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Recognition and Signaling by T oll-Like ReceptorsA.Phillip West,∗Anna Alicia Koblansky,∗and Sankar GhoshSection of Immunobiology and Department of Molecular Biophysics andBiochemistry,Yale University School of Medicine,New Haven,Connecticut 06520;email:phillip.west@,alicia.koblansky@,sankar.ghosh@Annu.Rev.Cell Dev.Biol.2006.22:409–37First published online as a Review in Advance on July 5,2006The Annual Review ofCell and Developmental Biology is online at This article’s doi:10.1146/annurev.cellbio.21.122303.115827Copyright c2006by Annual Reviews.All rights reserved1081-0706/06/1110-0409$20.00∗These authors contributed equally to this work.Key Wordsinnate immunity,leucine-rich repeat,TIR domain,signal transduction,MyD88,TRIFAbstractT oll-like receptors (TLRs)are transmembrane proteins that de-tect invading pathogens by binding conserved,microbially derived molecules and that induce signaling cascades for proinflammatory gene expression.A critical component of the innate immune sys-tem,TLRs utilize leucine-rich-repeat motifs for ligand binding and a shared cytoplasmic domain to recruit the adaptors MyD88,TRIF ,TIRAP ,and/or TRAM for downstream signaling.Despite signifi-cant domain conservation,TLRs induce gene programs that lead not only to the robust production of general proinflammatory me-diators but also to the production of unique effectors,which provide pathogen-tailored immune responses.Here we review the mecha-nisms by which TLRs recognize pathogens and induce distinct sig-naling cascades.409A n n u . R e v . C e l l D e v .B i o l . 2006.22:409-437. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b y H A R V A R D U N I V E R S I T Y o n 12/02/07. F o r p e r s o n a l u s e o n l y .ContentsINTRODUCTION.................410TLR LIGAND SPECIFICITIES....411TLR1,TLR2,and TLR6.........411TLR3...........................412TLR4...........................413TLR5...........................413TLR7and TLR8.................414TLR9...........................414TLR11..........................414DIVERSITY OF TLR LIGAND RECOGNITION................414Leucine-Rich-Repeat Diversity ...415Cooperative Interactions Between TLRs.........................416Coreceptors and TLR Binding Specificity.....................417TLR SIGNALING PATHWAYS....418MyD88-Dependent Signaling.....419MyD88-Independent/TRIF-Dependent Signaling..........421Additional TIR Adaptor Proteins..422TLR-Mediated Inductionof IRFs .......................424Chromatin Remodeling and TLR Signaling Specificity...........425NEGATIVE REGULATION OF TLR SIGNALING ..............425FINAL THOUGHTS ..............427INTRODUCTIONMetchnikoff first described the innate im-mune system more than a century ago,yet research into this critical arm of immunity was largely overshadowed by the discov-ery of antibodies,B and T cells,and other Innate immunity:the initial,rapidly induced immune response of multicellularorganisms;utilizes nonclonal,germline-encoded receptors for pathogenrecognition and the activation/recruitment of phagocytic cellscomponents of the adaptive immune sys-tem (Silverstein 2003).Recent work,how-ever,has yielded substantial insight into the composition and function of innate immu-nity,and it is now apparent that the innate immune system utilizes an intricate network of effector mechanisms to clear or moder-ate pathogen replication until the adaptiveimmune system can mount a more specific and robust response.In 1989,Janeway (1989)proposed that innate effector mechanisms are initiated via the specific detection of mi-crobes by germline-encoded,nonclonal re-ceptors,which are essential for the imme-diate detection and control of infection in mammals.These molecules,termed pattern-recognition receptors (PRRs),primarily func-tion in recognizing microbial structures re-ferred to as pathogen-associated molecular patterns (PAMPs).Building on this foundational hypothe-sis,researchers have shown that the innate immune system is phylogenetically con-served and is present in all multicellular or-ganisms (Hoffmann 1999,Medzhitov 2001,Medzhitov &Janeway 1997).As proposed by Janeway (1989),innate immunity is in fact predicated upon PRR detection of abun-dantly expressed PAMPs,which are con-served among broad classes of microor-ganisms.These molecules are critical for pathogen replication and/or survival and are unique to microorganisms.Thus,they are ab-sent from host cells and thereby endow the host with an efficient,nonself-reactive means to detect invading pathogens.Examples of such microbial products include lipopolysac-charide (LPS),lipoproteins,and viral or bac-terial nucleic acids.Signaling through PRRs induces a cascade of events including pro-duction of proinflammatory chemokines and cytokines,activation of complement,recruit-ment of phagocytic cells,and mobilization of professional antigen-presenting cells.In the late 1990s,three key discoveries significantly advanced the understanding and definition of PRR-mediated innate immunity.First,in 1996,the Drosophila melanogaster pro-tein T oll,identified previously for its role in dorso-ventral embryo patterning,was shown to be critical for effective immune responses in adult flies against the fungus Aspergillus fu-migatus (Lemaitre et al.1996).The following year,a mammalian homolog of the Drosophila T oll receptor,initially termed human T oll and now known as T oll-like receptor 4(TLR4),410West·Koblansky·GhoshA n n u . 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F o r p e r s o n a l u s e o n l y .was identified.A constitutively active form of this receptor could activate the transcrip-tion factor nuclear factor-kappa B (NF-κB),leading to the expression of proinflammatory genes encoding interleukin (IL)-1,IL-6,and IL-8and the upregulation of costimulatory molecules (Medzhitov et al.1997).Finally,identification of a point mutation in the Tlr4gene that rendered mice unresponsive to LPS challenge,and more susceptible to Gram-negative bacterial sepsis,definitively linked TLRs to innate immune responses (Poltorak et al.1998).TLRs are type I transmembrane proteins of the Interleukin-1receptor (IL-1R)fam-ily that possess an N-terminal leucine-rich-repeat (LRR)domain for ligand binding,a sin-gle transmembrane domain,and a C-terminal intracellular signaling domain.The TLR C terminus is homologous to the intracellular domain of the IL-1R and is thus referred to as the T oll/IL-1receptor (TIR)domain.TLRs are expressed at the cell membrane and in subcellular compartments such as the endo-some.TLRs are widely expressed in many cell types,including nonhematopoietic ep-ithelial and endothelial cells,although most cell types express only a select subset of these receptors.Hematopoietically derived sentinel cells,such as macrophages,neutrophils,and dendritic cells (DCs),however,express most of the TLRs,with some variation in differ-ent subsets,e.g.,between conventional DCs and plasmacytoid DCs.Thus far,13mam-malian TLRs,10in humans and 13in mice,have been identified (Beutler 2004).TLRs 1–9are conserved among humans and mice,yet TLR10is present only in humans and TLR11is functional only in mice.Although much is known about the ligands and signaling path-ways of TLRs 1–9and 11,the biological roles of TLRs 10,12,and 13remain unclear,as their expression patterns,ligands,and modes of signaling have yet to be defined.Over the past few years,several excellent reviews detailing the many aspects of TLR bi-ology have been published.T o avoid duplica-tion,we focus our review on the mechanismsPRR:patternrecognition receptor Pathogen-associatedmolecular pattern (PAMP):amolecular pattern common to classes of microorganisms but not found in host cellsLPS:lipopolysaccharide Cytokine:proteins such as TNF αand IL-6that affect the function and behavior ofneighboring cells via receptor-mediated signaling.Immune cytokines are often referred to as interleukins (IL)TLR:T oll-like receptorNuclearfactor-kappa B (NF-κB):an evolutionarily conservedtranscription factor critical for the production of effector cytokines,growth factors,and enzymes required for the initiation and resolution ofimmune responses LRR:leucine-rich repeatTIR:T oll/IL-1receptor homology domainby which TLRs recognize ligands and in-duce specific signaling cascades for unique an-timicrobial gene programs and general proin-flammatory responses.We provide a brief overview of known TLR ligands,a discussion of the mechanisms by which TLRs achieve diversity in ligand recognition,and a dis-cussion of our current knowledge about the molecular pathways that determine signaling specificity.TLR LIGAND SPECIFICITIES TLR1,TLR2,and TLR6LPS was originally considered to be the ligand for TLR2,but subsequent studies revealed that contaminating bacterial lipoprotein in LPS preparations is the actual ligand (Wetzler 2003).In agreement with these later findings,TLR2-deficient macrophages were found to be hyporesponsive to several Gram-positive bacterial cell wall components as well as Staphylococcus aureus peptidoglycan (T akeuchi et al.2000).Additional work has shown that TLR2is involved in the recognition of a wide range of microbial products and gen-erally functions as a heterodimer with either TLR1or TLR6(Ozinsky et al.2000,Wyllie et al.2000).The TLR2/TLR1heterodimer recognizes a variety of lipoproteins,includ-ing those from mycobacteria and meningo-cocci (T akeuchi et al.2002,Wetzler 2003),whereas the TLR2/TLR6complex recog-nizes mycoplasma lipoproteins and peptido-glycan (T akeuchi et al.2001).Recent reports have demonstrated that triacylated lipopro-teins from bacteria are preferentially recog-nized by the TLR1/TLR2complex,whereas diacylated lipoproteins are recognized by the TLR2/TLR6complex (T akeuchi et al.2002).However,additional TLR2ligands do not ap-pear to require TLR1or TLR6for signal-ing,implying that TLR2may recognize some ligands as homodimers or heterodimers with other non-TLR molecules.Such TLR2lig-ands include the Gram-positive cell wall com-ponent lipoteichoic acid;the mycobacterial •TLR Recognition and Signaling411A n n u . 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F o r p e r s o n a l u s e o n l y .Figure 1TLR ligand specificities.TLRs recognize a diverse array of PAMPs from bacteria,viruses,protozoa,and fungi.For detection of bacteria,heterodimeric TLR2/1binds triacyl lipopeptides,whereas TLR2/6dimers bind diacyl lipopeptides and lipoteichoic acid.Homodimeric TLR2binds peptidoglycan,atypical LPS,phenol-soluble modulin from Staphylococcus epidermidis ,and porin proteins from Neisseria .Inaddition,TLR4binds LPS,TLR5binds flagellin,and TLR9binds bacterial CpG DNA.TLR11detects an unidentified protein(s)from uropathogenic Escherichia coli .Viral dsRNA,RSV F protein,ssRNA,and unmethylated CpG DNA are sensed by TLRs 3,4,7/8,and 9,respectively.Finally,heterodimeric TLR2/6binds fungally derived zymosan and Trypanosoma cruzi GIPLs,whereas TLR11also senses a profilin-like protein from T oxoplasma gondii .Abbreviations:dsRNA,double-stranded RNA;LPS,lipopolysaccharide;GIPLs,glycolipids;PAMP ,pathogen-associated molecular pattern;RSV F ,respiratory syncytial virus fusion;ssRNA,single-stranded RNA;TLR,T oll-like receptor.cell wall component lipoarabinomannan;atypical LPS produced by Legionella ,Lep-tospira interrogans,Porphyromonas gingivitis,and Bordetella ;and porins present in the outer membrane of Neisseria (Figure 1)(Massari et al.2002,Wetzler 2003).In addition to bacterial PAMPs,TLR2het-ero/homodimers recognize fungal and pro-tozoan molecules.Zymosan (a crude mix-ture of glucans,mannan,proteins,chitin,and glycolipids extracted from the cell mem-brane of fungi)induces signaling through TLR2/6(Kataoka et al.2002,Underhillet al.1999a).In addition,glycosylphos-phatidylinositol (GPI)anchors and glycoinos-itolphospholipids from the parasitic protozoa Trypanosoma cruzi induce signaling through TLR2(Campos et al.2001).TLR3Double-stranded RNA (dsRNA)was long hypothesized to be a viral PAMP ,as it is pro-duced during the course of many viral in-fections.Expression of human TLR3con-fers responsiveness to purified dsRNA and412West·Koblansky·GhoshA n n u . 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F o r p e r s o n a l u s e o n l y .polyinosinic-polycytidylic acid [poly(I:C)]in HEK293cells,and TLR3-deficient mice dis-play impaired responses to these ligands.TLR3signaling results in the activation of NF-κB and interferon regulatory factor 3(IRF3),ultimately leading to the production of antiviral molecules,such as type I interfer-ons (IFN-α/β)(Alexopoulou et al.2001).Recent research,however,has demon-strated TLR3-independent recognition of viral dsRNA via the helicases RIG-I (retinoic acid–inducible gene I)and MDA5(melanoma-differentiation-associated gene 5),which are cytoplasmic PRRs expressed abundantly in multiple cell types (Andrejeva et al.2004,Y oneyama et al.2004).RIG-I and MDA5differentially recognize different groups of RNA viruses and are thus critical for a robust antiviral response (Kato et al.2006).These receptors contain a helicase domain for RNA binding and two caspase recruitment domains (CARDs)for signal transduction.Upon ligand binding,RIG-I and MDA5bind and activate the adaptor IPS-1(interferon-βpromoter stimulator 1;also termed CARDIF ,MAVS,and VISA)for NF-κB and IRF3activation and subsequent production of IFN-β(Kawai et al.2005,Meylan et al.2005,Seth et al.2005,Sun et al.2006,Xu et al.2005).The importance of RIG-I-and MDA5-mediated viral recog-nition is further supported by gene-targeting experiments demonstrating that TLR3and its adaptor TRIF are not required for type I IFN production in some virally infected cells,such as fibroblasts and conventional DCs (Honda et al.2003).However,plasmacytoid dendritic cells exclusively utilize TLR3/TRIF signaling for type I IFN production in re-sponse to RNA viruses and poly(I:C)(Kato et al.2005).TLR4LPS is the most thoroughly studied TLR lig-and.LPS,a glycolipid component of the outer membrane of Gram-negative bacteria,ex-hibits the most potent immunostimulatory ac-Polyinosinic:poly-cytidylic acid [poly(I:C)]:a synthetic,double-stranded RNA mimic that binds and signals through TLR3IRF:interferon regulatory factor Interferon (IFN):cytokines that block viral replication and infection ofsurrounding cellstivity among all known TLR ligands (Miyake 2004).T race amounts of LPS activate the in-nate immune system via TLR4,leading to the production of numerous proinflammatory mediators,such as TNF α,IL-1,and IL-6.As discussed below,TLR4alone is not suffi-cient for a robust response to LPS;additional components of the LPS recognition com-plex,CD14and MD-2,are required.Other TLR4ligands include Lipid A analogs (Lien et al.2000),taxol (Perera et al.2001),my-cobacterial components (Means et al.1999),Aspergillus hyphae (Mambula et al.2002),Cryptococcus neoformans capsular polysaccha-ride (Shoham et al.2001),and respiratory syn-cytial virus (RSV)F protein (Kurt-Jones et al.2000).TLR5Flagellin,a protein component of Gram-negative bacterial flagella,is the cognate lig-and for TLR5(Hayashi et al.2001).TLR5recognizes a highly conserved,central core structure of flagellin that is essential for protofilament assembly and bacterial motil-ity (Smith et al.2003).Interestingly,the TLR5recognition site is masked in the fil-amentous flagellar structure,thus indicat-ing that TLR5recognizes only monomeric flagellin (Smith et al.2003).Furthermore,flagellin appears to bind directly to TLR5at residues 386–407of the extracellular do-main (ED),as TLR5-ED mutants lacking this domain are unable to interact with flagellin in biochemical assays (Mizel et al.2003b).Recent articles have demonstrated TLR5-independent recognition of cytosolic Salmonella typhimurium flagellin via Ipaf,a member of the nucleotide-binding oligomer-ization domain (NOD)-LRR (Franchi et al.2006,Miao et al.2006).Ipaf-mediated recog-nition of cytosolic flagellin is critical for caspase-1activation and subsequent IL-1βse-cretion by macrophages,further highlighting the significance of TLR-independent pattern recognition in the overall complexity of the innate immune response. •TLR Recognition and Signaling413A n n u . 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F o r p e r s o n a l u s e o n l y .CpG DNA:short DNA sequences consisting ofcytosine-guanosine sequences flanked by additionalnucleotides that bind and signal via TLR9TLR7and TLR8Mouse TLR7recognizes a class of synthetic antiviral compounds,such as imidazoquino-lines and loxoribine (Hemmi et al.2002).Furthermore,TLR7and human TLR8de-tect the antiviral azoquinoline compound R-848(Jurk et al.2002).These synthetic compounds are structurally related to nu-cleic acids;TLR7and human TLR8rec-ognize guanosine-or uridine-rich single-stranded RNA (ssRNA)derived from RNA viruses (Diebold et al.2004,Heil et al.2003,Lund et al.2004).Interestingly,mammalian RNA,which contains many modified nu-cleosides,is significantly less stimulatory via TLRs 7and 8than bacterial RNA,suggest-ing that the mammalian host utilizes nucle-oside modification as a means to distinguish between endogenous and pathogen-derived RNA (Kariko et al.2005).Similar to the func-tion of TLR3,engagement of these receptors leads to the production of type I IFNs,which are obligate components of antiviral innate immunity.TLR9TLR9recognizes bacterial DNA contain-ing unmethylated CpG motifs,and TLR9-deficient mice are not responsive to CpG DNA challenge (Hemmi et al.2000).TLR9expressed in plasmacytoid DCs recognizes vi-rally derived CpG sequences for the induction of IFN-α(Krug et al.2004,Lund et al.2003,T akeshita et al.2001).The low frequency and high rate of methylation of CpG mo-tifs prevent recognition of mammalian DNA by TLR9under physiological circumstances.In certain autoimmune disorders,however,IgG2a/chromatin complexes containing CpG DNA can engage the B cell receptor and TLR9concomitantly,leading to the pro-duction of rheumatoid factor and other au-toreactive molecules (Leadbetter et al.2002,Viglianti et al.2003).A recent report indi-cated that the intracellular,endosomal restric-tion of TLR9is critical for discriminating be-tween self and nonself DNA,as host DNA,unlike microbial DNA,does not usually en-ter the endosomal compartment (Barton et al.2006).Finally,Coban et al.(2005)reported that TLR9recognizes a novel non-DNA lig-and called hemozoin,which is a hydrophobic heme polymer produced by malaria parasites as they digest host hemoglobin.In addition to TLR9-mediated detection of CpG DNA in the endosomal compartment,the mammalian innate immune system also responds to foreign DNA in the cytosol.This response is important for type I IFN produc-tion in response to viruses and intracellular pathogens,such as Listeria monocytogenes and Shigella flexneri ,that replicate in the cytoplasm (Ishii et al.2006,Stetson &Medzhitov 2006).As membrane restriction prevents TLRs from sampling the cytosol for PAMPs,cytosolic PRRs have evolved to provide comprehensive innate immune recognition that offsets TLR restriction.TLR11Gene-targeting studies revealed that TLR11-deficient mice are susceptible to uropathogenic Escherichia coli infection (Zhang et al.2004).Although the bacterial ligand for TLR11remains undiscovered,the stimulatory activity of uropathogenic bacteria can be destroyed by proteinase K treatment,suggesting that TLR11recognizes a protein ligand.TLR11also recognizes a class of profilin-like molecules expressed by apicomplexan protozoans,including T oxoplasma gondii (Yarovinsky et al.2005).DIVERSITY OF TLR LIGAND RECOGNITIONAs described above,TLRs sense an extremely diverse repertoire of molecular structures from pathogens.Because TLRs recognize conserved molecules shared among members of a particular class of microbes (e.g.,LPS from Gram-negative pathogens and ssRNA from RNA viruses),the entire pathogenic414West·Koblansky·GhoshA n n u . 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F o r p e r s o n a l u s e o n l y .universe can be readily sampled by a small group of receptors (Medzhitov &Janeway 1997).However,the mechanism by which this limited group of germline-encoded re-ceptors uniquely recognizes its ligands is still unclear,and it is remarkable that TLRs rec-ognize such varied molecules utilizing a con-served LRR motif.In addition,TLRs 2and 4are unique in their ability to recognize multiple,diverse PAMPs.In light of this di-versity of recognition,we discuss below the identified and otherwise possible mechanisms by which TLRs bind their ligands,namely through diversification of LRR motifs,coop-erative interactions between different TLRs,and the use of non-TLR coreceptors or accessory molecules.Leucine-Rich-Repeat DiversityThe basic LRR motif comprises 24amino acids that form a β-strand and an α-helix joined by a loop,and it is present in several prokaryotic and eukaryotic receptors (Kobe &Kajava 2001).TLRs contain a unique consen-sus LRR,xLxxLxLxx [N/L ]x +xx +xxxxFxxLx ,where x represents any amino acid and +rep-resents any hydrophobic amino acid (Bell et al.2003).This consensus sequence appears re-peatedly throughout the extracellular domain of TLRs,although other variants of this mo-tif are present.In a recent article,Bell et al.(2003)thoroughly characterize each LRR in TLRs 1–10and propose that nonconsensus LRRs containing insertions after residues 10and 15confer ligand specificity.Insertions in LRRs after the tenth amino acid are predicted to provide ligand binding sites at the concave surface of TLRs,whereas insertions at posi-tion 15are predicted to create binding sites at the convex surface.There are several compelling examples in support of this model,including TLR9and TLR5LRR insertions that potentially create binding sites for CpG DNA and flagellin,re-spectively.With regard to TLR9,insertions at position 10of LRRs 2,5,8,and 11occur on the concave face of the receptor and perhapscreate potential CpG binding sites.Notably,the insertion in the eighth LRR contains two cysteine-rich CXXC motifs,which mediate direct binding of CpG DNA by a transcrip-tional activator termed CpG-binding protein (Lee et al.2001).Thus,the location and sequence of these nonconsensus LRRs sug-gest that they function in TLR9-dependent CpG DNA binding,although this hypothe-sis has not been tested experimentally (Bell et al.2003,Lee et al.2001).As mentioned above,Mizel et al.(2003b)recently mapped the TLR5extracellular domain required for flagellin binding (amino acids 386–407),and they also showed that the domain encom-passes an LRR.Interestingly,the predicted flagellin binding site resides within LRR 14of the extracellular domain,and it contains a six-residue insertion after position 15,which provides further experimental support for the insertion specificity model (Bell et al.2003).Finally,mutational analysis of the TLR2extracellular domain illustrated that deletion of the first seven N-terminal LRRs did not drastically affect its ability to transduce sig-nals for NF-κB and mitogen-activated pro-tein kinase (MAPK)activation in 293cells;therefore,its ability to interact with microbial polypeptides and peptidoglycan remains un-affected (Meng et al.2003,Mitsuzawa et al.2001).However,N-terminal mutant TLR2displayed downstream signaling deficiencies in response to many other TLR2ligands,pro-viding evidence that specific LRR domains on TLR2interact with different PAMPs,thereby increasing recognition capacity.Therefore,LRRs on each TLR seem to function to create unique ligand specificities for the respective receptors,which aids in explaining the diverse array of PAMPs recognized by this family of PRRs.Most data regarding LRRs and ligand binding are predicated on some form of muta-tional analysis in which large portions of var-ious TLR ectodomains are removed and the degree of inhibition between PAMP/receptor interaction is observed biochemically.Although these experiments are highly •TLR Recognition and Signaling415A n n u . 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F o r p e r s o n a l u s e o n l y .informative,they do not address the concern that mutating portions of any protein may result in an overall altered conformation.Thus,the altered binding of ligands to mutated TLRs may not be the direct re-sult of removing the binding site per se.Ultimately,cocrystallographic studies are needed to provide concrete proof of specific PAMP/TLR interaction domains,but given the difficulty in purifying large amounts of TLR extracellular domains,this proof has been slow to arrive.Recently,Bell et al.(2005)and Choe et al.(2005)independently solved the crystal struc-ture of human TLR3-ED,providing a ma-jor breakthrough in the structural analysis of TLRs.Both groups concluded that the struc-ture of human TLR3is a horseshoe-shaped solenoid that is heavily glycosylated,and both identified possible dsRNA binding sites.Dis-agreeing with the current model of TLR lig-and binding,Choe et al.(2005)concluded that glycosylation and the overall negative charge of the concave surface would likely prevent dsRNA binding.They proposed that the binding site of dsRNA exists on the con-vex,glycosylation-free face of TLR3,which consequently has many basic residues to ac-commodate the net negative charge of nucleic acids.In contrast,Bell et al.(2005)argued that the binding site for dsRNA may reside in the concave region of the solenoid because sulfate ions that mimic the phosphate backbone of nucleic acids were found attached to the side chains of several amino acids on the concave face.These authors also proposed that loops created from insertions in LRRs 12and 20yield a pocket on the glycosylation-free,con-vex face of TLR3suitable for dsRNA bind-ing.Because these insertions are highly con-served across TLR3from different species,these sites likely participate in ligand binding (Bell et al.2005).Although no cocrystalliza-tion data were provided,the structural anal-yses present substantial insight into the po-tential means by which TLR3binds dsRNA.As the LRR sequence and crystal structure of TLR3indicate multiple ligand binding sites,it is interesting to consider whether this re-ceptor binds ligands in addition to dsRNA.Additional evidence is needed to determine whether this idea is correct.However,other TLRs bind multiple ligands,and the large sur-face area and spatial distribution of putative binding sites on TLR3may explain the di-verse ligand specificities of other TLRs.Cooperative Interactions Between TLRsAs described above,TLR2functions as a heterodimer with TLRs 1and 6for recog-nition of a diverse group of PAMPs.Al-though bacterial lipopeptides are important ligands for these heterodimeric pairs,data from many other groups suggest that TLR2/1or TLR2/6dimers recognize additional lig-ands,such as zymosan,Y ersinia V antigen (Sing et al.2002),and Escherichia coli en-terotoxins (Hajishengallis et al.2005).These ligands are structurally unrelated to bacte-rial lipopeptides,and the diversity of recog-nition is likely provided by the combination of LRR domains on each TLR.Recently,Omueti et al.(2005)showed that LRRs 9–12of human TLRs 1and 6mediate the abil-ity of these receptors to discriminate between acylated lipoproteins,as domain exchange be-tween the two receptors alters lipopeptide re-sponsiveness in transfected cells.Additionally,LRRs 7–10of TLR2were shown to be crit-ical for responses to tri-lauroylated lipopep-tides (Grabiec et al.2004,Meng et al.2003).T aken in combination,these data suggest that TLR2/1and TLR2/6heterodimers bind lipopeptide PAMPs cooperatively at regions predicted to lie on the convex,outer loops of both receptors (Omueti et al.2005).Although these studies clarify heterodimeric TLR re-sponses to lipopeptides,they do not readily explain the requirement of both TLR2and either TLR1or TLR6receptor pairs for the recognition of structurally unrelated PAMPs.Analysis of all three TLRs reveals that several nonconsensus LRRs containing insertions lie outside of the predicted lipopeptide binding416West·Koblansky·GhoshA n n u . R e v . C e l l D e v .B i o l . 2006.22:409-437. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b y H A R V A R D U N I V E R S I T Y o n 12/02/07. F o r p e r s o n a l u s e o n l y .。