Structure and Function of Toll Receptors and Their Ligands Nicholas J. Gay and Monique Gangloff Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom; email: [email protected], [email protected]Annu. Rev. Biochem. 2007. 76:141–65 First published online as a Review in Advance on March 15, 2007 The Annual Review of Biochemistry is online at biochem.annualreviews.org This article’s doi: 10.1146/annurev.biochem.76.060305.151318 Copyright c 2007 by Annual Reviews. All rights reserved 0066-4154/07/0707-0141$20.00 Key Words leucine-rich repeat, pathway cross talk, signaling mechanism, Toll-IL-1 receptor identity region Abstract The Toll family of class I transmembrane receptors recognizes and responds to diverse structures associated with pathogenic microor- ganisms. These receptors mediate initial responses in innate immu- nity and are required for the development of the adaptive immune response. Toll receptor signaling pathways are also implicated in serious autoimmune diseases such as endotoxic shock and thus are important therapeutic targets. In this review we discuss how micro- bial structures as different as nucleic acids and lipoproteins can be recognized by the extracellular domains of Toll receptors. We review recent evidence that the mechanism of signal transduction is complex and involves sequential changes in the conformation of the receptor induced by binding of the ligand. Finally, we assess the emerging area of cross talk in the Toll pathways. Recent work suggests that signaling through TLR4 in response to endotoxin is modified by inputs from at least two other pathways acting through β2 integrins and protein kinase Cε. 141 Annu. Rev. Biochem. 2007.76:141-165. Downloaded from www.annualreviews.org by University of Chicago Libraries on 03/17/13. For personal use only.
Transcript
ANRV313-BI76-07 ARI 8 June 2007 18:27
Structure and Functionof Toll Receptors andTheir LigandsNicholas J. Gay and Monique GangloffDepartment of Biochemistry, University of Cambridge, Cambridge CB2 1GA,United Kingdom; email: [email protected], [email protected]
Annu. Rev. Biochem. 2007. 76:141–65
First published online as a Review in Advance onMarch 15, 2007
The Annual Review of Biochemistry is online atbiochem.annualreviews.org
This article’s doi:10.1146/annurev.biochem.76.060305.151318
leucine-rich repeat, pathway cross talk, signaling mechanism,Toll-IL-1 receptor identity region
AbstractThe Toll family of class I transmembrane receptors recognizes andresponds to diverse structures associated with pathogenic microor-ganisms. These receptors mediate initial responses in innate immu-nity and are required for the development of the adaptive immuneresponse. Toll receptor signaling pathways are also implicated inserious autoimmune diseases such as endotoxic shock and thus areimportant therapeutic targets. In this review we discuss how micro-bial structures as different as nucleic acids and lipoproteins can berecognized by the extracellular domains of Toll receptors. We reviewrecent evidence that the mechanism of signal transduction is complexand involves sequential changes in the conformation of the receptorinduced by binding of the ligand. Finally, we assess the emergingarea of cross talk in the Toll pathways. Recent work suggests thatsignaling through TLR4 in response to endotoxin is modified byinputs from at least two other pathways acting through β2 integrinsand protein kinase Cε.
The German word Toll is difficult to renderprecisely in English but approximates to fan-tastic, mad, or amazing. In a scientific context,Nusslein-Volhard and Anderson first used theword Toll to name a gene that they discoveredin a genetic screen of Drosophila, the pheno-type of which they thought to be Toll (1, 2). In
fact, this pioneering work in the early 1980sidentified a group of 10 different genes, all ofwhich produced qualitatively similar maternaleffect phenotypes, now known as the dorsalgroup. Null mutations in any of these genesresult in the failure to differentiate pattern el-ements on the dorsoventral axis and lead toearly embryonic lethality. During the follow-ing 10 years, all the dorsal group genes werecloned, and a compelling picture emerged ofhow dorsoventral patterning occurred in theDrosophila embryo. Shortly after the fertiliza-tion of the embryo, a ventrally restricted sig-nal associated with an extracellular membranestructure activates a protease cascade, and theterminal member Easter then processes an in-active precursor of Spatzle, an endogenousprotein ligand of Toll, a class I transmembranereceptor. By this mechanism Toll is activatedat ventral positions in the embryos, and an in-tracellular signaling pathway then causes therelocalization of a transcription factor, dorsal,from the cytoplasm to nuclei located at ven-tral positions in the embryo. This results inthe formation of a ventral-dorsal gradient ofthe transcription factor, and the informationcontained in this morphogenetic gradient isthen used to direct the subsequent differenti-ation of the dorsoventral body axis (3).
The sequence of Toll determined in 1988revealed a tripartite structure with an N-terminal region containing tandem arrays of ashort leucine-rich motif, the leucine-rich re-peat (LRR), a sequence likely to form a sin-gle transmembrane helix and a C-terminal do-main of unknown structure and function (4).An important development in the early 1990swas the recognition that this C-terminal do-main was significantly related to that of thevertebrate interleukin-1 receptor (IL-1R) (5,6). IL-1R is activated by a cytokine formerlyknown as endogenous pyrogen that is nownamed IL-1. IL-1 is part of the acute phaseresponse to infection characterized by feverand the secretion of defense proteins into thecirculation by the liver. This discovery was im-portant as it suggested that this domain, nowknown as the Toll-interleukin receptor (TIR)
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domain, was involved in signaling processesnot only in the restricted context of insectdevelopment but also in the generation of ini-tial responses to infection by human immune-system cells. This conclusion received sig-nificant support in 1995 when Hoffmann’sgroup (7) in Strasbourg discovered that Tolland some other members of the dorsal groupfunctioned in innate immune responses topathogenic fungi and bacteria, as well as inembryonic patterning in the fly. Shortly af-ter this important discovery, the progress ofgenome projects led to the identification ofapproximately 10 receptors in vertebrates thatwere direct homologs of Toll with relatedextracellular domains as well as cytoplasmicTIRs, and these became known as the Toll-like receptors (TLRs) (8).
Until this time vertebrate immunologiststended to focus on the adaptive response andthe generation of diversity in antibody reper-toires [see Di Noia & Neuberger (8a) in thisvolume]. By contrast immunologists consid-ered innate responses to be a less importantpart of the immune response and studied themlittle, except perhaps in the context of com-plement. It took both the identification ofthe TLRs and the prescience of Janeway tochange this picture. Janeway (9) formulatedthe problem as the “immunologist’s dirty lit-tle secret,” that is, the well-known fact that anadaptive response is only produced if an ad-juvant containing a microbial extract is pre-sented to the immune system together withthe specific antigen. This observation impliedthat antibody responses are tightly coupled toa second largely unknown process of activa-tion mediated by the microbial extracts in theadjuvant. Two key results published in 1998demonstrated the important roles played byTLRs in the recognition of microbial prod-ucts and the coupling of innate to adaptiveimmunity. First, activation of the TLR4 path-way induced the expression of costimulatorymolecules required for T cell activation in re-sponse to antigen (10). Second, cell signal-ing induced by a powerful immunostimula-tory component of gram-negative bacteria,
Class Itransmembranereceptor: a family ofsignaling moleculesthat consists of anextracellular domain(ectodomain), asingle-membrane-spanning α-helix,and a cytoplasmicdomain
Cross talk:modification of asignaling response byinputs from distinctsignaling pathways
lipopolysaccharide (LPS), or endotoxin re-quired TLR4 (11). These findings led to arapid expansion of research into innate im-munity, and the TLRs are now established asbona fide pattern-recognition receptors thatrespond to specific molecules derived frombacteria and viruses (12) (see Table 1).
The aim of this review is not to providea comprehensive overview of TLR research,but rather to focus on three specific but inter-related problems of particular interest to bio-chemists. First, how are microbial ligands rec-ognized by the TLRs, and what is the mech-anism of signal transduction? Second, whatare the molecular events that regulate down-stream signaling? Third, what are the na-ture and significance of cross talk between theTLRs and other signaling pathways in cells ofthe immune system?
OVERVIEW OF TOLLSIGNALING PATHWAYS:EVOLUTIONARYCONSERVATION ANDDIVERGENCE
Although elements of the Toll pathways frominsects and vertebrates have been conservedin evolution, it is also clear that there are sub-stantial differences in the way that the ligandis detected and in the regulatory networks thatcontrol the signaling process.
The Extracellular Pathwaysin Insects and Mammals
Insects use specific recognition proteins todetect peptidoglycan derived from the cellwall of gram-positive bacteria (Figure 1). Aglucanase, gram-negative binding protein 1(GNBP1), is thought to fragment the peptido-glycan and then present it to a peptidoglycanrecognition protein (PGRP-SA) with whichit forms a complex. Recent work suggests thatconformational changes in the PGRP/GNBPcomplex caused by the breakdown and trans-fer of peptidoglycan lead to a self-limiting ac-tivation of a serine protease cascade (13). In
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Table 1 Ligands for the Toll-like receptors (TLRs)
Figure 1Conservation and divergence in the Toll signaling pathways. Components of the Drosophila Toll (left) andthe human Toll-like receptor (right) pathways are illustrated schematically. Evolutionarily or functionallyconserved elements in the two pathways are illustrated in the same color. Abbreviations: CD14, anextrinsic, PI-glycan modified membrane protein; Dif, dorsal-related immunity factor; dMyD88,Drosophila homolog of MyD88; dToll, Drosophila Toll receptor; dTRAF2, Drosophila homolog of TNFreceptor-associated factor 2; GNBP1, gram-negative binding protein 1; IFN, interferon; IKK, IκB kinase;LBP, LPS-binding protein; IRAK, interleukin-1 receptor-associated kinase; IRF3, interferon responsefactor 3; LPS, lipopolysaccharide; MD-2, co-receptor of TLR4; Mal, MyD88 adaptor-like; MyD88,myeloid differentiation primary response protein 88; NF-κB, nuclear factor κ B; Pelle, product of theDrosophila pelle gene a protein kinase and homolog of IRAK; PGRP-SA, a peptidoglycan recognitionprotein; SPE, Spatzle processing enzyme; P, phosphorylation of IRF3; Psh, Persephone, a Drosophilaserine protease; TLR4, Toll-like receptor 4; TRAF6, TNF receptor-associated factor 6; TRAM,TRIF-related adaptor molecule; TRIF, TIR domain–containing adaptor protein inducing interferon-β.
the immune response, the number and iden-tity of these proteases have not been estab-lished, but recent work points to a clip domainserine protease named Spatzle processing en-zyme (SPE) as the terminal member (14). SPEacts on the precursor form of Spatzle to gen-erate an activating ligand for the Toll receptor.
The Drosophila Toll pathway also responds tothe presence of pathogenic fungi, and in thiscase a different and unidentified recognitionprotein is required for the response. Antifun-gal responses also require SPE and a secondserine protease, Persephone, that is not nec-essary for antibacterial responses (15). Thus
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Figure 2Activating ligands of the Toll and Toll-like receptor family. (a) HumanTLR3 ectodomain and Drosophila Toll ligand, Spatzle C-106 are shown inprotein structures, with β-sheets, α-helices, and loops in ribbonrepresentation. Disulphide bonds are also shown. (b) TLR3 ligand,double-stranded RNA (dsRNA) (152) is displayed as a cross section andalong its long axis in stick representation. In nucleic acid and theglycosylations attached to TLR3, oxygen atoms, nitrogen, and phosphateare shown. (c) Imiquimod, Pam3CSK4, and lipid A, the active part ofLPS, are displayed in Chemdraw representations. The structures ofdiverse ligands for Toll receptors are illustrated to scale. Abbreviations:LPS, lipopolysaccharide; Pam3CSK4, a tri-palmitoylated lipopeptide.
microbial signals derived from gram-positivebacteria and fungi converge on a protease thatthen generates an active protein ligand for theToll receptor Spatzle.
The Spatzle precursor is synthesized andsecreted as a dimer held together by a singleintermolecular disulphide bond (Figure 2).SPE, or in dorsoventral patterning Easter,protease cleaves the precursor at an arginineresidue 106 amino acids from the C termi-nus to generate a prodomain of approximately200 amino acids and an active dimeric ligand,C-106. C-106 but not the proprotein bindstwo molecules of the Toll receptor withhigh affinity and stimulates downstream sig-nal transduction (16). Spatzle C-106 is re-lated structurally to a family of vertebrategrowth factors, the neurotrophins, such asnerve growth factor (NGF) (17). These pro-teins have a cystine knot fold that has a longantiparallel β-sheet held together by threeintersecting intramolecular disulphide bonds(Figure 2). Recent work has shown that af-ter cleavage the prodomain of Spatzle re-mains stably associated with C-106 but is dis-placed by binding to the Toll ectodomain.This suggests that prior to proteolysis, theprodomain sequesters residues in C-106 thatconfer specific binding to Toll. After process-ing, a conformational change occurs that ex-poses these determinants, an activation mech-anism analogous to that of protease zymogens(153).
In view of the evolutionary relatedness ofinsect Toll and the human TLRs, there wasinitially an expectation that the mechanismsused for sensing microbial products and forthe generation of ligands would be conserved(18). Protease cascades are particularly suit-able for this type of process as they can am-plify initially small stimuli and can also beregulated by negative feedback loops, causingthe response to be spatially and temporally re-stricted. Thus it was thought that the humangenome would encode functional homologsof Spatzle as corresponding endogenousprotein ligands for the TLRs, but on com-pletion of the genome-sequence project, none
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could be found. In fact activation of TLRsis completely different and involves the sens-ing of stimuli either directly by the receptorsthemselves or by receptor/accessory proteincomplexes (see below and Table 1).
The Postreceptor Signaling Pathway
In contrast to extracellular recognition,postreceptor signal transduction by Toll andTLRs requires the function of several con-served proteins (Figure 1). Unlike other classI receptors, such as the tyrosine kinases, thecytoplasmic domains of Toll and TLRs do nothave an enzymatic activity. Instead stimulus-
Adaptor:cytoplasmic proteinthat links differentelements in asignaling pathway,e.g., an activatedreceptor and proteinkinase
induced dimerization or oligomerization isthought to rearrange the TIR domains suchthat they act as a scaffold for the recruit-ment of downstream adaptor proteins (seebelow). In Drosophila, the immediate postre-ceptor events involve the proteins dMyD88,tube, and pelle. dMyD88 is a modular adap-tor protein with a TIR domain and a C-terminal death domain (19). Before signalinitiation, dMyD88 is present in the cell asa heterodimer with a second adaptor pro-tein tube, and this complex is localized byan unknown mechanism to the plasma mem-brane. Tube has a death domain (Figure 3)but no TIR, and it associates specifically
Figure 3Structures and motifs involved in Toll signaling. (a) N-terminal capping structure taken from theToll-like receptor 3 (TLR3) ectodomain structure (2A0Z). (b) A single leucine-rich repeat (LRR) unittaken from the TLR3, LRR20 (see Figure 2). The short β-sheet (yellow) forms the inside surface of thesolenoid. The conserved leucine residues point into the structure to form a hydrophobic core.(c) C-terminal capping structure (taken from TLR3) (see Figure 2). (d) The Toll-interleukin receptor(TIR) domain from TLR2. The positions of the BB and DD loops are indicated. (e) The death domain ofthe pelle protein kinase showing the arrangement of the six α-helical segments.
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Protein kinase:enzyme thattransfers phosphategroups from ATP toserine, threonine,and tyrosine residuesin other proteins
with dMyD88 by death domain–death do-main interactions. Once the receptor is ac-tivated, the dMyD88/Tube dimer is recruitedinto a ternary complex with the Toll/Spatzleheterotrimer and this in turn recruits pelle(20). Pelle is a serine/threonine protein ki-nase and also has an N-terminal death do-main. The current evidence suggests thatthe tube death domain is bivalent and hasbinding sites for both dMyD88 and pelledeath domains. Presumably the pelle bind-ing site of tube only becomes available whenthe tube/MyD88 (myeloid differentiation pri-mary response protein 88) dimer is associ-ated with activated receptor. The signalingcomplex that contains pelle, tube, dMyD88,and Toll is short lived, and recruitment mayactivate the pelle protein kinase, causingautophosphorylation and as a consequencethe dissociation of pelle from the receptorcomplex (21).
The events that lie downstream of thepelle protein kinase activity are less wellunderstood in Drosophila. However, there isevidence that activated pelle can bind andphosphorylate Drosophila tumor necrosisfactor (TNF) receptor–associated factor 2(dTRAF2) and that dTRAF2 is required forthe activation of the downstream pathway,ultimately causing the relocalization of dorsalto the nucleus (22). dTRAF2 has both zincfinger and RING finger domains. The latterusually function as an E3 ubiquitin ligase, andTRAF2 appears to be autoubiquitinated. It isnot known whether this modification leads toproteasome-mediated degradation of TRAF2or whether, like its mammalian counterpartTRAF6 (see below), this modification is re-quired for signal transmission. In mammals,signaling by activated TRAF6 involves thekinase TGFβ-activated kinase (TAK1) andthe adaptors TAB1 and TAB2. Homologs ofthese proteins are encoded by Drosophila andare known to function in an insect pathwaysimilar to that of vertebrate TNF (23) and inresponses to gram-negative bacteria mediatedby the immune deficiency (IMD) pathway(24). However, they are not essential for Toll
signaling, which suggests either that there isredundancy in the pathway or that unidenti-fied components link dTRAF2 to activationof the dorsal/cactus complex. The final step inToll signaling involves the regulated nuclearrelocalization of the dorsal and dorsal-relatedimmunity factor (DIF) transcriptional reg-ulators to the nucleus. Dorsal and DIF arerelated to mammalian NF-κB and are held inthe cytoplasm as a complex with cactus, equiv-alent to IκB in mammals. Signal-inducedphosphorylation likely tags cactus for ubiqui-tination and degradation by the proteasomepathway, although at present a cactus kinasehas not been characterized. However, thereare homologs of the mammalian IKKs β, γ,and ε encoded by Drosophila, but neither β
nor γ is required for Toll signaling (25).Postreceptor signal transduction by the
mammalian TLRs uses several proteins thatare functional homologs of those describedabove for the insect pathway (Figure 1).However, during evolution there clearly havebeen duplication, diversification, and a sub-stantial increase in complexity in these path-ways. For example, researchers have iden-tified approximately 15 different negativeregulators in the TLR pathway (26), and thereis growing evidence for cross talk between theTLRs and other pathways (see below). As inDrosophila, the first step in TLR signaling isstimulus-induced dimerization or oligomer-ization. Similarly, the next step is the re-cruitment of adaptors to the TIR domainsof the dimerized receptor, but in contrast toDrosophila, TLRs use a total of five adap-tor proteins—MyD88, Mal/TIRAP (MyD88adaptor-like/TIR domain–containing adap-tor molecule), TRAM (TRIF-related adaptormolecule), TRIF (TIR domain–containingadaptor protein inducing interferon-β), andSARM (27). MyD88 is a direct homolog ofdMyD88, but there is no equivalent of tubein mammals. MyD88 is used exclusively byfour of the TLRs (5, 7, 8, and 9), whereasTLR2 requires both Mal and MyD88 (28).TLR4 can use either Mal/TIRAP and MyD88or TRAM and TRIF to signal to NF-κB or
Next in the signaling hierarchy arethe interleukin-1 receptor-associated kinases(IRAKs). These are homologs of pelle ki-nase, and there are four of them in mam-mals. Two, IRAK2 and IRAKM, are not ac-tive phosphotransferases (31) and appear tofunction as negative regulators. IRAK4, con-versely, fulfils a role similar to that of pelle,and humans lacking functional IRAK4 areprofoundly sensitive to bacterial infections(32–34). Similar to pelle, both IRAK4 andIRAK1 autophosphorylate, and the evidencesuggests that they are recruited by associa-tion with MyD88 into the postreceptor sig-naling complex (35; N.J. Gay, unpublishedresults). The precise events that lead to down-stream signaling are not understood, but onlykinase-inactive forms of IRAK4 can form astable complex with MyD88 (36). This sug-gests a mechanism similar to that for pellefor the binding, activation, autophosphory-lation, and dissociation from the postrecep-tor complex. In the related IL-1 pathway,there is evidence that the initial activation ofIRAK4 causes the cross-phosphorylation andactivation of IRAK1. IRAK1 then becomesautophosphorylated at multiple sites, dissoci-ates from the postreceptor complex, and bindswith TRAF6. However, other studies showthat the kinase activity of IRAK4 is not nec-essary for IL-1 signaling, but the lack of bothIRAK1 and -4 abolishes IL-1 signaling (37),suggesting the functional redundancy of thetwo kinases.
Signaling downstream of the postreceptorcomplex is critically dependent on TRAF6.As with dTRAF2, TRAF6 has a RING fin-ger domain that functions as an E3 ubiquitinligase and that can interact with the ubiquitinconjugating complex (E2) Ubc13/Uev1, re-sulting in the synthesis of polyubiquitin linkedthrough lysine 63 (38, 39). The activation ofTRAF6 also causes it to oligomerize throughthe C-terminal TRAF domain, and this in
IRAK: interleukin-1receptor-associatedkinase
turn results in the transfer of the lysine 63polyubiquitin chains to the TRAF6 oligomer.The polyubiquitin chains do not appear totarget TRAF6 for degradation by the pro-teasome. The next step downstream involvesa heterotrimer of the protein kinase TAK1and the adaptor molecules TAB1 and TAB2(sometimes called TRIKA2) (40). TAK1 is amember of the MAP kinase kinase kinase fam-ily and is required for activation of NF-κBby TNF, TGFβ, as well as by TLRs (41).The roles played by TAB1, TAB2, and a thirdfamily member, TAB3, remain unclear as cellsfrom knockout mice respond normally to LPSstimulation. Again, this may indicate redun-dancy in the pathway. The TAK/TAB trimerdoes not itself become stably ubiquitinatedbut may be activated by noncovalent associ-ation with the ubiquitin conjugated TRAF6complexes (42). Thus in this case a stable pro-tein modification different from phosphory-lation is essential for signal transduction. Thefinal step in this arm of the TLR pathway is theactivation of the IκB kinase (IKK) by TAK1.The IκB kinase consists of the two subunitsIKKβ and the regulatory IKKγ or NEMO(40). Phosphorylation by IKKβγ targets IκBfor degradation by the proteasome, and thisreleases NF-κB, which then relocalizes to thenucleus. TAK1 is also able to activate a secondbranch of the TLR pathway mediated throughJun N-terminal kinase ( JNK) to the transcrip-tion factor AP-1.
In contrast to insects, mammalian cellsare able to activate a second pathway inresponse to some TLR stimuli that leadsto antiviral responses such as the produc-tion of interferons, chemokines, and anti-inflammatory cytokines. In the case of TLR4,these signals require the adaptor moleculesTRAM and TRIF; for TLR3, they requireTRIF alone (29, 30, 43). Engagement ofthese adaptors results in the activation of acomplex containing two related protein ki-nases, TANK binding kinase 1 (TBK1) andIKKε, and the adaptor TANK (TNF path-way with ankyrin repeats) (44, 45). Recentresults also suggest that the TRIF adaptor is
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linked to the TBK/IKKε/TANK complex byTRAF3, a homolog of TRAF6 (46). The ac-tive TBK/IKKε kinases then phosphorylatethe interferon-responsive factors IRF3 and -7,which causes them to dimerize and enter thenucleus (47). IRF3 is ubiquitously expressedand induces the production of β-type inter-ferons. By contrast, IRF7 acts on interferon-αpromoters and has a more restricted expres-sion pattern. It is strongly expressed in plas-macytoid dendritic cells (DCs) and is inducedin other cell types by interferon-β (48). Acti-vation of IRF7 is mediated by a subset of theTLRs (7, 8, and 9) that responds to nonself-nucleic acids. This subset also induces NF-κBand requires only the MyD88 adaptor. IRF7binds to the death domain of MyD88, whichresults in the activation of the interferon-αpromoters. IRF7 also binds to TRAF6, andthe ubiquitin ligase activity of TRAF6 is re-quired for the activation process (49). Con-versely, a more recent paper suggests thatTRAF3 is in fact responsible for the activationof IRF7, perhaps by coordinating the assem-bly of TBK1 into activated receptor complex(50). In this view TRAF6 would primarily me-diate signals to NF-κB and the production ofproinflammatory cytokines.
STRUCTURAL BASIS OFSIGNALING SPECIFICITY INTHE TOLL AND TOLL-LIKERECEPTOR PATHWAYS
Although only limited progress has beenmade toward understanding the structural ba-sis of Toll signaling, it is nevertheless pos-sible to derive some general principles fromthe fragmentary information available. In thepast few years a much clearer picture hasemerged about the nature of endogenous pro-tein ligands and microbial products that stim-ulate TLR signaling (see Table 1). A certainamount of confusion had been engendered bythe use of impure preparations of bacterialproducts such as peptidoglycan, but most ofthese problems have now been resolved by theuse of chemically synthesized materials.
In view of the clear evolutionary conser-vation of the Toll and TLR pathways and el-ements in the signaling cascade, it is perhapssurprising that the activating ligands shouldbe so diverse. Figure 2 presents the structuresof some of these molecules to scale and the ex-tracellular domain of TLR3 for comparativepurposes. How then can large protein ligandssuch as Spatzle and small-molecule stimulisuch as imidazoquinolines induce a function-ally equivalent dimerization of Toll and TLRectodomains?
Protein Ligands
Spatzle, the cytokine ligand of the Drosophilapathway, is well characterized at the biochem-ical level, although no structure of the lig-and/receptor complex has been solved (16,51). However, the structure of NGF boundto the p75 and Trk neurotrophin recep-tors provides a useful analogy (52, 53). Inboth cases, NGF makes multiple hydropho-bic, hydrogen-bonded, and electrostatic con-tacts with the receptor chains, burying a largesurface area of more than 2000 A2. This isreflected by the high affinities of these in-teractions, between approximately 0.1 and 1nM (54). Spatzle binds Toll with a similaraffinity (0.4 nM), and the alignment of theSpatzle sequence with that of NGF shows thatmany of the amino acids involved in bindingare conserved or are charge reversals (153).Thus the interaction of Spatzle with the Tollectodomain likely involves an extensive bind-ing area that buries a large hydrophobic sur-face, stabilized by charge complementarityprovided by salt bridges and hydrogen bonds.The two symmetrically related binding sites inSpatzle allow the formation of a high-affinitycross-linked complex at the membrane sur-face consisting of two receptor chains and oneSpatzle dimer, and this initiates signal trans-duction (see Molecular Mechanism of SignalTransduction, below).
It does not appear that endogenous cy-tokines equivalent to Spatzle function in ver-tebrate TLR signaling, but two microbially
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derived proteins, flagellin and respiratory syn-cytial virus (RSV) fusion protein, can activateTLR5 and TLR4, respectively (55, 56). Flag-ellin is a protein that assembles into the proto-filaments that constitute the flagellum, struc-tures that confer on the bacterium motility.Flagellae are also virulence factors requiredfor tissue invasion. TLR5 is activated by ahighly conserved sequence in flagellin that isessential for function and is sequestered inthe protofilament in assembled flagellae. ThusTLR5 is activated only by monomeric flag-ellin and responds at low concentrations (lessthan 100 fM), suggesting a high binding affin-ity (57). The interaction between flagellin andTLR5 appears to be direct, and the bindingsite has been mapped to a single LRR in thereceptor ectodomain (58). A second microbialprotein that may be a direct ligand for TLR4is the fusion protein of the RSV, a significantpathogen in infants. However, there is no evi-dence for direct binding, and the MD-2 core-ceptor protein is also required for the response(59) (see below).
Lipids and Lipopeptides
The second major class of ligands for theTLRs comprises bacterial lipids and lipopep-tides. LPSs from the outer membrane ofgram-negative bacteria are the principal ago-nists for TLR4. LPS derived from Escherichiacoli consists of six acyl chains of variablelength linked together by a phosphorylateddiglucosamine head group (lipid A), and thisis linked to a polysaccharide chain of vari-able length and composition (Figure 2). Thelipid A moiety of LPS is required to activatethe TLR4 signaling pathway in conjunctionwith the coreceptor protein MD-2. MD-2 isa secreted glycoprotein of 160 amino acidsand is required for LPS signaling via TLR4(60). MD-2 interacts with the ectodomain ofTLR4 in a constitutive manner (61), and theTLR4/MD-2 complexes form in the endo-plasmic reticulum and at the cell surface. Incontrast to TLR4, MD-2 binds LPS directly(62, 63). MD-2 belongs to a novel family of
proteins, the MD-2-related lipid-recognitionfamily (64). All members of this family thathave been characterized biochemically bindto lipids. They are characterized by a single-domain architecture, belonging to the all-betaclass and displaying an immunoglobulin-likeβ-sandwich fold. It is thought that acyl chainsof lipid A intercalate into the β-sandwichstructure and that this promotes cross-linkingof two TLR4 receptor chains either directlyor by inducing a conformational change thatallows the association to occur (65). Thusin contrast to Drosophila Toll, which bindsan endogenous protein ligand, in TLR4 theectodomain has evolved to form a complexwith a specific coreceptor protein (66).
The agonists that signal through TLR2have obvious similarities to LPS (Figure 2).In fact, as well as detecting a variety of lipids,lipoproteins, and lipopeptides derived prin-cipally from gram-positive bacteria and para-sites, TLR2 can respond to atypical LPS fromcertain gram-negative bacteria, for example,Leptospira interrogans (67). In general TLR2agonists tend to be less complex than LPS.Lipoteichoic acid (LTA) and macrophage ac-tivating lipopeptide 2 (MALP2) have diacylchains with a glucosamine diphosphate andpeptide head groups, respectively. By contrastPAM3CSK4 has three acyl chains and is con-nected to a tetra-lysine peptide chain by a cys-teine residue (Figure 2). Responses to thesemolecules are mediated by heterodimericcombinations of TLR2 with TLR1 and -6.In general, diacylated molecules require theTLR2/6 combination, whereas the triacy-lated lipopeptides signal through TLR2/1, al-though in some in vitro assays TLR2 alonecan signal in response to MALP2 and LTA(68–70). The chirality of these molecules alsoseems important for specificity; for example,the R but not S diasteroisomer of MALP2is active. Domain-exchange experiments haveshown that the ability of the closely relatedTLR1 and TLR6 to discriminate differentagonists resides in LRRs 9–12 in the middleof the ectodomain (71). There are also severaldistinct coreceptor proteins that are able to
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enhance the responsiveness of TLR1/2/6 tostimuli. CD14, an extrinsic membrane pro-tein that also functions in the TLR4 pathway,is thought to bind lipopeptides at the cell sur-face and deliver them to TLR2 (72). Similarly,the lectin dectin 1 increases the responsive-ness of TLR2 to fungal cell-wall components,especially β-glucans (73). Finally, the scav-enger receptor CD36 is required for mousemacrophages to respond to low concentra-tions of MALP2, LTA, PAM3CSK4, and zy-mosan (a fungal extract) (74).
At present, it is unclear whether TLR1,-2, and -6 bind directly to these diverseligands, although the domain-swap experi-ments outlined above point to this conclu-sion for the lipopeptides (71). There is alsosome uncertainty whether peptidoglycan is abona fide agonist for TLR2 (Table 1), ow-ing to possible contamination of the prepa-rations. Clearly the structural information ofTLR1/2/6 ectodomains in complex with de-fined ligands would be useful for understand-ing the signaling mechanism at a molecularlevel.
Nucleic Acids
The third major class of molecules thatact as specific agonists of TLR pathwayscomprises nucleic acids and small-moleculedrugs that have some structural resemblanceto nucleic-acid building blocks, for exam-ple, immunostimulatory guanosine analogsand imidazoquinoline compounds (Figure 2)(75). The nucleic-acid-responsive subgroupof the TLRs comprises TLR3, -7, -8, and -9,and these receptors are more closely re-lated to each other than to the other Tolls.TLR3 responds to double-stranded (ds)RNAmolecules produced during viral infectionand also synthetic copolymers such as polyIC(inosine/cytosine) (76). The natural ligandfor both TLR7 and TLR8 is U-rich single-stranded RNA exposed to the receptor as aresult of viral infection (77, 78). TLR7 and -8also respond to the imidazoquinolines, small-molecule drugs used to treat genital warts
caused by infection with human papillomavirus and basal-cell carcinomas (79). A num-ber of imidazoquinoline derivatives have beencharacterized, and some of these display speci-ficity for either the TLR7 or TLR8 path-ways in humans (80). Finally, DNA with un-methylated CpG dinucleotides is the agonistfor TLR9 (81). In mammals, these sequencesare usually methylated at the seven position ofcytosine, whereas viral and bacterial DNA donot on the whole have this modification.
An important property of this TLR sub-group is that it functions in acidified intra-cellular compartments, late endosomes, andlysosomes and that reduced pH is required forsignaling function (82). This has led to thesuggestion that the protonation of ionizablegroups, either histidine residues of the recep-tor ectodomain or the ligands themselves, isnecessary for signaling (83).
Leucine-Rich Repeats and theStructure of the ReceptorEctodomains
It is clear from the diverse nature of the ligandsthat TLR ectodomains must have featuresthat allow highly variable modes of molec-ular recognition to evolve from a commonstructural framework. The key to this prob-lem is the nature of LRR structures. LRRsare units of approximately 24 amino acidslength that are characterized by a conservedpattern of hydrophobic residues (Figure 3).LRRs are found in a large and diverse groupof proteins (approximately 254 in the humangenome) with functions ranging from RNAprocessing (snRNP U2A′) and transcriptionalregulation (CIITA, activator of MHCII ex-pression) to cell adhesion (chaoptin, which isrequired for eye development in the fly), bac-terial pathogenesis (Yop M virulence factorfrom Yersinia pestis), and signal transduction(TLRs and many other type I and 7TM re-ceptors) (84).
Each LRR folds into a secondary structureconsisting of a short parallel β-sheet, a turn,and a more variable region. The conserved
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hydrophobic residues form the core of thissecondary structure (Figure 3). The blocksof repeats form a curved, solenoidal structurewith the short parallel β-sheets formingthe inner (convex) surface of the structure(Figure 2). Specific molecular recognitionis often achieved by interactions mediatedthrough the side chains of variable residuesprotruding from the short parallel β-strands,contributed by each LRR, that form the inner(concave) surface of the solenoid. These sidechains point out of the structure and canbe viewed as a combinatorial code that hasevolved to bind specific ligands. Interest-ingly, chordates, an early divergence of thevertebrate lineage, have evolved an adaptiveimmune response that uses LRRs instead ofthe immunoglobulin fold (85). This findingemphasizes the ability of LRR-containingproteins to evolve highly diverse bindingmodes for proteins and other biologicalmolecules. LRR blocks are often flanked bycysteine-rich domains (86, 87). These do-mains are called LRRNT and LRRCT whenlocated at the amino- and carboxy-terminalends of LRR regions, respectively (Figure 3).They are crucial capping structures, bury-ing the otherwise exposed hydrophobicresidues at the ends of LRR superhelices.N-capping sequences are likely to form aβ-hairpin, whereas C-terminal caps adopta α-β structure. In both cases the fold isstabilized by the formation of disulphidebonds involving pairs of conserved cysteineresidues.
The Transmembrane andToll-Interleukin Receptor Domains
As with other type I receptors, theectodomains of the TLRs are connectedto the cytoplasmic TIRs by a single trans-membrane α-helix. Although there is nostriking pattern of sequence conservation inthese segments (other than hydrophobicity),the transmembrane and juxtamembranesequence are likely to play critical roles inreceptor activation.
Our current understanding of the TIR do-main fold is informed by the crystal structuresof TLR1 and TLR2 proteins (Figure 3) (88).The core sequence adopts an overall β/α-foldcomprising a central five-stranded parallel β-sheet surrounded by five α-helices. As withthe LRRs, sequence conservation seems to re-flect the structural requirements of the fold.Conversely, the surface properties of differentreceptor and adaptor TIRs are highly diverse,and this diversity may underlie the functionalspecificity of different modules in the signal-ing process (89).
A similar fold is seen in CheY, a bacte-rial chemotaxis protein (8, 90). CheY pro-teins consist of a single regulatory domainactivated by conformational change inducedby a phospho-transfer reaction from a histi-dine kinase to a conserved aspartyl residue.Structural superposition of CheY, TLR1, andTLR2 shows remarkable topological and ter-tiary structure conservation, especially overthe central β-sheet region. Investigators haveidentified many similar signal response reg-ulators with a β/α-fold in different species,suggesting this could be an evolutionarily con-served signal regulatory conformation.
Death Domains
The multitypic postreceptor complexesformed by the TIRs from receptors andadaptors are coupled to downstream signal-ing by a second protein-protein interactionmotif, the death domain, so named becauseit is also found in signaling pathways leadingto apoptosis (91). The adaptors MyD88 andtube have death domains, as do the IRAK andpelle kinases (33). The tube and pelle deathdomains associate together to form stableheterodimers, and this association is requiredfor signaling (92, 93). Death domains consistof approximately 120 amino acids and arecharacterized by a conserved six α-helicalbundle fold (Figure 3) (91, 94). A crystalstructure of the tube/pelle death domainheterodimer (95) and more recently struc-tures of the isolated pelle and IRAK4 death
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domains have been solved (96, 97). Togetherthese studies reveal that the binding interfacebetween tube and pelle death domains isextensive, involving at least 27 of the residuesin the fold. Helix four of pelle interacts witha groove formed by helices one, two, and sixof tube. Additionally, the C-terminal tail oftube interacts with a groove on pelle createdby helices two through five. Tube also bindsthe dMyD88 death domain at a separate site;this interaction is much more stable than thatbetween tube and pelle (Kd = 150 nM and2 μM, respectively; N.J. Gay & M. Gangloff,unpublished results). To date structuralanalysis of MyD88/IRAK4 death domaincomplexes has not been achieved, and theabsence of tube in the vertebrate pathwayssuggests that there are significant differencesin the signaling mechanism. It is also unclearhow at a structural level autophosphorylationinduced during the signaling process cancause the dissociation of death domain–deathdomain complexes.
MOLECULAR MECHANISMOF SIGNAL TRANSDUCTION
As with other type I transmembrane receptorssuch as the receptor tyrosine kinases, the basicmechanism for signal transmission by TLRsis ligand-induced dimerization or oligomer-ization. For example, Spatzle, a symmetricaldimer, cross-links two molecules of DrosophilaToll ectodomain into a heterotrimeric com-plex, and this binding event is necessary andsufficient to induce signal transduction (16).The activation of the vertebrate TLRs likelyrequires a similar process of ligand-induceddimerization. The evidence for this comesfirst from domain-swap experiments in whichthe ectodomain of TLR4 is replaced by thatof Drosophila Toll. The chimeric receptors areactivated by treatment with Spatzle, suggest-ing that TLR4 will signal if two receptormolecules are arranged in an equivalent sym-metrical complex (51). Second, some mono-clonal antibodies directed against TLR3 andTLR4 can produce cross-linked complexes
that are unable to signal (98). This suggeststhat the receptor chains have to be arrangedin the correct orientation with respect to eachother for signaling to occur.
Recent work also suggests that dimer-ization promotes conformational changes inthe receptor ectodomains and induces stableprotein-protein interactions between the re-ceptor chains. In the case of Drosophila Toll,the immediate juxtamembrane regions of theectodomain appear to mediate these receptor-receptor interactions, and ligand binding maybe relieving a constitutive autoinhibition thatprevents the signaling-competent associationof unactivated receptors (51, 99, 100).
The dimerization of receptor ectodomainsmust also couple to cytoplasmic signaling, en-abling the recruitment of cognate adaptorsinto a postreceptor complex. Thus, receptordimerization must create a new arrangementof the TIR domains and thereby provide thespecificity required for adaptor binding. Twoproperties of the activated receptor complexesmay be relevant to this situation. First, at leastin the case of Drosophila Toll and probablyalso TLR4, the receptor complexes are sym-metrical. Second, the connections betweenthe transmembrane α-helix, the ectodomain,and TIR are likely rigid, so the ectodomainand TIR cannot rotate independently of eachother. As illustrated in Figure 4 for TLR4,this suggests that symmetry-related surfacesof the receptor TIRs are forced to associateand may undergo a structural reorganizationas a consequence. In the case of TLR4, a sur-face structural feature called the BB loop (seeFigure 3) that links the second α-helix to thesecond β-sheet of the fold may be of particularimportance for receptor homodimerization.LPS hyporesponsiveness in C3H/HeJ miceis caused by a mutation of a proline residueto histidine in the BB loop of TLR4, andthese mutant receptors have dominant neg-ative effects on TLR4 signaling (11). Thissuggests that they are able to form inactivedimers with wild-type TLR4 that are un-able to support downstream signaling. Thedimerized receptor TIRs now provide a new
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scaffold, which allows the recruitment of thespecific adaptor proteins. Although the foldis conserved in both receptor and adaptorTIR domains, the surface electrostatic prop-erties are distinct. TLR2 and TLR4 surfacesare rather basic, whereas the Mal/TIRAPand TRAM adaptors have similar negativelycharged surfaces. Thus specificity may be de-termined by the electrostatic complementar-ity of the receptor and adaptor TIR domains(89).
The mechanism shown in Figure 4 canalso account for the inhibitory properties oflipid A variants such as the tetra-acylated lipidIVa. Early experiments carried out before theidentification of TLR4 as the LPS receptorshowed that lipid IVa acted as a competitiveinhibitor of LPS signaling in human mono-cytes. This suggests that it binds to the samesite on TLR4/MD-2 as hexa-acylated lipid A
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 4Model for signal transduction by Toll-likereceptor 4 (TLR4). The curved ectodomains(ECDs) are illustrated with the coreceptor proteinMD-2. Two surfaces of the Toll-interleukinreceptor (TIR) domains are shown rotatedthrough 180◦. The surface containing the BB loopis perpendicular to the plane of the paper. (a) Priorto activation, receptor molecules are able todiffuse in the membrane and may form transientdimers. The ectodomains are rigidly connected tothe cytoplasmic TIRs by the transmembrane helix.(b) Activation by lipopolysaccharide (LPS) bindingto MD-2 leads to the dimerization of ectodomains,probably stabilized by receptor-receptorinteractions in the juxtamembrane region. Byanalogy with Drosophila Toll, which is activated bya dimeric protein ligand, the receptor complexesare likely symmetrical. Thus the TIR domains areconstrained to associate by interactions betweenequivalent surfaces on the TIR to form asymmetrical dimer. The evidence suggests that thesurface centered on the flexible BB loop may bethe dimerization interface and that association isstabilized by a conformational rearrangementinvolving this structure. (c) The dimerized TIRsprovide a new molecular surface that can bind toadaptor molecules TRAM and/or Mal/TIRAPwith high affinity. Abbreviations: B, BB loop; L,LPS; M, membrane.
and with comparable affinity (100a). A morerecent study demonstrated that hexa-acylatedlipid A induces cross-linking of TLR4/MD-2.By contrast, lipid IVa binds to TLR4/MD-2but does not cause dimerization or oligomer-ization (100b). Thus, signal transduction byTLR4 depends critically on the ability of lipidA to bind simultaneously to two moleculesof MD-2 or alternatively to induce a con-formational change in MD-2 that facilitatesassociation of two receptor molecules into aheterotetrameric complex. Synthetic antag-onists of TLR4 based on lipid IVa, such asE5531, are effective at blocking responses toendotoxin in vivo and are now in Phase IIIclinical trials for treatment of sepsis (100c,100d).
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The structure of the isolated TLR3ectodomain, solved recently by X-ray crystal-lography, also provides support for the activa-tion scheme outlined above (101, 102). As ex-pected, the TLR3 LRRs fold into an extendedsolenoid, and the surface is extensively mod-ified by N-linked glycans (Figure 2). Theectodomain behaves as a monomer in so-lution, but two molecules are arranged asdimers in the crystals used to solve the struc-ture. The dimer interface is on the convex(outer) surface at the C terminus, and thisarrangement could represent the conforma-tion adopted during the signaling process.However, as pointed out previously by Kirk& Bazan (103), this interface may be an ar-tifact generated during the crystallization ofthe protein and therefore may be of uncer-tain biological significance. Recent data fromSegal’s lab (104) show that only two residues(H539 and N541) are required for the bind-ing of dsRNA by the TLR3 ectodomain.These are positioned close to the C termi-nus in LRR20 on a lateral surface that isfree of glycan (Figure 2). On the basis ofthese results, the authors propose that bind-ing is mediated by the formation of a hydro-gen bond between N541 of TLR3 and the 2′
OH group of a ribose in the dsRNA. Theyfurther suggest that dsRNA would be able tocross-link two TLR3 ectodomains to form asymmetrical complex, with the first moleculerelated to the second by a 180o rotation and ashort translation. In principal, a single dsRNAmolecule could bind together a number ofTLR3 ectodomains to produce higher-orderoligomers. The binding of dsRNA may alsopromote protein-protein contacts betweentwo TLR3 ectodomains, although it is notknown whether this would induce conforma-tional changes of the type proposed above.Thus, although Spatzle is different chemi-cally from dsRNA, both molecules are able topromote a precise symmetrical arrangementof the receptor ectodomain. Clearly, high-resolution crystal structures of TLR3 in com-plex with dsRNA and Spatzle bound to Tollare required to address these questions.
In contrast to the situation with Tolland TLR4, recent work suggests that het-erodimeric complexes of TLR2 with TLR1induced by triacyl lipopetides may have anasymmetrical arrangement of the two recep-tor chains. These studies identified a clus-ter of residues in TLR2 located toward theC terminus in the DD loop (connecting thefourth α-helix and β-sheet) and αD regionthat are required for signaling by PAM3CSK4,and corresponding residues in TLR1 were lo-cated in the BB loop. Docking studies alsosuggest that the dimerization of the recep-tor TIRs is mediated by interactions betweenthe BB loop of TLR1 and the DD loop ofTLR2 (see Figure 3) (105). This implies thatthe ectodomains also will be arranged in anasymmetric manner by the binding of the tri-acyl lipopeptide. Another recent study sug-gests that a different region, the conservedαE helix, is required for homodimerizationof TLR2 induced by diacyl lipopeptides (106).This study also revealed a site in the N termi-nus of the MyD88 TIR that is required foradaptor recruitment and suggests two differ-ent modes of engagement between TLR2 andMyD88.
Taken together, these results emphasizethe potential of receptor and adaptor TIR do-mains to mediate a range of signal-inducedprotein-protein interactions, and this plastic-ity probably underlies the complexity gen-erated during the TLR signaling process.Our understanding of these mechanismsis currently restrained by the absence ofstructural studies of receptor and adaptorcomplexes.
CROSS TALK IN THETOLL-LIKE RECEPTORPATHWAYS
An emerging theme in Toll signaling is theregulatory role played by pathway cross talk.Thus, the outcomes of TLR-mediated sig-nals can be modified depending on the celltype and the other stimuli present in theenvironment.
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The Role of Protein Kinase Cε andIntegrins in TLR4 Signaling
As described above, the activation of TLR4results in the recruitment of four adaptor pro-teins (Figure 5). Mal/TIRAP is thought tobind directly to the receptor TIRs and acts asa bridging adaptor for recruitment of MyD88.This arm of the pathway mediates rapid acti-vation of NF-κB responses within a few min-utes of stimulation. By contrast, TRAM is
thought to bridge the binding of TRIF to thepostreceptor complex, and these adaptors ac-tivate the MyD88 independent pathway, lead-ing to the activation of IRF3 and delayed NF-κB responses (29, 107). At this stage, it is notknown if both sets of adaptors can bind simul-taneously or whether binding to the dimer-ized receptor TIRs is mutually exclusive. BothMal/TIRAP and TRAM are localized to theplasma membrane in unstimulated cells, and
Figure 5Cross talk in the TLR4 pathway. Model for the modulation of TLR4 signaling by inputs from β2 integrinand Fcγ receptors. Depending on the inputs received, there will be a bias toward signaling throughMal/MyD88 or TRAM/TRIF, leading to the production of different cytokine profiles. TRIF-directedsignaling also favors cross talk to the death pathway through RIP1/RIP3 [adaptors in the tumor necrosisfactor (TNF) signaling pathway]. Abbreviations: AP-1, activating protein-1; CD14, an extrinsicmembrane protein; FADD, Fas-associated death domain; IFN, interferon; IKK, IκB kinase; IRAK,interleukin-1 receptor-associated kinase; IRF3, interferon response factor 3; JNK, Jun N-terminal kinase;LPS, lipopolysaccharide; MAL, Mal/TIRAP (MyD88 adaptor like), MD-2, a secreted glycoprotein;MyD88, myeloid differentiation primary response protein 88; NF-κB, Nuclear factor κ B; PKCε, proteinkinase C ε; PLC, phospholipase C; PI3K and PI5K, phosphatidyl inositol 3 kinase and phosphatidylinositol 5 kinase; TAB1 and TAB2/3, adaptor molecules; TAK1, TGFβ-activated kinase; TLR4, Toll-likereceptor 4; TNFR, tumor necrosis factor receptor; TRAF6, TNF receptor-associated factor 6; TRAM,TRIF-related adaptor molecule; TRIF, TIR domain–containing adaptor protein inducing interferon-β.
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recent studies have shed light on the natureand functional significance of this association.
In the case of TRAM, constitutive bind-ing to the cytoplasmic membrane is caused bythe addition of myristate, a 14-carbon fatty-acid chain, to a glycine residue located at theN-terminus of the protein (108). TRAM istherefore one of a group of myristoyl pro-teins that also includes the src nonreceptortyrosine kinase, some small G proteins, anda protein kinase C (PKC) substrate protein,MARCKS (109). The anchoring of proteinsto the membrane by myristoyl groups is of-ten stabilized by additional electrostatic in-teractions between basic amino-acid residuesclose to the site of modification and the headgroups of phospholipids in the membrane.Thus, the membrane localization of myris-toyl proteins may be reversed by disruptingthese stabilizing electrostatic interactions, aso-called myristoyl electrostatic switch (110).In the case of TRAM, a myristoyl electrostaticswitch is operated by the PKC isoform PKCε
(111).PKCε phosphorylates a serine residue
close to the N terminus of TRAM, which dis-rupts the electrostatic contacts that stabilizemembrane association. The release of TRAMfrom the membrane is required for function,and this suggests that the activation of this armof the TLR4 pathway requires a second sig-nal from protein PKCε. Consistent with thisidea, mice lacking PKCε are hyporesponsivewhen stimulated by LPS (112). PKCε activ-ity is regulated by diacyl glycerol producedby rises in intracellular calcium concentration(113), but at this stage it is not known whichintrinsic or extrinsic stimuli regulate PKCε inimmune-system cells. One possibility, how-ever, is the Fcγ receptor family. These re-ceptors are expressed on many immune-celltypes, including B cells, macrophages, andDCs. They signal in response to the Fc regionof immunoglobulin G molecules and inducerises in intracellular calcium, leading to the ac-tivation of PKC family kinases (114). Ligationof FcγR receptors is known to cause a changein the profile of cytokines produced by treat-
ment with LPS (115). Thus the treatment ofmacrophages with immune complexes causesa polarization of T cell responses away fromTh1 toward Th2 (116). A plausible explana-tion for this effect is that the activity level ofPKCε will determine the profile of cytokinesproduced by TLR4 through the slow NF-κB and IRF3 responses (TRAM dependent)and the fast NF-κB activation (Mal/TIRAPdependent). One member of the Fcγ recep-tor family, FcγRIIb, has an inhibitory effecton calcium responses and may exert an an-tagonistic effect on TRAM-dependent LPSsignaling.
The Mal/TIRAP adaptor is also lo-calized to the cytoplasmic plasma mem-brane but uses a different mechanism toTRAM. Recent work shows that the N ter-minus of Mal/TIRAP contains a bindingsite for the inositol phospholipid PIP2 andthat Mal/TIRAP binds to the membranein a PIP2-dependent manner (117). Mem-brane localization is required for MyD88-dependent signaling through TLR4, andremarkably grafting the Mal/TIRAP PIP2binding site on to MyD88 confers the abil-ity to signal independently from Mal/TIRAP.This suggests that Mal/TIRAP functions totarget MyD88 to the membrane, acting as asorting adaptor rather than a bridging adap-tor as suggested above. The level of PIP2in the membrane is regulated by β2 integrinsignaling through the small G protein ARF6and the PI5 kinase. Membrane localizationand signaling by Mal/TIRAP but not TRAMare defective in macrophages lacking β2 in-tegrin. Interestingly, Fcγ receptors activatephospholipase C, which acts on PIP2 to gen-erate IP3 and PI3 kinases that convert PIP2to PIP3. Thus Fcγ receptors could exert anegative effect on Mal/TIRAP-mediated sig-naling, as well as a positive effect on TRAM.In this context, it is also interesting to notethat PIP3 is responsible for recruiting pro-teins containing PH domains to the mem-brane. These include the Bruton tyrosine ki-nase, which phosphorylates Mal/TIRAP atthree sites, and phospho-Mal/TIRAP cannot
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form complexes with TLR4 and MyD88 butmay be targeted for degradation (118, 118a).
The results described above and summa-rized in Figure 5 show that signaling in re-sponse to LPS can be modified by at leasttwo other signaling systems in response toboth endogenous and exogenous conditions.Similar complexity likely exists in other TLRpathways.
Toll-Like Receptor Signalingand Apoptosis
Under certain conditions and in some celltypes, TLR signaling can induce apoptosis;therefore it must have the capacity to crosstalk with the death signaling pathways. In par-ticular, there is a clear analogy with signalingthrough the TNF pathway that can both acti-vate NF-κB and induce apoptotic cell death.For example, in insects, responses to gram-negative bacteria are sensed by the IMD path-way. This pathway uses homologs of TNFpathway components such as Fas-associateddeath domain (FADD), DREDD (caspase 8),and relish (NF-κB), although the IMD path-way itself does not lead to apoptosis (119).Similarly, components of the vertebrate TNFpathway are used promiscuously in TLR sig-naling (e.g., TRAF6, TAK, and IKK kinases)(41).
The activation of TLR2 with several bac-terial lipoproteins can induce apoptosis inboth transfected human embryonic kidney(HEK293) cells and a monocyte-derived cellline (MHP1) (120). However, a related studywith HEK293 cells did not observe cell death,suggesting that the activation of apoptosis issensitive to culture conditions and the na-ture of the treatment (121). It appears thatapoptosis induced by TLR2 signaling involvesan interaction between the death domains ofMyD88 and the TNF-pathway-componentFADD, leading in turn to the activation ofcaspase 8 (122). Thus the TLR2 pathway bi-furcates at the level of MyD88 with branchesleading to either the activation of NF-κB orapoptotic cell death. In addition to TLR2,
TLR3 and TLR4 can induce cell death bothin vivo and in culture cells. For example, LPScan induce both the maturation and apop-tosis of DCs. DCs are professional antigen-presenting cells that couple innate and adap-tive immunity. Apoptosis may be an importantway of limiting infection and downregulat-ing DC-mediated immune responses in sep-sis. Recent work has shown that in contrast toTLR2, apoptosis mediated by both TLR3 andTLR4 requires the adaptor TRIF (123, 124).TRIF is a large adaptor protein with a TRAF6interacting domain and a receptor interactingprotein homotypic interaction motif (RHIM),as well as a TIR domain. In DCs, the RHIMdomain is required for TLR4-mediated apop-tosis, and it interacts with two proteins im-plicated in TNF signaling, RIP1 and RIP3.As RIP1 is involved in activating NF-κB, itis unclear how the RIP1/3/TRIF complexpromotes apoptosis. One possibility is thatRIP1, which has a death domain, can recruitFADD when in complex with RIP3 and TRIF.The involvement of TRIF in LPS-inducedcell death is intriguing as it suggests thatthe activation of PKCε, which should chan-nel LPS signaling through TRAM/TRIF, mayalso bias cells toward apoptosis.
CONCLUSIONS ANDPERSPECTIVE
The discovery of the TLRs 10 years ago ledto the development of a large research fieldand has changed our understanding of thefunction and importance of innate processesin the human immune response. The initialphase of characterization driven by reverse ge-netics in the mouse and the use of in vitroculture-based signaling systems are close tocompletion. However, this knowledge pro-vides a basis to address more difficult ques-tions of mechanism at the molecular level andthe complexities of regulation and pathwaycross talk in vivo. In this regard, Toll recep-tors and pathway components have provedespecially challenging as targets for struc-tural studies, and the picture obtained to date
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remains partial and fragmentary. However, aswith other biochemical systems, the evidencesuggests that the signaling process is dynamicand involves concerted and sequential pro-tein conformational changes. Another chal-lenge for the future is to elucidate further the
complexity of pathway cross talk for TLR4and other TLRs. It is likely that effective in-tervention in endotoxic shock, for example,requires a more detailed understanding of theother signaling inputs that drive responses toLPS in a pathological direction.
SUMMARY POINTS
1. Toll receptors have evolved to recognize highly diverse ligands derived frompathogenic microorganisms.
2. Insect Toll receptors are activated by an endogenous protein ligand generated indi-rectly by exposure to microbial stimuli. Vertebrate TLRs probably bind directly tomicrobial ligands.
3. The signaling mechanism of Toll and TLRs involves the dimerization of two receptorchains induced by ligand.
4. Ligand binding is likely to induce conformational changes that facilitate receptor-receptor interactions.
5. The association of the cytoplasmic TIR domains of the activated receptors producesa scaffold that can bind specifically to postreceptor adaptor proteins.
6. The signaling output of TLR4, the endotoxin receptor, can be modified by cross talkwith other pathways.
7. Cross talk may determine the cytokine profile produced by immune system cells andthus the type of adaptive immune response that is generated.
FUTURE ISSUES
1. Structural studies of receptor ectodomains in complex with ligands are required toestablish the principles of molecular recognition by TLRs.
2. Membrane biochemistry and structural analysis of activated whole receptor complexesare needed. Structures of isolated ecto- and TIR domains are unlikely to elucidate theconformational changes that provide specificity to the signaling process.
3. Further elucidation of cross talk in the TLR and other pathways is needed, and thesignificance of cross talk for immune system function in vivo should be explored.
4. Biochemical and structural information should be exploited for the developmentof therapy in TLR-associated diseases (e.g., endotoxic shock, autoimmunity, viralinfection).
LITERATURE CITED
1. Anderson KV, Bokla L, Nusslein-Volhard C. 1985. Cell 42:791–982. Anderson KV, Nusslein-Volhard C. 1984. Nature 311:223–273. Belvin MP, Anderson KV. 1996. Annu. Rev. Cell Dev. Biol. 12:393–416
160 Gay · Gangloff
Ann
u. R
ev. B
ioch
em. 2
007.
76:1
41-1
65. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Chi
cago
Lib
rari
es o
n 03
/17/
13. F
or p
erso
nal u
se o
nly.
ANRV313-BI76-07 ARI 8 June 2007 18:27
4. Hashimoto C, Hudson KL, Anderson KV. 1988. Cell 52:269–795. Schneider DS, Hudson KL, Lin TY, Anderson KV. 1991. Genes Dev. 5:797–8076. Gay NJ, Keith FJ. 1991. Nature 351:355–567. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. 1996. Cell 86:973–838. Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF. 1998. Proc. Natl. Acad. Sci.
USA 95:588–938a. Di Noia JM, Neuberger MS. 2007. Annu. Rev. Biochem. 76:1–229. Janeway CAJ. 1989. Cold Spring Harb. Symp. Quant. Biol. 54(Pt. 1):1–13
10. Medzhitov R, Preston-Hurlburt P, Janeway CA. 1997. Nature 388:394–9711. Poltorak A, He XL, Smirnova I, Liu MY, Van Huffel C, et al. 1998. Science 282:2085–8812. Akira S, Takeda K. 2004. Nat. Rev. Immunol. 4:499–51113. Wang L, Weber A, Atilano M, Filipe S, Gay NJ, Ligoxygakis P. 2006. EMBO J. 26:5005–
1414. Jang IH, Chosa N, Kim SH, Nam HJ, Lemaitre B, et al. 2006. Dev. Cell 10:45–5515. Ligoxygakis P, Pelte N, Hoffmann JA, Reichhart JM. 2002. Science 297:114–1616. Weber A, Tauszig-Delamasure S, Hoffmann J, Lelievre E, Gascan H, et al. 2003. Nat.
munol. 3:91–9720. Sun H, Towb P, Chiem DN, Foster BA, Wasserman SA. 2004. EMBO J. 23:100–1021. Shen B, Manley JL. 2002. Development 129:1925–3322. Shen B, Liu H, Skolnik EY, Manley JL. 2001. Proc. Natl. Acad. Sci. USA 98:8596–60123. Geuking P, Narasimamurthy R, Basler K. 2005. Genetics 171:1683–9424. Hoffmann JA. 2003. Nature 426:33–3825. Rutschmann S, Jung AC, Zhou R, Silverman N, Hoffmann JA, Ferrandon D. 2000. Nat.
Immunol. 1:342–4726. Liew FY, Xu D, Brint EK, O’Neill LA. 2005. Nat. Rev. Immunol. 5:446–5827. McGettrick AF, O’Neill LA. 2004. Mol. Immunol. 41:577–8228. Vogel SN, Fitzgerald KA, Fenton MJ. 2003. Mol. Interv. 3:466–7729. Fitzgerald KA, Rowe DC, Barnes BJ, Caffrey DR, Visintin A, et al. 2003. J. Exp. Med.
198:1043–5530. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, et al. 2003. Science 301:640–4331. Janssens S, Beyaert R. 2003. Mol. Cell 11:293–30232. Picard C, Puel A, Bonnet M, Ku CL, Bustamante J, et al. 2003. Science 299:2076–7933. Suzuki N, Suzuki S, Yeh WC. 2002. Trends Immunol. 23:503–634. Medvedev AE, Thomas K, Awomoyi A, Kuhns DB, Gallin JI, et al. 2005. J. Immunol.
174:6587–9135. Kollewe C, Mackensen AC, Neumann D, Knop J, Cao P, et al. 2004. J. Biol. Chem.
279:5227–3636. Li SY, Strelow A, Fontana EJ, Wesche H. 2002. Proc. Natl. Acad. Sci. USA 99:5567–7237. Qin J, Jiang Z, Qian Y, Casanova JL, Li X. 2004. J. Biol. Chem. 279:26748–5338. Deng L, Wang C, Spencer E, Yang L, Braun A, et al. 2000. Cell 103:351–6139. Wooff J, Pastushok L, Hanna M, Fu Y, Xiao W. 2004. FEBS Lett. 566:229–3340. Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. 2001. Nature 412:346–5141. Shim JH, Xiao C, Paschal AE, Bailey ST, Rao P, et al. 2005. Genes Dev. 19:2668–8142. Kanayama A, Seth RB, Sun L, Ea CK, Hong M, et al. 2004. Mol. Cell 15:535–48
www.annualreviews.org • Toll Receptor Signal Transduction 161
Ann
u. R
ev. B
ioch
em. 2
007.
76:1
41-1
65. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Chi
cago
Lib
rari
es o
n 03
/17/
13. F
or p
erso
nal u
se o
nly.
ANRV313-BI76-07 ARI 8 June 2007 18:27
43. Hoebe K, Beutler B. 2004. J. Endotoxin Res. 10:130–3644. Hemmi H, Takeuchi O, Sato S, Yamamoto M, Kaisho T, et al. 2004. J. Exp. Med.
199:1641–5045. Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, et al. 2003. Nat. Immunol.
4:491–9646. Oganesyan G, Saha SK, Guo B, He JQ, Shahangian A, et al. 2006. Nature 439:208–1147. Mori M, Yoneyama M, Ito T, Takahashi K, Inagaki F, Fujita T. 2004. J. Biol. Chem.
279:9698–70248. Sato M, Suemori H, Hata N, Asagiri M, Ogasawara K, et al. 2000. Immunity 13:539–4849. Kawai T, Sato S, Ishii KJ, Coban C, Hemmi H, et al. 2004. Nat. Immunol. 5:1061–6850. Hacker H, Redecke V, Blagoev B, Kratchmarova I, Hsu LC, et al. 2006. Nature 439:204–
751. Weber AN, Moncrieffe MC, Gangloff M, Imler JL, Gay NJ. 2005. J. Biol. Chem.
280:22793–9952. He XL, Garcia KC. 2004. Science 304:870–7553. Wiesmann C, Ultsch MH, Bass SH, de Vos AM. 1999. Nature 401:184–8854. Bibel M, Barde YA. 2000. Genes Dev. 14:2919–3755. Haynes LM, Moore DD, Kurt-Jones EA, Finberg RW, Anderson LJ, Tripp RA. 2001.
J. Virol. 75:10730–3756. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, et al. 2001. Nature 410:1099–10357. Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA, et al. 2003. Nat.
Immunol. 4:1247–5358. Mizel SB, West AP, Hantgan RR. 2003. J. Biol. Chem. 278:23624–2959. Rallabhandi P, Bell J, Boukhvalova MS, Medvedev A, Lorenz E, et al. 2006. J. Immunol.
177:322–3260. Schromm AB, Lien E, Henneke P, Chow JC, Yoshimura A, et al. 2001. J. Exp. Med.
194:79–8861. Re F, Strominger JL. 2002. J. Biol. Chem. 277:23427–3262. Correia JD, Ulevitch RJ. 2002. J. Biol. Chem. 277:1845–5463. Viriyakosol S, Tobias PS, Kitchens RL, Kirkland TN. 2001. J. Biol. Chem. 276:38044–5164. Inohara N, Nunez G. 2002. Trends Biochem. Sci. 27:219–2165. Gangloff M, Gay NJ. 2004. Trends Biochem. Sci. 29:294–30066. Lien E, Means TK, Heine H, Yoshimura A, Kusumoto S, et al. 2000. J. Clin. Invest.
310:1970–7386. Kajava AV. 1998. J. Mol. Biol. 277:519–2787. Kobe B, Deisenhofer J. 1994. Trends Biochem. Sci. 19:415–2188. Xu YW, Tao X, Shen BH, Horng T, Medzhitov R, et al. 2000. Nature 408:111–1589. Dunne A, Ejdeback M, Ludidi P, O’Neill LAJ, Gay NJ. 2003. J. Biol. Chem. 278:41443–
5190. Stock AM, Mottonen JM, Stock JB, Schutt CE. 1989. Nature 337:745–4991. Fesik SW. 2000. Cell 103:273–8292. Towb P, Galindo RL, Wasserman SA. 1998. Development 125:2443–5093. Schiffmann DA, White JHM, Cooper A, Nutley MA, Harding SE, et al. 1999. Biochem-
istry 38:11722–3394. Aravind L, Dixit VM, Koonin EV. 1999. Trends Biochem. Sci. 24:47–5395. Xiao T, Towb P, Wasserman SA, Sprang SR. 1999. Cell 99:545–5596. Moncrieffe MC, Stott KM, Gay NJ. 2005. FEBS Lett. 579:3920–2697. Lasker MV, Gajjar MM, Nair SK. 2005. J. Immunol. 175:4175–7998. Matsumoto M, Kikkawa S, Kohase M, Miyake K, Seya T. 2002. Biochem. Biophys. Res.
Commun. 293:1364–6999. Hu X, Yagi Y, Tanji T, Zhou S, Ip YT. 2004. Proc. Natl. Acad. Sci. USA 101:9369–74
100. Gay N, Gangloff M, Weber A. 2006. Nat. Rev. Immunol. 6:693–98100a. Golenbock DT, Hampton RY, Qureshi N, Takayama K, Raetz CRH. 1991. J. Biol.
Chem. 266:19490–98100b. Saitoh S, Akashi S, Yamada T, Tanimura N, Kobayashi M, et al. 2004. Int. Immunol.
16:961–69100c. Christ WJ, Asano O, Robidoux AL, Perez M, Wang Y, et al. 1995. Science 265:80–83100d. Lynn M, Rossignol DP, Wheeler JL, Kao RJ, Perdomo CA, et al. 2003. J. Infect. Dis.
187:31–39101. Bell JK, Botos I, Hall PR, Askins J, Shiloach J, et al. 2005. Proc. Natl. Acad. Sci. USA
102:10976–80102. Choe J, Kelker MS, Wilson IA. 2005. Science 309:581–85103. Kirk P, Bazan JF. 2005. Immunity 23:347–50104. Bell JK, Askins J, Hall PR, Davies DR, Segal DM. 2006. Proc. Natl. Acad. Sci. USA
103:8792–97
www.annualreviews.org • Toll Receptor Signal Transduction 163
Ann
u. R
ev. B
ioch
em. 2
007.
76:1
41-1
65. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Chi
cago
Lib
rari
es o
n 03
/17/
13. F
or p
erso
nal u
se o
nly.
ANRV313-BI76-07 ARI 8 June 2007 18:27
105. Gautam JK, Ashish, Comeau LD, Krueger JK, Smith MFJ. 2006. J. Biol. Chem.281:30132–42
106. Jiang Z, Georgel P, Li C, Choe J, Crozat K, et al. 2006. Proc. Natl. Acad. Sci. USA103:10961–66
107. Yamamoto M, Sato S, Hemmi H, Uematsu S, Hoshino K, et al. 2003. Nat. Immunol.4:1144–50
108. Rowe DC, McGettrick AF, Latz E, Monks BG, Gay NJ, et al. 2006. Proc. Natl. Acad.Sci. USA 103:6299–304
109. Johnson DR, Bhatnagar RS, Knoll LJ, Gordon JI. 1994. Annu. Rev. Biochem. 63:869–914110. McLaughlin S, Aderem A. 1995. Trends Biochem. Sci. 20:272–76111. McGettrick AF, Brint EK, Palsson-McDermott EM, Rowe DC, Golenbock DT, et al.
2006. Proc. Natl. Acad. Sci. USA 103:9196–201112. Castrillo A, Pennington DJ, Otto F, Parker PJ, Owen MJ, Bosca L. 2001. J. Exp. Med.
194:1231–42113. Tan SL, Parker PJ. 2003. Biochem. J. 376:545–52114. Ravetch JV, Bolland S. 2001. Annu. Rev. Immunol. 19:275–90115. Sutterwala FS, Noel GJ, Salgame P, Mosser DM. 1998. J. Exp. Med. 188:217–22116. Anderson CF, Mosser DM. 2002. J. Leukoc. Biol. 72:101–6117. Kagan JC, Medzhitov R. 2006. Cell 125:943–55118. Gray P, Dunne A, Brikos C, Jefferies CA, Doyle SL, O’Neill LA. 2006. J. Biol. Chem.
281:10489–95118a. Mansell A, Smith R, Doyle SL, Gray P, Fenner JE, et al. 2006. Nat. Immunol. 7:148–55119. Zhou R, Silverman N, Hong M, Liao DS, Chung Y, et al. 2005. J. Biol. Chem. 280:34048–
55120. Aliprantis AO, Yang RB, Mark MR, Suggett S, Devaux B, et al. 1999. Science 285:736–39121. Brightbill HD, Libraty DH, Krutzik SR, Yang RB, Belisle JT, et al. 1999. Science
285:732–36122. Aliprantis AO, Yang RB, Weiss DS, Godowski P, Zychlinsky A. 2000. EMBO J. 19:3325–
36123. Kaiser WJ, Offermann MK. 2005. J. Immunol. 174:4942–52124. De Trez C, Pajak B, Brait M, Glaichenhaus N, Urbain J, et al. 2005. J. Immunol. 175:839–
46125. Wyllie DH, Kiss-Toth E, Visintin A, Smith SC, Boussouf S, et al. 2000. J. Immunol.
165:7125–32126. Massari P, Henneke P, Ho Y, Latz E, Golenbock DT, Wetzler LM. 2002. J. Immunol.
168:1533–37127. Buwitt-Beckmann U, Heine H, Wiesmuller KH, Jung G, Brock R, et al. 2005. Eur.
J. Immunol. 35:282–89128. Schwandner R, Dziarski R, Wesche H, Rothe M, Kirschning CJ. 1999. J. Biol. Chem.
274:17406–9129. Underhill DM, Ozinsky A, Smith KD, Aderem A. 1999. Proc. Natl. Acad. Sci. USA
96:14459–63130. Hajjar AM, O’Mahony DS, Ozinsky A, Underhill DM, Aderem A, et al. 2001. J. Immunol.
166:15–19131. Campos MA, Almeida IC, Takeuchi O, Akira S, Valente EP, et al. 2001. J. Immunol.
167:416–23132. Opitz B, Schroder NWJ, Spreitzer I, Michelsen KS, Kirschning CJ, et al. 2001. J. Biol.
Chem. 276:22041–47
164 Gay · Gangloff
Ann
u. R
ev. B
ioch
em. 2
007.
76:1
41-1
65. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by U
nive
rsity
of
Chi
cago
Lib
rari
es o
n 03
/17/
13. F
or p
erso
nal u
se o
nly.
ANRV313-BI76-07 ARI 8 June 2007 18:27
133. Hirschfeld M, Weis JJ, Toshchakov V, Salkowski CA, Cody MJ, et al. 2001. Infect. Immun.69:1477–82
134. Asea A, Rehli M, Kabingu E, Boch JA, Bare O, et al. 2002. J. Biol. Chem. 277:15028–34135. Scheibner KA, Lutz MA, Boodoo S, Fenton MJ, Powell JD, Horton MR. 2006.
J. Immunol. 177:1272–81136. Bieback K, Lien E, Klagge IM, Avota E, Schneider-Schaulies J, et al. 2002. J. Virol.
76:8729–36137. Gomi K, Kawasaki K, Kawai Y, Shiozaki M, Nishijima M. 2002. J. Immunol. 168:2939–
43138. Kawasaki K, Akashi S, Shimazu R, Yoshida T, Miyake K, Nishijima M. 2000. J. Biol.
Chem. 275:2251–54139. Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP, et al. 2000. Nat. Immunol.
1:398–401140. Rassa JC, Meyers JL, Zhang Y, Kudaravalli R, Ross SR. 2002. Proc. Natl. Acad. Sci. USA
99:2281–86141. Bulut Y, Faure E, Thomas L, Karahashi H, Michelsen KS, et al. 2002. J. Immunol.
168:1435–40142. Ohashi K, Burkart V, Flohe S, Kolb H. 2000. J. Immunol. 164:558–61143. Okamura Y, Watari M, Jerud ES, Young DW, Ishizaka ST, et al. 2001. J. Biol. Chem.
276:10229–33144. Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, et al. 2002. J. Exp. Med. 195:99–111145. Johnson GB, Brunn GJ, Kodaira Y, Platt JL. 2002. J. Immunol. 168:5233–39146. Smiley ST, King JA, Hancock WW. 2001. J. Immunol. 167:2887–94147. Roelofs MF, Boelens WC, Joosten LA, Abdollahi-Roodsaz S, Geurts J, et al. 2006.
J. Immunol. 176:7021–27148. Eaves-Pyles TD, Wong HR, Odoms K, Pyles RB. 2001. J. Immunol. 167:7009–16149. Akira S, Hemmi H. 2003. Immunol. Lett. 85:85–95150. Jurk M, Heil F, Vollmer J, Schetter C, Krieg AM, et al. 2002. Nat. Immunol. 3:499151. Leadbetter EA, Rifkin IR, Hohlbaum AM, Beaudette BC, Shlomchik MJ, Marshak-
Rothstein A. 2002. Nature 416:603–7152. Klosterman PS, Shah SA, Steitz TA. 1999. Biochemistry 38:14784–92153. Weber ANR, Gangloff M, Moncrieffe MC, Hyvert Y, Imler JL, Gay NJ. 2007. J. Biol.
Chem. 282: In press. doi:10.1074/jbc.M700068200
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