Engineered Mutation 'Ticam2tm1Btlr'
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Mutation Name Ticam2tm1Btlr
Common Name Ticam2 KO
Mutation Type Knockout
Strain of Origin C57BL/6J
Gene Symbol Ticam2
Gene Nametoll-like receptor adaptor molecule 2
SynonymsTirp, TRAM
Accession Numbers

NCBI RefSeq: NM_173394; MGI: 3040056

Alleles Listed at MGI

All alleles(2) : Targeted, knock-out(2)

Chromosomal Location46559155-46574533 bp(-)

MMRRC: 030018-UCD

Phenotypic Description

Peritoneal macrophages from homozygous Tram KO mice produce no type I interferon (IFN), and reduced amounts of tumor necrosis factor (TNF)-α in response to lipopolysaccharide (LPS), a Toll-like receptor (TLR) 4 ligand. Tram KO macrophages produce normal levels of type I IFN in response to poly I:C (TLR3 ligand).

Nature of Mutation
Ticam2 (Genbank genomic region NC_000084 for linear DNA sequence of Ticam2), located on Chromosome 18, contains 3 exons. To generate a null allele, exon 3, which encompasses the entire coding region of Ticam2, was replaced with a neomycin resistance selection cassette. No Ticam2 transcript is detected in Tram KO tissue. The mutant allele was placed in embryonic stem cells of C57BL/6J origin.
Protein Prediction
Ticam2 [Toll-interleukin 1 receptor (TIR) domain-containing adaptor molecule-2; also Tram (Trif-related adaptor molecule)] is a 232 amino acid protein adaptor in TLR4 signaling. Ticam2 is most closely related to the TLR adaptor Trif (TIR domain-containing adaptor inducing IFN-β) (1;2). Like the other adaptors for TLR signaling, Ticam2 contains a central Toll/IL-1 receptor (TIR) domain (amino acids 78-171), a conserved region of approximately 200 amino acids which mediates homo- and heterotypic protein interactions during signal transduction (1;3). TIR domains in TLRs, IL receptors and the adapter MyD88 contain 3 conserved boxes (boxes 1, 2 and 3), which are required for signaling (4). However, conserved sequences in boxes 1, 2 and 3 are lacking in Ticam2 (5). Specifically, the (F/Y)D in box 1, RD in box 2 and FW in box 3 are missing in Ticam2 (5). TIR domains contain six α-helices (αA, αB, αC, αC’, αD and αE) and five β-strands (βA, βB, βC, βD and βE) which are connected by seven loops. The crystal structures of the TLR1 and TLR2 TIR domains revealed that they fold into a structure with a central five-stranded parallel β-sheet surrounded by five helices (6). Many of the α-helices and connecting loops in the TIR domains of TLR1 and TLR2 are predicted to participate in binding partner recognition, and their mutation is expected to abrogate specific binding interactions. This is true of a proline to histidine mutation in the BB loop of TLR4, which abolishes MyD88 binding (7) and LPS-induced signaling in mice (8). This proline is not conserved in Ticam2, where instead a cysteine residue exists (3). A proline is found adjacent to this cysteine (3). Ticam2 differs from MyD88 in that it lacks a death domain, which in MyD88 recruits IL-1 receptor associated kinase (IRAK) family proteins (9).
Northern blot analysis detected Ticam2 transcript in most tissues examined, including spleen, prostate, testis, uterus, small intestine, colon, peripheral blood leukocytes, heart, placenta, lung, liver, skeletal muscle, pancreas, lymph node, thyroid and trachea (1;5). RT-PCR analysis revealed Ticam2 transcript in peripheral blood immature dendritic cells (DC), macrophages and natural killer (NK) cells (5). Three transcripts of different sizes were differentially expressed in each tissue (1). Ticam2 is localized in the cytoplasm (3).
TLRs are transmembrane receptors that sense molecules of microbial origin and trigger host cell responses. The twelve mouse TLRs and ten human TLRs recognize a wide range of structurally distinct molecules, and all signal through only four adaptor proteins known to date: MyD88, Tirap (Mal), TICAM-1 (TRIF) and TRAM (10). TLR signaling through these adaptors initiates a cascade of signaling events involving various kinases, adaptors and ubiquitin ligases, ultimately leading to transcriptional activation of cytokine [e.g. TNF-α, interleukin (IL)-1, IL-6] and other genes through the transcription factors NF-κB, AP-1, interferon responsive factor (IRF)-3, and IRF-7. Two branches of signaling are known to exist, one defined by early NF-κB activation (MyD88-dependent pathway, mediated by MyD88), and another distinguished by late NF-κB activation as well as IRF-3 activation leading to type I IFN production and costimulatory molecule upregulation (MyD88-independent pathway, mediated by Trif) (11-13). The TLRs may utilize one or both of these pathways to elicit a ligand-specific response. TLRs -3 and -4 are known to activate both NF-κB and IRF-3, and do so using the MyD88-independent pathway (for TLR3) or both pathways (for TLR4) (2;14).
Ticam2 was first identified by sequence homology search for TIR domain-containing adaptors (1). In this study, overexpression of Ticam2 (called TIRP) in HEK 293 cells was found to activate a reporter for NF-κB, but not for an IFN-β promoter (1). Ticam2 could also potentiate IL-1R- or IL-1RAcP-dependent NF-κB activation (1). No interaction was detected between Ticam2 and either TLR4 or TLR2, and the authors suggested that Ticam2 functions in IL-1R-mediated NF-κB activation pathways (1).
Ticam2 was also identified as a Chromosome 18 Trif homologue (by tBLASTn search using Trif as a query) in a study of the Lps2 mutant strain, which harbors a frameshift mutation in Trif that replaces the 24 C-terminal residues with 11 unrelated amino acids. In this report, a population of homozygous Lps2 macrophages, expected to have little to no response to TLR4 ligands, was observed to produce low levels of TNF-α in response to LPS (2). This response was attributed to an unknown “adaptor X” and hypothesized to be the Trif homologue identified on mouse Chromosome 18 (2).
Two subsequent reports also identified Ticam2 by sequence homology searches for TIR domain-containing proteins, and demonstrated that in fact, Ticam2 activates NF-κB, IRF-3 and IRF-7 downstream of TLR4 (3;5). Overexpression of Ticam2 in HEK 293 cells resulted in expression of the IFN-inducible genes IFN-β, RANTES and IP-10, and activation of an NF-κB reporter, suggesting that Ticam2 may function in the TLR4 and/or TLR3 pathways (Figure 1) (3;5). A dominant negative Ticam2 construct (Ticam2 C113H) or siRNA silencing of Ticam2 expression, inhibited TLR4-, but not TLR3-dependent, RANTES promoter activation and NF-κB activation in HEK 293 or U373-CD14 cells (3;5). These data supported the conclusion that Ticam2 is specifically required for LPS-induced TLR4 signaling.
Putative Mechanism
Generation of Ticam2-/- mice and analysis of TLR signaling in their cells confirmed that Ticam2 signals specifically in the MyD88-independent TLR4 pathway, filling the role of “adaptor X” (2;15). IL-1β-induced NF-κB and JNK activation are normal in Ticam2-/- cells, indicating that Ticam2 is not required for IL-1R-mediated signaling (15). Ticam2-/- macrophages fail to produce TNF-α and IL-6 in response to LPS, but not crude peptidoglycan preparations (which activate the TLR2/6 complex), R-848 (TLR7 ligand), or unmethylated CpG oligodeoxynucleotides (CpG ODN, TLR9 ligand) (15). Upregulation of costimulatory molecules CD69, CD86 and major histocompatibility complex (MHC) class II molecules is also impaired in Ticam2-/- B220-positive splenocytes in response to LPS, but not α-IgM (15). While LPS-stimulated early NF-κB activation is normal in Ticam2-/- cells, late phase NF-κB activation appears diminished, as does JNK activation (15). In addition, expression of IFN-inducible genes is abrogated in response to LPS. In contrast, both NF-κB activation and IFN-inducible gene expression are completely normal in response to poly I:C (TLR3 ligand) in Ticam2-/- cells (15). Thus, Ticam2 specifically mediates the MyD88-independent pathway of TLR4 signaling.
In addition to LPS, the TLR4-Ticam signaling pathway is specifically activated by at least one other stimulus, the vesicular stomatitis virus (VSV) glycoprotein G (gpG), a surface protein on the virus envelope (16). TLR4 engagement by VSV gpG requires the coreceptor CD14, and signals predominantly through Ticam2 (16). Neither MyD88 nor Tirap are required for signaling, although there is a partial requirement for Trif (16). Downstream from Ticam2, VSV gpG leads to a type I IFN response via IRF-7, but does not activate NF-κB. The data suggest that Ticam2 may in some instances function alone, rather than in combination with Trif (see below). Interestingly, this pathway functions in myeloid dendritic cells and macrophages rather than plasmacytoid dendritic cells (16).
A new study has recently implicated Ticam2 in TLR2/6 signaling (17). This report demonstrated that IL-6 production by Ticam2-/- mouse embryo fibroblasts is significantly reduced in response to MALP-2 and lipoteichoic acid (TLR2/6 ligands) (17). In addition, dominant negative Ticam2 inhibited lipoteichoic acid-induced IL-6 production by synovial fibroblasts and human umbilical endothelial cells (17). The reason for discrepancy with previous data on Ticam2-null cells is unknown, but may be due to testing of different cell types which express different species or levels of receptor cofactors (17).
Ticam2 has been shown to bind to TLR4 when coexpressed in cells (3;5) and evidence suggests that Ticam2 cooperates with Trif, serving as a bridging molecule linking TLR4 to Trif (5). Ticam2 and Trif interact in a yeast two-hybrid system, and studies using dominant negative proteins transfected with reporters into HEK 293 cells, demonstrate that dominant negative Trif can block Ticam2-mediated NF-κB and IFN-β promoter activation, but not vice versa (3;5). These results suggest that Ticam2 facilitates Trif function such that Trif cannot signal optimally without Ticam2 (3;5).
Initial evidence suggested that the MyD88-independent pathway mediates NF-κB activation by converging with the MyD88-dependent pathway at TRAF6 (TNF receptor-associated factor-6), a signaling protein distal to MyD88 and required for NF-κB activation. Both Trif (18;19) and Ticam2 (1) bind to TRAF6 when coexpressed in heterologous cells. However, using TRAF6-/- cells, one study demonstrated that TRAF6 is not required for MyD88-independent signaling (late NF-κB activation and IFN-inducible gene expression), and the convergence of the MyD88-dependent and –independent pathways leading to NF-κB activation must lie downstream of TRAF6 (20).
The noncanonical IκB kinases (IKK), IKK-ε and TBK1 [Traf-family-member-associated NF-κB activator (TANK)-binding kinase 1], phosphorylate and activate IRF3 in response to viral infection or activation of TLR3 or Trif signaling pathways (21-23). Notably, Ticam2-dependent IFN-β promoter activation of a reporter could be inhibited by co-expression of dominant negative mutants of IKKε or TBK1, suggesting that these kinases may also function downstream of Ticam2 (3).

PCR is used to detect the presence of exon 3 and/or the neomycin resistance cassette in Ticam2.


Run separate reactions for primers 1 and 2, and primers 3 and 4.



 PCR Reaction Mix

Volume (uL)
DNA Sample
10x Buffer (contains 15mM MgCl2)
dNTPs (25mM stock)
Primer 1 (20 uM stock)
Primer 2 (20 uM stock)
Primer 3 (20 uM stock)
Primer 4 (20 uM stock)
Taq Polymerase (JumpStart RED AccuTaq LA DNA Polymerase)
25 ul


PCR Program

Temp (°C)
Time (min:sec)
94   *HOT START*
60 (annealing)
Repeat steps (2-4) 34X


Gel electrophoresis
Run samples approximately 20 minutes on 1.2% agarose gel at 100 volts.


Primer combination
1 and 2
495 bp
3 and 4
700 bp





Synonyms: Ticam2tm1Btlr, Tram-

AuthorMichael Berger, Bruce Beutler, Xin Du, Philippe Georgel
Science WriterEva Marie Y. Moresco