An Alanine-to-Proline Mutation in the BB-Loop of TLR3 Toll/IL-1R Domain Switches Signalling Adaptor Specificity from TRIF to MyD88 This information is current as of September 28, 2021. Brett Verstak, Christopher J. Arnot and Nicholas J. Gay J Immunol 2013; 191:6101-6109; Prepublished online 6 November 2013; doi: 10.4049/jimmunol.1300849 http://www.jimmunol.org/content/191/12/6101 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

An Alanine-to-Proline Mutation in the BB-Loop of TLR3 Toll/IL-1R Domain Switches Signalling Adaptor Specificity from TRIF to MyD88

Brett Verstak, Christopher J. Arnot, and Nicholas J. Gay

A functionally important proline residue is highly conserved in the cytosolic Toll/IL-1R signaling domains of human TLRs. The antiviral Toll, TLR3, is unusual because it has alanine instead of proline at this position and is the only human TLR that associates directly with the adaptor molecule TIR domain–containing adaptor inducing IFN-b (TRIF) rather than MyD88. In this article, we report that a mutant TLR3 that substitutes the BB-loop alanine for proline (A795P) enhances NF-kB activation but is incapable of mediating TRIF-dependent IFN response factor 3 responses. Wild-type and A795P TLR3 associate constitutively with both TRIF and MyD88, and activation induces additional binding of TRIF to the wild-type and of MyD88 to the A795P mutant receptors, Downloaded from respectively. In addition, activation of A795P, but not wild-type TLR3, leads to the recruitment of TRAF6, a downstream signal transducer of the MyD88-dependent pathway. These results show that adaptor specificity can be conferred by minimal determi- nants of the Toll/IL-1R domain. The Journal of Immunology, 2013, 191: 6101–6109.

oll-like receptors (TLRs) play an important role in innate interacting protein-1 (RIP-1) can bind to the C terminus of TRIF, immunity by recognizing specific molecular patterns ex- causing both Fas-associated death domain–dependent apoptosis http://www.jimmunol.org/ T pressed by microbes. TLR signaling requires dimerization and activation of NF-kB by the IkB kinase complex. The role of of the extracellular domains, mediated by microbial pro- TRAF6 in TRIF signaling is controversial. Although some studies ducts, leading to the dimerization of the receptor cytoplasmic Toll/ found that TRAF6 functions exclusively in MyD88-directed sig- IL-1R (TIR) domains (1, 2). The activated conformation of the naling, others suggest a secondary role in TRIF-mediated activa- receptor provides a scaffold for the recruitment of the downstream tion of NF-kB (5–7). signaling molecules that activate transcription factors including The TIR domains of the TLRs and their cognate adaptors are NF-kB and IFN response factors (IRFs), leading to the production ∼200 aa and fold into a characteristic a-b structure (8). The loops of proinflammatory and type 1 IFNs. connecting these secondary structure elements mediate receptor–

TLRs use four signaling adaptor proteins (3). Of these, TIR adaptor interactions. Of particular interest is the BB-loop that by guest on September 28, 2021 domain–containing adaptor inducing IFN-b (TRIF) and MyD88 links the second b-sheet to the second a-helix of the TIR. A nat- adaptor molecules are required for the activation of IRF3/7 and urally occurring mutation of the conserved proline residue in the BB NF-kB, respectively. TRIF is the largest member of the adaptor loop of TLR4 was identified as the cause of LPS hyporesponsive- family and is required for TLR3-induced signaling and, together ness in C3H/HeJ mice. This missense mutation substituted proline with TRIF-related adaptor molecule (TRAM), the MyD88-inde- with a histidine residue (P712H) and displayed dominant-negative pendent signaling pathway of TLR4, whereas MyD88 couples to properties (9, 10). The importance of the BB-loop in TLR and adaptor all the human TLRs except TLR3. Acting as a platform, TRIF function is further emphasized by the inhibitory properties of cell- accumulates other signaling molecules to the “TRIF signalosome” permeable peptide mimetics (11, 12). and activates a range of cellular responses mediated by other The BB-loop proline is conserved in all human TLRs except for modules in TRIF, as well as the TIR (4). TRAF3 binds to the N TLR3, which has alanine at this position, and previous studies terminus of TRIF leading to phosphorylation of IRF3 and IRF7 suggest that this residue plays an important role in recruiting TRIF by the TANK-binding kinase-1 (TBK-1). In contrast, receptor- into the signaling complex (13). To test this idea further, we have studied the signaling characteristics of a mutant TLR3 that re- places the BB-loop alanine for proline. We show that this single Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United amino acid change switches TLR3 from a TRIF to a MyD88- Kingdom directed signaling pathway. Received for publication March 27, 2013. Accepted for publication October 3, 2013. This work was supported by Medical Research Council UK Programme Grant G1000133-E01 (to N.J.G.). Materials and Methods Address correspondence and reprint requests to Brett Verstak or Prof. Nicholas Plasmids and reagents J. Gay, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, U.K. E-mail addresses: [email protected] (B.V.) or [email protected] (N.J.G.) Full-length pCMV-Myc-Mal, pCMV-MyD88-Myc, and pcDNA3.1-TLR7 The online version of this article contains supplemental material. were a kind gift from Dr. Alexander Weber (University of Tu¨bingen, Tu¨bingen, Germany); pcDNA3.1-Myc-TRIF was a kind gift from Abbreviations used in this article: HEK, human embryonic kidney; IRF, IFN response Dr. Rongtuan Lin (McGill University, Montreal, QC, Canada); pcDNA3.1- factor; Mal, MyD88 adaptor-like; NT, nontargeting; poly(I:C), polyinosinic-polycy- Flag-TLR2,-Flag-TLR3, -Flag-Mal, and -HA-Mal were a kind gift from tidylic acid; qPCR, quantitative PCR; RIP-1, receptor-interacting protein-1; siRNA, small interfering RNA; TBK-1, TANK-binding kinase-1; TIR, Toll/IL-1R; TRAM, Dr. Ashley Mansell (Monash Institute of Medical Research, Clayton, VIC, TRIF-related adaptor molecule; TRIF, TIR-domain-containing adapter-inducing IFN-b; Australia); pcDNA3.1-TBK1-Myc was a kind gift from Dr. Ivan Dikic UAS, upstream activation sequence. (Institute of Biochemistry, Frankfurt Medical School, Frankfurt, Ger- many); pcDNA3-Myc-TRAF6 was a kind gift from Dr. William Dalton Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 (Moffit Cancer Centre, Tampa, FL); pcDNA.I–RIP-1–Myc was a kind gift www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300849 6102 TLR ADAPTOR SPECIFICITY from Dr. Michelle Kelliher (University of Massachusetts Medical School, The cDNA served as template for real-time qPCR analysis to determine Worcester, MA), and Gal4-IRF3,-IRF5, -IRF7, and Gal4-luciferase con- the level of gene knockdown using Maxima SYBR Green/ROX qPCR structs were a kind gift from Dr. Kate Fitzgerald (University of Massa- SuperMix (Thermo Scientific). Relative gene levels of TRIF were determined chusetts Medical School). Vector pEYFPN1-TLR5-YFP was obtained and normalized against internal control of GAPDH to determine the relative from Clontech, and reporter gene constructs NF-kB–, IFN-b–luciferase, gene knockdown using Pfaffl method. The gene-specific primers for TRIF: and Renilla luciferase-thymidine kinase encoding plasmid were a kind gift 59-TCCTCCCTGTTCCCTTCC-39,59-CCTGGAAATCCTCGCAGA-39; from Dr. Clare Bryant (University of Cambridge, Cambridge, U.K.). Myc- and GAPDH: 59-GAAGGTGAAGGTCGGAGTC-39;59-GAAGATGGTG- TRAM was cloned into the pCMV-Myc vector at sites HindIII and KpnI, ATGGGATTTC-39 were designed to span the exon–exon junction to elim- and Flag-TRAF6 into the pEFBOS vector at sites XhoI and HindIII. All inate amplification of genomic DNA. BB-loop mutants were generated using the QuikChange II site-directed mutagenesis kit with PFU-Turbo (Stratagene) using the above mentioned plasmids. Results The BB-loop alanine residue of TLR3 confers specificity for Cell culture and reagents TRIF-directed activation of IRF-3 and IFN-b Human embryonic kidney (HEK) 293, HEK293T, and HEK293-derived The 10 human TLRs all have a proline-glycine dipeptide in the BB- MyD88-deficient (I3A) (14) cells were cultured in DMEM medium sup- plemented with 10% FCS (Invitrogen) and 100 U/ml penicillin/strepto- loop except for TLR3, which has alanine-glycine (Supplemental mycin, and maintained in a 37˚C humidified atmosphere. Pam3Cys4K Fig. 1). In view of this, we asked whether the BB-loop alanine and was obtained from EMC microcollections and CL097 imidazoquinoline proline residues played a role in receptor selectivity for TRIF and compound, FLA-ST Ultrapure Flagellin from Salmonella typhimurium and MyD88, respectively. To this end, we mutated A795 of TLR3 to polyinosinic-polycytidylic acid [poly(I:C)] HMW synthetic analog of dsRNA; all were sourced from Invivogen. a proline and also to histidine, a mutation previously shown to disrupt TRIF recruitment and IRF3-directed signaling (13). HEK293 Downloaded from Luciferase reporter assay cells were transfected with either wild-type TLR3, TLR3 A795P, or HEK293 or HEK293 MyD88-deficient (I3A) cells were seeded at 1 3 105 TLR3 A795H, and downstream activation of the IFN-b and NF-kB cells/well in a 96-well plate 36 h before transfection with jetPEI (Poly- reporter genes was assayed. After poly(I:C) stimulation, wild-type plus). NF-kB–, IFN-b–, or upstream activation sequence (UAS)-Gal4– TLR3 strongly activated the IFN-b promoter, whereas mutants dependent gene expression was determined using luciferase reporter con- A795P and A795H were not responsive (Fig. 1A). As expected, structs concomitantly with indicated vectors. Gal4-fusions IRF3, IRF5, and k http://www.jimmunol.org/ IRF7 were used to analyze activation of IRF activity. The Renilla lucif- wild-type TLR3 supports a small but sustained induction of NF- B, erase-thymidine kinase encoding plasmid was used to normalize for trans- but surprisingly the A795P potentiated this response by almost 20- fection efficiency, and pcDNA3.1 empty vector was used to maintain fold (Fig. 1B). A795H was unable to activate the NF-kB–driven constant DNA. Transfected cells were lysed using Passive lysis buffer reporter and exerted a dominant negative effect on the endogenous (Promega), and assayed for luciferase and Renilla activity using luciferase assay reagent (Promega). Luminescence readings were corrected for Renilla response to poly(I:C). activity and expressed as fold increase over nonstimulated control values. We next sought to investigate the effect of the TLR3 A795P Data are presented as mean 6 SE of one of three independent experiments. mutant on downstream IRF activity. Because TLR3 signaling Statistical analysis was performed using two-way ANOVA where signifi- activates IRF3 and IRF7, but not IRF5 (15), we measured acti- , , cance is relative to wild-type TLR plasmid transfection: *p 0.05, **p vation of these transcription factors by wild-type and A795P

0.01, ***p , 0.001. by guest on September 28, 2021 mutant. For these experiments, we used an in vivo reporter assay Immunoprecipitation and immunoblot analysis that uses hybrid proteins consisting of the yeast Gal4 DNA binding HEK293T cells were seeded at 1.5 3 105 cells/well in a 6-well plate and domain (16). UAS(Gal)-driven luciferase reporter gene was used to were transfected using JetPEI with the indicated plasmids where the total measure activation of the IRF fusion proteins. As expected, wild- amount of DNA (3 mg/well) was kept constant. Twenty-four hours after type TLR3 activated IRF3 and IRF7, but not IRF5. In contrast, the transfection, cells were lysed in KalB buffer as described (15). EZview TLR3 A795P mutant inhibited poly(I:C)-induced activation of IRF3 Red Anti-Flag M2 affinity beads (25 ml, 50% slurry; Sigma) were incu- bated with the cell lysates for 2 h at 4˚C. The immune complexes were and IRF7, but activated IRF5 when compared with wild-type TLR3 precipitated, washed, and eluted by addition of sample buffer for resolution (Fig. 1C–E). by SDS-PAGE and immunoblotting with either anti-Myc (Abcam), anti- HA (Abcam), or anti-Flag Ab (Sigma). Immunocomplexes were visualized Constitutive and inducible binding of MyD88 and TRIF to by using SuperSignal West Pico chemiluminescent substrate solution (Pierce) wild-type and TLR3 A795P mutant followed by exposure to X-ray film (Hyperfilm ECL, Amersham Pharmacia) We next asked whether a switch in adaptor specificity from TRIF to to detect chemiluminescence. k Densitometry analysis for immunoprecipitated Western band intensities MyD88 accounts for the observed enhancement of NF- B activation was calculated using the Totallab software (Nonlinear Dynamics), verifying by TLR3 A795P. Both wild-type and A795P, but not A795H TLR3, for nonsaturation and subtracting background. Values were expressed as a associate with MyD88 in the absence of stimulation. However, percentage of the total area of each band normalized to the strongest band. treatment with poly(I:C) induces a 2-fold increase in MyD88 Small interfering RNA knockdown of TRIF, RNA extraction, binding to A795P, but not to wild-type TLR3 (Fig. 2A, 2B). To and quantitative PCR investigate the adaptor specificity of the A795P receptor further, we used a derivative of the HEK293 cell line (I3A) that does not express The 25-bp duplex of targeting stealth small interfering RNA (siRNA) or nontargeting (NT) siRNA (Invitrogen) was transfected into subconfluent MyD88. As shown in Fig. 2C, A795P mutant induces a 2.5-fold HEK293/293T cells, using siRNA jetPRIME (Polyplus). Double-stranded increase in NF-kB activity, but the 20- to 30-fold enhancement seen siRNAs containing equal parts of the following antisense sequences was in Fig. 1B is not observed. Interestingly, reconstitution of MyD88 used to knock down TRIF: siRNA2, 59-CCCAUUGACGGUGUUUCGGA- into the HEK293 I3A cells caused a suppression of this low-level 9 9 9 CUGGA-3 ; siRNA3, 5 -CCAUCACUUCCUAGCGCCUUCGACA-3 .The response by both the wild-type and A795P TLR3. This suggests that NT siRNA were low GC and medium GC stealth RNAi negative control duplexes (Invitrogen). Forty-eight hours after transfection, the cells were overexpression of MyD88 exerts a dominant negative effect on either transfected with the relevant constructs or analyzed by real-time TRIF-mediated activation of NF-kB, an effect that was also dem- quantitative PCR (qPCR) or Western blot using anti-TRIF (Cell Signaling) onstratedinanotherstudyinwhichMyD88negativelyregulated and b-actin (Ambion). TLR3/TRIF-induced corneal inflammation (17). Total RNA from cells were purified using RNeasy Plus kit and QiaShredder (Qiagen). First-strand cDNA was synthesized from the total To further explore the mechanism by which the A795P TLR3 RNA using SuperScript II reverse transcriptase (Invitrogen) with random mutant upregulates NF-kB activation, we asked whether the adaptor hexamer (Promega) as described in the manufacturers’ instructions. molecule TRIF can still associate with this mutant receptor. These The Journal of Immunology 6103 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. BB-loop alanine-proline mutant of TLR3 is defective for TRIF signaling but enhances NF-kB activation. (A and B) HEK293 cells were transfected with empty vector or expression plasmid for TLR3, TLR3 A795P, or TLR3 A795H mutants (10 ng) with the IFN-b (25 ng) or NF-kB (10 ng) promoter reporter. After 24 h, cells were stimulated with 200 (A)or20mg/ml (B) poly(I:C) for 4, 8, and 24 h. (C–E) TLR3 or TLR3 A795P mutant (5 ng) was transfected with either 15 ng Gal4-IRF3, Gal4-IRF5, or Gal4-IRF7 along with UAS-Gal4-luc reporter (30 ng). After stimulation, cell lysates were harvested for assessment of luciferase reporter gene activity. All results were comparative to a constitutively expressed “reference” reporter gene, TK- Renilla, and fold induction relative to unstimulated cells. Data are presented as mean 6 SEM of one of three independent experiments. Statistical analysis was performed using two-way ANOVA where significance is relative to WT TLR3 plasmid transfection: **p , 0.01, ***p , 0.001. experiments show that TRIF can bind constitutively to both wild- TRIF mRNA as measured by qPCR also demonstrated a significant type and A795P TLR3. However, after stimulation, TRIF binding to reduction when compared with NT siRNA control levels (Fig. 3D). the wild-type TLR3 is enhanced 2.5-fold compared with the mutant As shown in Fig. 3E and 3F, silencing of TRIF abolished signaling A795P, with only a 1.5-fold increase. This is the reciprocal pattern to both IFN-b and NF-kB by wild-type TLR3 in response to treat- to that observed with MyD88 (Fig. 3A, 3B, compared with Fig. 2A, ment with ligand. By contrast, A795P TLR3 retained the ability to 2B). To determine whether the enhanced signaling to NF-kBby activate NF-kB in the absence of TRIF protein, a finding that sup- A795P TLR3 is TRIF dependent, activation of NF-kBandIFN-b ports the conclusion that the mutant receptor signals to NF-kBby was examined in the presence and absence of targeted functional a MyD88-dependent pathway. gene silencing of TRIF. In HEK293 cells, endogenous TRIF protein We also examined the association of other signal transducers that was constitutively expressed in the presence of an NT siRNA con- are involved in the TRIF- and MyD88-directed pathways. We found trol, whereas TRIF protein levels were markedly reduced after that the death-domain kinase RIP-1 that is required for TRIF- treatment with two TRIF-targeted siRNAs (Fig. 3C). Expression of dependent activation of NF-kB binds constitutively to wild-type 6104 TLR ADAPTOR SPECIFICITY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 FIGURE 2. Constitutive and inducible binding of MyD88 to the wild-type and A795P TLR3. (A) HEK293T cells were transfected with 3 mg total DNA containing 0.5 mg full-length Myc-tagged MyD88 with either 2.5 mg Flag-TLR3 (lanes 1, 2), Flag-TLR3 A795P (lanes 3, 4), or Flag-TLR3 A795H (lanes 5, 6) in the presence or absence of poly(I:C) (100 mg/ml) for 30 min. Lysates were immunoprecipitated with a-M2 Flag agarose beads (top panel) and detected by Western blot. Results are representative of three independent experiments. (B) Densitometry analysis for immunoprecipitated Western band intensities. (C) HEK293 MyD88-deficient (I3A) cells were cotransfected with empty vector or expression plasmid for wild-type TLR3 or TLR3 A795P mutant (10 ng) and wild-type MyD88 (5 ng) with the NF-kB promoter reporter (10 ng). After 24 h, cells were stimulated with poly(I:C) (20 mg/ml) for 8 and 24 h followed by harvesting of the cell lysates and assessment of luciferase reporter gene activity. *p , 0.05, ***p , 0.001. and A795P TLR3 (Fig. 4A, 4B). In contrast, TBK-1, required for time points when compared with wild-type TLR2. By contrast, TRIF-dependent IRF3 activation, is present at much lower levels IFN-b–driven reporter gene activity for both mutant TLR2 con- in complexes with the A795P mutant (Fig. 4C, 4D). Interestingly, structs showed no response after Pam3CSK4 stimulation, whereas TRAF6, an E3-ubiquitin ligase recruited to the myddosome com- a minor increase over time was observed for wild-type TLR2 (Fig. plex in MyD88-directed signaling (5), formed a complex with the 5B). For TLR5, an expected NF-kB response was induced by A795P receptor upon poly(I:C) stimulation, whereas wild-type flagellin, and like TLR2 P681A mutant, TLR5 P736A mutant also TLR3 did not (Fig. 4E, 4F). The bridging adaptors MyD88 significantly enhanced NF-kB reporter gene activity, whereas mutant adaptor-like (Mal) and TRAM were unable to bind either wild- TLR5 P736H was at levels similar to that of wild-type TLR5 re- type TLR3 or the A795P/H mutants (Fig. 4G, 4H). Taken together, sponse (Fig. 5C). Both TLR5 mutants induced a small IFN-b signal, these results indicate that the mutant A795P shifts the adaptor but wild-type TLR5 remained unresponsive (Fig. 5D). In the case of specificity of TLR3 toward MyD88. TLR7, neither BB-loop mutant drove NF-kBorIFN-b activity in BB-loop mutations within the TIR domain of other TLRs alter response to imidazoquinolines, whereas wild-type receptor signaled signaling normally (Fig. 5E, 5F). These results demonstrate that a BB-loop alanine is not sufficient to confer TRIF-directed signaling to MyD88- In view of the striking properties of the TLR3 A795P mutant and dependent TLRs. a recent finding that TRIF can mediate activation of NF-kB and MAPKs by TLR5 (18), we investigated the effect of mutating the Discussion BB-loop proline residue in other TLRs. As expected, upon treat- The activity of human TLR3 is required for protection against en- ment of HEK293 cells with the TLR2 agonist Pam3CSK4, cephalitis caused by HSV-1, and although not essential, probably transfected wild-type TLR2 induced strong NF-kB reporter gene participates in host defense against viral infection in other tissues activity over time, whereas the BB-loop TLR2 P681H mutant (19, 20). Activation of TLR3 by viral nucleic acid leads to the inhibited this response (Fig. 5A). Interestingly, the TLR2 P681A production of IFN-b and -l, which inhibit viral replication. The mutant significantly enhanced NF-kB activity by 3-fold at early antiviral functions of TLR3 are mediated by IRFs that require The Journal of Immunology 6105 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. TLR3 A795P signaling response is not TRIF dependent. (A) HEK293T cells were transfected with 3 mg total DNA containing 1 mg full- length Myc-tagged TRIF with either 2.0 mg Flag-TLR3 (lanes 1, 2), Flag-TLR3 A795P (lanes 3, 4), or Flag-TLR3 A795H (lanes 5, 6) in the presence or absence of poly(I:C) (100 mg/ml) for 30 min. Results are representative of three independent experiments. (B) Densitometry analysis for immunopreci- pitated Western band intensities. (C–F) Two different TRIF-targeted siRNAs or NT siRNA at 10 nM were transfected into HEK293 cells and left for 48 h. (C) After siRNA transfection, cell lysates were probed with anti-TRIF Ab for endogenous TRIF detection. (D) Total RNA was extracted, and relative expression levels of TRIF were determined by qPCR. (E and F) HEK293 cells were transfected with empty vector or expression plasmid for TLR3, TLR3 A795P, or TLR3 A795H mutants (10 ng) with the NF-kB (10 ng) or IFN-b (25 ng) promoter reporter. After 24 h, cells were stimulated with 20 (E) or 200 mg/ml (F) poly(I:C) for 8 h followed by harvesting of the cell lysates and assessment of luciferase reporter gene activity. ***p , 0.001.

TRIF for activation. In this article, we report the remarkable finding microdomains (21). Treatment with the ligand poly(I:C) enhances that the specificity of TLR3 for TRIF depends critically on a single the binding of TRIF to wild-type TLR3, and MyD88 to the A795P residue A795 in the TIR domain “BB-loop.” Interestingly, IRF-5, mutant receptor, providing further evidence that this mutation alters which is not a target of the TLR3 signaling pathway (15), was ac- adaptor specificity. Furthermore, the activation of NF-kBbythe tivated by the A795P mutant, a response that is also observed in A795P receptor was found to be dependent on MyD88 and largely MyD88-dependent signaling by TLR7 and TLR8. We also show that independent of TRIF. The partial inhibition observed in NF-kB the mutant receptor associates with TRIF and MyD88 constitutively, activity induced by the A795P mutant in cells targeted with TRIF either directly or perhaps as part of detergent-resistant membrane siRNAs (Fig. 3E) suggests that this receptor may retain the ability to 6106 TLR ADAPTOR SPECIFICITY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. TLR3 A795P suppresses TBK-1 but enhances TRAF6 recruitment. HEK293T were transfected with 3 mg total DNA containing either (A) 0.5 mg full-length Myc-tagged RIP-1 and Myc-TRIF, (C) 0.5 mg Myc-tagged TBK-1 and Myc-TRIF, (E) 0.5 mg Myc-tagged TRAF6 and Myc-TRIF, (G) 0.2 mg Myc-tagged Mal, or (H)1mg Myc-tagged TRAM, with either 2.0 mg Flag-TLR3 (lanes 1, 2), Flag-TLR3 A795P (lanes 3, 4), or Flag-TLR3 A795H [lanes 5, 6 for Mal and TRAM (G, H) only] in the presence or absence of poly(I:C) (100 mg/ml) for 30 min. Results are representative of three independent experiments. (B, D, and F) Quantitation by densitometry. activate NF-kB by the TRIF pathway, or that the presence of specificity, the A795P receptor, but not the wild-type, recruits TRIF enhances the MyD88-directed response. The inability of the downstream signal transducer TRAF6, further evidence of TLR3 A795H to complex with TRIF confirms another study in a switch to MyD88-directed signaling. Previous studies showed which this mutant was unable to recruit TRIF, highlighting that TRAF6 is essential for MyD88signalinginmurinemac- the BB-loop of TLR3 as a critical determinant for TRIF–TIR rophages, but not for TLR3 signaling. In contrast, the related interaction (13). In addition to alterations in TIR adaptor signal transducer TRAF3, but not TRAF6, is essential for the The Journal of Immunology 6107

FIGURE 5. BB-loop proline-alanine mutant is not sufficient to confer TRIF-directed signaling. HEK293 cells were transfected with empty vector (control) or expression plasmid for wild-type TLR2, TLR2 P681A, or TLR2 P681H mutants (10 ng) (A, B); wild-type TLR5, TLR5 736A, or TLR5 P736H mutants (10 ng) (C, D); wild-type TLR7, TLR7 P935A, or TLR7 P935H mutants (10 ng) (E, F), with either the NF-kB promoter reporter (10 ng) or IFN-b promoter reporter (25 ng). After 24 h, cells were stimulated with either 50 (A)or

200 ng/ml (B)Pam3CysK4,60(C) or 100 ng/ml fla- Downloaded from gellin (D), or 3 (E)or6mg/ml CL097 (F) for 4, 8, and 24 h, and the cell lysates harvested for assessment of luciferase reporter gene activity. Statistical analysis was performed using two-way ANOVA where significance is relative to each WT TLR plasmid transfection: *p , 0.05, **p , 0.01, ***p , 0.001. http://www.jimmunol.org/ by guest on September 28, 2021 induction of type 1 IFNs and the anti-inflammatory naling response against a particular pathogen (21). Therefore, our IL-10 (5, 7). results may suggest that in the absence of stimulus, TLR3 is Our finding that MyD88 binds constitutively to TLR3 indicates primed in microdomains with MyD88, TRIF, or both adaptors, and that adaptor usage by the TLRs is more promiscuous than previously that binding of ligand leads to the formation of an active signal- thought. In this regard, recent reports have provided evidence that osome with TRIF and wild-type TLR3, or with MyD88 in the case Mal and MyD88 have regulatory roles in normal TLR3 signaling (17, of the A795P mutant receptor. 22). In cells lacking MyD88, TRIF–TLR3 activation of IFN-b is Currently, the molecular mechanisms by which postreceptor com- significantly enhanced, although TRAM-TRIF signaling by TLR4 is plexes form during TLR signaling are unclear. The variability of suppressed (22). Thus, the repressive function of MyD88 is specific different receptor and adaptor TIR surfaces and potential electro- for TLR3, and our finding that MyD88 is constitutively associated static complementarity may underlie the functional specificity of withTLR3suggeststhatthisdominantnegativeeffectactsatthe different modules in the TLR signaling response (27). In this regard, level of adaptor recruitment. Another study revealed that the absence we describe BB-loop mutants of TLR2 and TLR5 that exhibit of Mal also enhances signaling by TLR3, consistent with Mal similar upregulation of NF-kBtothatseenwiththeTLR3A795P serving as a bridging function to bring MyD88 to the membrane mutant. It may be that the adaptor molecules bound to these mutants (23), although in this study we could not detect a direct interaction undergo a conformational change that favors enhanced recruitment of Mal with TLR3. A possible mediator of TLR3 regulation by Mal/ of downstream molecules. Conversely, the mutation may also inhibit MyD88 is IRAK-2. IRAK-2 can associate with MyD88 and TRAF6 or restrict further recruitment of other adaptor molecules that neg- to activate the transcription factor NF-kB (24, 25) and also indi- atively regulate NF-kB.BecausebothTLR2andTLR5donotsignal rectly associates with TLR3 (26). Thus, formation of TLR3–TRIF from endosomes, it may be that altering their BB-loop to an alanine complexes may allow for recruitment of TRAF6, which then forms is not sufficient for it to switch adaptor specificity with TRIF. By a complex with IRAK-2 and MyD88, and signals to NF-kB. In the contrast, in the case of endosomal TLR7, both mutations within the case of the A795P mutant, a conformational change in TRIF might BB-loop disrupted NF-kB signaling. However, the P935A mutant inhibit TBK-1 binding and allow for enhanced TRAF6 recruitment, also inhibited IFN-b activity, suggesting that switching to TRIF which, in turn, recruits more IRAK-2 and MyD88 resulting in a may require more than just a single amino acid substitution. Crystal strong upregulation of NF-kB activity. However, our finding that structures of several isolated TIR domains are known, but there are TRIF is not required for NF-kB activation by the A795P receptor no structures of receptor–adaptor TIR complexes (28). However, argues against such an indirect model. modeling studies suggest that ligand binding to the receptor ecto- Different stoichiometric combinations of receptors and adaptors domain causes dimerization of the TIR domains in a symmetrical cluster into membrane microdomains and regulate a specific sig- arrangement (29–31). The receptor BB-loops are predicted to form 6108 TLR ADAPTOR SPECIFICITY a critical part of this homodimerization interface, a conclusion receptor signalling through distinct effector functions of TRAF3 and TRAF6. Nature 439: 204–207. that is supported by site-directed mutagenesis and studies with 6. Sato, S., M. Sugiyama, M. Yamamoto, Y. Watanabe, T. Kawai, K. Takeda, and cell-permeable peptide inhibitors. Comparison of the crystal S. Akira. 2003. 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