to an apical caspase in invertebrate cells and therefore suggests that the Toll-mediated pathway of caspase activation may be the evolutionary ancestor of the death receptor-mediated pathway for apoptosis induction in mammals.

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Figures

Figure 1
Figure 1
DMyD88 domain structure and signaling. (A) dMyD88 consists of a death domain (DD, amino acids 82–175), a middle domain (MD, amino acids 176–237), and a TIR domain (amino acids 238–375), as well as an N-terminal region (N, amino acids 1–81) and a C-terminal region (C, amino acids 375–537) of unknown functions. (B and C) dMyD88 induces a Drosomycin, but not an Attacin, reporter gene. S2 cells were transiently transfected with 2.0 μg of empty plasmid, dMyD88, or Toll 10b, a constitutively active form of Toll-1, and 0.1 μg of either a Drosomycin (B) or an Attacin (C) luciferase reporter gene.
Figure 2
Figure 2
Signaling by dMyD88. (A) A Cactus mutant that cannot be phosphorylated inhibits induction of a Drosomycin reporter by dMyD88. S2 cells were transfected with either 2.0 μg of empty plasmid or 0.2 μg of dMyD88 with or without 0.9 μg of the Cactus mutant and appropriate amounts of filler DNA. (B) Activation of a Drosomycin reporter gene by dMyD88 deletion constructs. dMyD88 (1), (1), and (375) did not induce expression of a Drosomycin reporter gene, whereas TIR domain-containing constructs dMyD88 (237) and (176) retained some residual activity.
Figure 3
Figure 3
dMyD88 is downstream of Toll but upstream of Pelle. (A) S2 cells were transfected with either 2.0 μg of empty plasmid or 0.25 μg of Toll 10b and 1.75 μg of the dMyD88 deletion constructs. dMyD88 (1) completely abolished Toll-mediated Drosomycin induction. (B) dMyD88 1–237 inhibits Toll-mediated Drosomycin induction in a dose-dependent manner. S2 cells were transfected with 0.25 μg of Toll and increasing concentrations of dMyD88 (1), as indicated. (C) PelleN, a dominant-negative form of Pelle, inhibits activation of a Drosomycin reporter by dMyD88. (D) dMyD88 (1) does not inhibit Pelle-induced Drosomycin activation, indicating that Pelle is downstream of dMyD88. Cells were transfected with 0.5 μg of dMyD88 (C) or Pelle (D) and the indicated concentrations of the inhibitors.
Figure 4
Figure 4
dMyD88 associates with Toll and Pelle. (A) S2 cells were transiently transfected with dMyD88 (176) tagged with a V5 epitope and Toll 10b. Cell lysates were incubated overnight with Protein G Sepharose and either Toll-specific rabbit immune serum or rabbit serum of irrelevant specificity. Immunoprecipitates were analyzed by SDS/PAGE immunoblot with a V5-specific antibody. dMyD88 (176) can be detected in anti-Toll immunoprecipitates (Top, lane 2) but not in control immunoprecipitates from cells transfected with dMyD88 alone (Top, lane 1) or in controls immunoprecipitated with the irrelevant antiserum (Top, lanes 3 and 4). Whole cell lysates (50 μg/lane) immunoblotted with V5-specific antibody (Bottom) and Toll-specific antiserum (Bottom) confirm expression of the indicated proteins. dMyD88 therefore can be detected in active Toll receptor complexes and the middle, TIR, and C-terminal domains are sufficient for association of dMyD88 with Toll. (B) S2 cells were transfected with dMyD88 (1) tagged with a V5 epitope and Flag-tagged Pelle. Lysates were incubated overnight with anti-Flag M2 agarose beads, and immunoprecipitates were analyzed by SDS/PAGE followed by immunoblotting with V5 antibody and Flag antibody. dMyD88 (1) coimmunoprecipitated with Flag Pelle (Top, lane 3). No dMyD88 (1) could be detected in immunoprecipitated lysates of untransfected cells (Top, lane 1) or cells transfected with dMyD88 (1) alone (Top, lane 2). Immunoblotting of whole cell lysates show appropriate expression of proteins (bottom two sections). The death domain and middle domain of dMyD88 are therefore sufficient for interaction with Pelle.
Figure 5
Figure 5
dMyD88 interacts with dFADD and Dredd. (A) dMyD88 (1) associates with dFADD. Cells were transfected with either V5 dMyD88 (1) or V5 dMyD88 (1) and Flag dFADD. Lysates were subjected to immunoprecipitation by using anti-Flag beads as before. dMyD88 (1) strongly associates with dFADD, presumably through their respective death domains (Top, lane 3). Immunoblots of whole cell lysates show appropriate expression of all proteins (bottom two sections). (B) dFADD associates with the caspase Dredd. S2 cells were transfected with the indicated constructs. Full length V5-tagged Dredd is coimmunoprecipitated with Flag dFADD (Top, lane 3).
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References

    1. Medzhitov R, Janeway C A., Jr Cell. 1997;91:295–298. - PubMed
    1. Anderson K V. Curr Opin Immunol. 2000;12:13–19. - PubMed
    1. Imler J L, Hoffmann J A. Curr Opin Microbiol. 2000;3:16–22. - PubMed
    1. Medzhitov R, Preston-Hurlburt P, Kopp E, Stadlen A, Chen C, Ghosh S, Janeway C A., Jr Mol Cell. 1998;2:253–258. - PubMed
    1. Muzio M, Natoli G, Saccani S, Levrero M, Mantovani A. J Exp Med. 1998;187:2097–2101. - PMC - PubMed

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