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TIRP: a novel TIR domain-containing adaptor proteininvolved in Toll/interleulin-1 receptor signaling

Liang-Hua Bin*, Liang-Guo Xu*, and Hong-Bing Shu*#¶

*Department of ImmunologyNational Jewish Medical and Research CenterUniversity of Colorado Health Sciences Center

1400 Jackson StreetDenver, CO 80206

#Department of Cell Biology and GeneticsCollege of Life Sciences

Peking UniversityBeijing, China

¶Address correspondence to:

Hong-Bing ShuNational Jewish Medical and Research Center

1400 Jackson Street, k516cDenver, CO 80206

Tel: 303-398-1329Fax: 303-398-1396

Email: shuh@njc.org

This work was supported by grants from the National Institutes of Health (AI49992), theEllison Medical Foundation, the National Natural Science Foundation of China(#39925016 and 30100097), the Chinese High-Technology Program (#2001AA221281),and the Special Funds for Major State Basic Research of China (G19990539) to H.-B.Shu.

Running Title: A TIR domain-containing adapter.

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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Abstract

The Toll/interleukin-1 receptor (TIR) family members play important roles in host

defense. These receptors signal through TIR domain-containing adapter proteins. In this

report, we identified a novel TIR domain-containing adapter protein designated as TIRP.

C0-immunoprecipitation experiments suggest that TIRP is associated with IL-1 receptors.

TIRP also interacts with kinase inactive mutants of IRAK and IRAK-4, IRAK-2, IRAK-

M, and TRAF6. Overexpression of TIRP activates NF-κB and potentiates IL-1 receptor-

mediated NF-κB activation. A dominant negative mutant of TIRP inhibits IL-1-, but not

TNF-triggered NF-κB activation. Moreover, TIRP-mediated NF-κB activation is

inhibited by dominant negative mutants of IRAK, IRAK-2, TRAF6 and IKKβ. Our

findings suggest that TIRP is involved in IL-1-triggered NF-κB activation and functions

upstream of IRAK, IRAK-2, TRAF6 and IKKβ.

Key Words: TIRP, TIR, IRAK, interleukin-1, NF-κB

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Introduction

The Toll/interleukin-1 receptor (TIR)1 family members are evolutionary

conserved proteins that are critically involved in host defense from plants to humans (1-

5). The TIR family is divided into two subfamilies, the IL-1 receptor (IL-1R) and Toll-

like receptor (TLR) subfamilies. In mammals, the IL-1R subfamily members are

important mediators of inflammation and adaptive immune responses, while the TLR

subfamily members recognize microbial products termed PAMPs (pathogen-associated

molecular patterns) and are critical components of the innate immune system (1-5).

The IL-1R and TLR subfamilies are distinguished through structural divergence

in the extracellular domains. The IL-1R subfamily members contain immunoglobulin-

like motifs, while the TLR subfamily members contain leucine-rich repeats. However,

both subfamily members share a conserved cytoplasmic domain, the TIR domain (1-5).

Various studies have shown that the TIR domain is required for TIR family member-

mediated signaling that leads to activation of transcription factors NF-κB, AP-1 and

ATF-2, and subsequent induction of various chemokines and cytokines that are involved

in host defense against the pathogens (1-5).

The signaling pathways initiated by IL-1 receptors have become a paradigm on

how other TIR family receptors signal. Upon IL-1 stimulation, IL-1R1 forms a complex

with IL-1RAcP, a co-receptor (6-9). This leads to recruitment of the adapter protein

MyD88 to the receptor signaling complex (10-12). MyD88, in turn, recruits the

serine/threonine kinase IRAK (11). Once IRAK is recruited to the receptor complex, it is

activated and then dissociated from the receptor complex (13). Dissociated IRAK

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interacts with TRAF6, which then activates NF-κB through a kinase cascade containing

TAK1 and IKK (13-16).

In addition to IL-1 receptor signaling, MyD88 is also involved in signaling by

TLRs (1-5). Gene knock-out studies have suggested that MyD88 is required for cytokine

induction triggered by a variety of ligands that signal through the TIR family members,

such as IL-1β, IL-18, LPS, MALP-2, CpG-DNA and poly (I:C) (17). However, it has

been shown that Poly(I:C) and LPS-induced NF-κB and JNK activation was delayed, but

not abolished in MyD88-/- cells. In addition, Poly(I:C) and LPS still triggered dentricic

cell maturation (17). These studies suggest that MyD88-independent pathways exist.

Recently, two additional TIR domain-containing adapter molecules, TIRAP/Mal and

TRIF/TICAM, were identified (18-21). TIRAP is associated with TLR4. A dominant

negative mutant of TIRAP inhibits NF-κB activation triggered by TLR4, but not by

TLR9 or IL-1R (18, 19). Furthermore, gene knock-out studies have indicated that TIRAP

is essential for LPS-induced cytokine production, suggesting TIRAP is required for TLR4

signaling (22, 23). It has also been shown that TIRAP is involved in signaling triggered

by ligands for TLR1, TRL2, and TLR6, but not by IL-1, IL-18, and ligands for TLR3,

TLR7 and TLR9 (22, 23).

TRIF is the third identified TIR domain-containing adaptor molecule. In addition

to NF-κB, TRIF also activates IFN-β promoter (20, 21). A dominant negative mutant of

TRIF inhibits TLR2, TLR3, TLR4, and TLR7-mediated NF-κB activation, and TLR3-

mediated activation of IFN-β promoter (20, 21). So far, TRIF is the only TIR-containing

adapter protein that is implied in TLR3-mediated production of IFN-β, suggesting that

TRIF is involved in TLR3 activates NF-κB as well as IFN-β (20, 21).

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Similar with the TIR domain-containing adapter proteins, four IRAK-like

molecules have been identified (24). Although IRAK was firstly identified as a

component of the IL-1R signaling complex and later shown to be recruited to the TLR

signaling complexes upon ligand stimulation, gene knock-out studies have indicated that

IRAK deficiency has a relatively mild effect on IL-1R and TLR signaling (25, 26). It

has been shown that the kinase activity of IRAK is dispensable for IRAK’s function as a

mediator of TIR-triggered NF-κB activation (27). IRAK-2 and IRAK-M, two other

members of the IRAK family, have no kinase activity but activate NF-κB when

overexpressed in 293 cells and partially restore IL-1 signaling in IRAK-/- cells (10, 28,

29).

IRAK-4, the fourth member of the IRAK family, has unique features that

distinguish it from all other IRAK proteins. Firstly, overexpression of IRAK-4 does not

result in robust NF-κB activation. Second, expression of a kinase-inactive mutant of

IRAK-4 is sufficient to inhibit IL-1-mediated NF-κB activation in cells (30). By

contrast, corresponding point mutations of IRAK, IRAK-2, or IRAK-M do not have the

same dominant negative effects (10, 28, 31). Third, IRAK is a direct substrate of IRAK-

4, but IRAK-4 can not be phosphorylated by IRAK (30). Finally, gene knock-out studies

indicate that IRAK-4 is required for signaling triggered by IL-1R and TLR engagement

(32). These results suggest that IRAK-4, as well as its kinase activity, is required for TIR

signaling and that IRAK-4 functions upstream of IRAK.

Recently, it has been shown that IRAK-M prevents dissociation of IRAK and

IRAK-4 from MyD88 and formation of IRAK-TRAF6 complexes (28). IRAK-M-/- cells

exhibited increased cytokine production upon TLR/IL-1 stimulation and bacterial

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challenge. IRAK-M-/- mice showed increased inflammatory responses to bacterial

infection and decreased tolerance to endotoxin shock (28). These data suggest that

IRAK-M is a negative regulator of signaling by the TIR family members.

In the present study, we identified a novel TIR domain-containing adapter protein,

designated as TIRP (for TIR-containing protein). Our findings suggest that TIRP

interacts with various proteins involved in the TIR signaling pathways and plays a role in

NF-κB activation triggered by the IL-1 receptors.

Experimental Procedures

Reagents--Recombinant TNFα and IL1 (R&D Systems, Minneapoplis, MN), monoclonal

antibodies against FLAG and the HA (Sigma, St. Luis, MO) epitopes were purchased

from the indicated resources.

Northern blot hybridization--Human multiple tissue mRNA blots were purchased from

Clontech (Palo Alto, CA). The blots were hybridized with p32-dCTP- and p32-dATP-

labeled cDNA probe corresponding to TIRP coding sequence. The hybridization was

performed in the Rapid Hybridization Buffer (Clontech, Palo Alto, CA) under high-

stringency condition.

Constructs--NF-κB luciferase reporter construct (Dr. Gary Johnson, University of

Colorado Health Sciences Center), mammalian expression plasmids for FLAG-TRAF6,

FLAG-TRAF6(289-522), IKKβ, IκBα(SS/AA) (Dr. David Goeddel, Tularik Inc.),

FLAG-IRAK, FLAG-IRAK (K239S), FLAG-IRAK-4, FLAG-IRAK-4-KA, FLAG-

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IRAK-4(1-191), FLAG-IRAK-M, FLAG-IL1R1, FLAG-IL1RAcP, Myc-MyD88 (Dr.

Zhaodan Cao, Tularik Inc.), pCMV1-FLAG-TLR2 and pCMV1-FLAG-TLR4 (Dr. Bruce

Beutler, Scripps Research Institute) were provided by the indicated investigators.

Mammalian expression plasmids for HA-TIRP and its deletion mutants, FLAG-tagged

IRAK-2, IRAK(1-215), IRAK-2(97-590), TRIF, TIRAP were constructed by PCR

amplification of the corresponding cDNA fragments and subsequently cloning into a

CMV promoter-based vector containing a 5’-HA or FLAG tag. IFN-β luciferase reporter

was constructed by PCR amplification of the human IFN-β promoter fragment (-300 to +

25) and cloning into pGL3-Basic vector (Promega, Madison, WI).

Cell transfection and reporter gene assays--293 cells (2x105) were seeded in 6-well

dishes and transfected the following day by the standard calcium phosphate precipitation

(33). Within the same experiment, each transfection was performed in triplicate, and

where necessary, empty control plasmid was added to ensure that each transfection

receives the same amount of total DNA. To normalize for transfection efficiency, 0.2 µg

of RSV-β-gal luciferase reporter plasmid was added to each transfection.

Approximately sixteen hours after transfection, luciferase reporter assays were

performed using a luciferase assay kit (BD PharMingen, San Diego, CA) by following

the manufacturer’s protocol. β-galactosidase activity was measured using the Galacto-

Light chemiluminescent kit (TROPIX, Bedford, MA). Luciferase activities were

normalized on the basis of β-gal luciferase expression levels.

All reporter gene assays were repeated for at least three times. Data shown were

from one representative experiment.

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Co-immunoprecipitation and Western blot analysis--For transient transfection and co-

immunoprecipitation experiments, 293 cells (2x106) were transfected for 24 hours.

Transfected cells were lysed in 1 ml of lysis buffer (15 mM Tris, 120 mM NaCl, 1%

Triton, 25 mM KCl, 2 mM EGTA, 2 mM EDTA, 0.1 mM DTT, 0.5% Triton X-100, 10

µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, pH 7.5). For each

immunoprecipitation, a 0.4 ml aliquot of lysate was incubated with 0.5 µg of the

indicated monoclonal antibody or control mouse IgG and 25 µl of a 1:1 slurry of

GammaBind G Plus-Sepharose (Amersham Pharmacia, Piscataway, NJ) for 2 hours. The

sepharose beads were washed three times with 1 ml of lysis buffer with 500 mM NaCl.

The precipitates were fractionated on SDS-PAGE and subsequent Western blot analysis

was performed as described.

All immunoprecipitation experiments were repeated for at least three times andsimilar data were obtained.

Results

Identification and cloning of TIRP

It has been suggested that several TIR domain-containing adapters are involved in

the TIR signaling pathways and differential use of these adapters provides the specificity

in the TIR signaling pathways (1-5). We reasoned that additional TIR domain-containing

adapters might exist. Therefore, we searched the GeneBank EST databases for sequences

that encode TIR domain-containing proteins. This effort identified multiple human EST

clones that encode a protein designated as TIRP. GeneBank accession number for the

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longest EST clone is BQ438847. Further BLAST searches also identified mouse

ortholog of human TIRP. Sequence comparisons suggest that human and mouse TIRPs

share approximately 70% identity at amino acid level (Fig. 1A). TIRP contains a TIR

domain at the middle, which is mostly conserved with that of TRIF/TICAM (18-21) (Fig.

1B).

Northern blot analysis suggests that human TIRP mRNA is expressed in most

examined tissues, including the spleen, prostate, testis, uterus, small intestine, colon,

peripheral blood leukocytes, heart, placenta, lung, liver, skeletal muscle, and pancreas

(Fig. 1C). Three transcripts of different sizes, approximately 3.8, 3.6 and 2.0 kb

respectively, were detected and these transcripts were differentially expressed in the

tissues (Fig. 1C).

TIRP interacts with IL-1R1 and IL-1RAcP

Previously, it has been shown that the TIR domain-containing adapter proteins

interact with TIR receptors (1-5). We determined whether TIRP interacts with IL-1R1,

IL-1RAcP, and TLR2. To do this, we transfected expression plasmids for C-terminal

FLAG-tagged receptors and N-terminal HA-tagged TIRP into 293 cells. Co-

immunoprecipitation experiments indicated that TIRP interacted with IL-1R1 and IL-

1RAcP, but not with TLR2 and TLR4 (Fig. 2A). These data suggest that TIRP is a

component of the IL-1 receptor signaling complex.

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TIRP interacts with other TIR-containing adapter proteins

It has been reported that TIR-containing adapter proteins can form heterodimers

(1-5). To test whether TIRP can interact with known TIR-containing adapter proteins,

we transfected expression plasmids for TIRP and MyD88, TIRAP, and TRIF into 293

cells respectively. Co-immunoprecipitation experiments suggest that TIRP interacts with

TIRAP and TRIF (Fig. 2B), but not with MyD88 (data not shown). These data suggest

that TIRP selectively interacts with other TIR-containing adapter proteins.

TIRP interacts with IRAKs

MyD88, TIRAP and TRIF function as adapters to recruit IRAKs into the

signaling complexes of TIR family members (10-13, 20-23). We examined whether

TIRP also interacts with IRAKs. In transient transfection and co-immunoprecipitation

experiments, TIRP did not interact with wild-type IRAK and IRAK-4, but interacted with

kinase inactive mutants of IRAK and IRAK-4 (Fig. 3A). There results are similar to

MyD88, which has been shown to interact with kinase inactive mutant but not wild type

IRAK.

In transient transfection and co-immunoprecipitation experiments, TIRP also

interacted with wild-type IRAK-2 and IRAK-M (Fig. 3A), two IRAK family members

that have no kinase activity. Taken together, these data suggest that TIRP only interacts

with kinase inactive IRAKs.

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TIRP interacts with TRAF6

It has been shown that TRAF6 is associated with activated IRAK (13). We

examined whether TIRP is associated with TRAF6. To do this, we transfected FLAG-

tagged TRAF6 and HA-tagged TIRP into 293 cells and performed co-

immunoprecipitation experiments. These experiments indicated that TIRP interacted

with TRAF6, but not TRAF5 (Fig. 3B). Also, the TRAF domain of TRAF6 was

sufficient for its interaction with TIRP (Fig. 3B). These results suggest that TIRP

specifically interacts with TRAF6.

We next determined which domain of TIRP is responsible for its interaction with

TRAF6. TIRP contains a conserved TIR domain at the middle flanked by unconserved

N- and C-terminal domains. We made HA-tagged TIRP deletion mutants containing one

individual domain or combinations of different domains. Transient transfection and co-

immunoprecipitation experiments suggest that the TIR domain is required and sufficient

for TIRP’s interaction with TRAF6 (Fig. 3C). Interestingly, we found that the TIR

domain of TIRP contains a PRERT motif at aa181, which is conserved with the defined

TRAF6 binding motif PxExx (34).

Overexpression of TIRP activates NF-κκκκB but not IFN-ββββ promoter

Since TIRP interacts with proteins involved in signaling by TIR family members,

we determined whether TIRP activates NF-κB. We transfected a mammalian expression

plasmid for TIRP into 293 cells and performed NF-κB luciferase reporter gene assays.

These experiments indicated that TIRP could activate NF-κB (Fig. 4A). However, TIRP

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activated NF-κB to a lesser degree than MyD88, TRIF and TIRAP in the same

experiments (Fig.4A).

Previously, it has been shown that TRIF can potently activate IFN-β promoter.

To examine whether TIRP can activate IFN-β promoter, we transfected 293 cells (2x105)

with various amounts of TIRP plasmid ranging from 0.01 to 3.2 µg, and then performed

IFN-β luciferase reporter gene assays. The result indicated that TIRP could not activate

IFN-β promoter regardless of the transfected TIRP plasmid concentration (data not

shown and Fig. 4B). In these experiments, MyD88 and TIRAP also did not activate IFN-

β promoter, while TRIF potently activated IFN-β promoter (Fig. 4B).

Since TIRP can weakly activate NF-κB, we determined whether it potentiates IL-

1R1 and IL1AcP-mediated NF-κB activation. Previously, it has been shown that

overexpression of IL-1R1 or IL1RAcP alone weakly activates NF-κB. In reporter gene

assays, we found that TIRP could synergy with IL-1R1 or IL1-RAcP to activate NF-κB

(Fig. 4C).

A TIRP dominant negative mutant inhibits NF-κκκκB activation mediated by IL1

receptors but not by TRAF6 and IKKββββ

Previously, it has been shown that the TIR domains of MyD88, TRIF, and TIRAP

function as dominant negative mutants to inhibit signaling by the TIR family members

(10-13, 20-23). We examined whether the TIR domain of TIRP can inhibit NF-κB

activation triggered by IL-1 receptors. In reporter gene assays, we found that the TIR

domain of TIRP, TIRP(78-171), significantly inhibited NF-κB activation mediated by

overexpression of IL1R1 and IL1RacP, but not by overexpression of TRAF6 and IKKβ

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(Fig. 5A). In these experiments, TIRP(78-171) did not inhibit TLR4 and only slightly

inhibited TLR2-mediated NF-κB activation (Fig. 5A).

In reporter gene assays, TIRP(78-171) also inhibited NF-κB activation triggered

by IL1, but not by TNF (Fig. 5B). These data suggest that TIRP is specifically involved

in IL-1-triggered NF-κB activation.

NF-κκκκB activation by TIRP is inhibited by dominant negative mutants of IRAK,

IRAK-2, TRAF6 and IKKββββ

To determine the molecular order of TIRP in the IL-1 signaling pathways, we

determined whether TIRP-mediated NF-κB activation is inhibited by dominant negative

mutants of proteins involved in IL-1 signaling pathways. In reporter gene assays, we

found that dominant negative mutants of IRAK, IRAK-2, TRAF6, IKKβ, as well as IκBα

undegradable mutant, potently inhibited TIRP-induced NF-κB activation (Fig. 5C). In

these experiments, dominant negative mutants of MyD88 and IRAK-4 did not inhibit

TIRP-mediated NF-κB activation (Fig. 5C). These data suggest that TIRP functions

upstream of IRAK, IRAK-2, TRAF6 and IKKβ in IL-1 receptor-mediated NF-κB

activation pathways.

Discussion

Previously, three TIR-containing adapter proteins have been reported, including

MyD88, TIRAP/Mal, and TRIF/TICAM. In this paper, we describe the identification of

TIRP, the fourth member of this family.

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TIRP shares many similar properties with the other three TIR-containing adapter

proteins. It contains a conserved TIR domain and is associated with IL-1RI and IL-

1RAcP, suggesting that TIRP plays a role in IL-1 signaling.

Previous studies have shown that MyD interacts with kinase-inactive mutant of

IRAK, but not wild-type IRAK. It has been proposed that MyD88 recruits inactive IRAK

to IL-1R complex. Once IRAK is activated in the complex, it dissociates with MyD88

and then interacts with TRAF6 (10-12). Similar to MyD88, TIRP interacts with kinase

inactive mutants of IRAK and IRAK-4, but not with their wild-type counterparts. These

data suggest that TIRP may also interact with inactive IRAK and IRAK-4 in cells and

dissociate when they are activated.

In addition to kinase-inactive IRAK and IRAK-4 mutants, TIRP also interacts

with IRAK-2 and IRAK-M, two IRAK family members that are kinase inactive. These

data further support our hypothesis that TIRP interacts with kinase-inactive IRAKs.

In addition to IRAKs, TIRP also interacts with TRAF6. This interaction is

mediated by the TRAF domain of TRAF6. TIRP does not interact wit TRAF5, a TRAF

family member that is involved in signaling by TNF receptor family members. The TIR

domain of TIRP is required and sufficient for its interaction with TRAF6. These data

suggest that TIRP specifically interacts with TRAF6 and other signaling proteins

involved in IL-1 signaling pathways.

Overexpression of TIRP activates NF-κB but not IFN-β promoter. A dominant

negative mutant of TIRP (TIRP78-171) inhibits IL-1-, but not TNF-induced NF-κB

activation. Conversely, dominant negative mutants of IRAK, IRAK-2, TRAF6 and

IKK β inhibited TIRP-mediated NF-κB activation. These data suggest that TIRP

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functions upstream of IRAK, IRAK-2, TRAF6 and IKKβ in the IL-1-induced NF-κB

activation pathway. Since IL-1 is unable to activate NF-κB in certain MyD88 deficient

cells, it is possible that these cells do not express TIRP. Alternatively, TIRP is not able to

complement MyD88 deficiency and both proteins are required for IL-1-induced NF-κB

activation.

Although TIRP interacts with kinase inactive mutant of IRAK-4, a dominant

negative mutant of IRAK-4 did not inhibit TIRP-induced NF-κB activation. It is possible

that IRAK-4 functions upstream of TIRP in IL-1 signaling.

In conclusion, we identified TIRP, a novel TIR domain containing adapter protein

that is involved in IL-1 triggered NF-κB activation pathways. It should be pointed out

that our characterization of TIRP was based on the mammalian overexpression systems.

Further experiments, such as analysis of TIRP knock-out mice, will be needed to confirm

a role of TIRP in IL-1 or other TIR signaling pathways.

Acknowledgement

We thank Drs. David Goeddel, Zhaodan Cao, Gary Johnson and Bruce Beutler for

reagents.

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1The abbreviations used are:

TIR, Toll/interleukin-1 receptor; TIRP, TIR domain-containing protein; TLR,

Toll like receptor; IRAK, interleukin-1 receptor associated kinase; TRAF, TNF receptor

associated factor; IKK, IκB kinase; NF-κB, nuclear factor kappa B.

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Figure Legends

Fig. 1. Identification and tissue expression of TIRP. A. Alignment of amino acid

sequences of human and mouse TIRPs. The putative TIR domain is underlined. hTIRP,

human TIRP; mTIRP, mouse TIRP. The GeneBank accession numbers for human and

mouse TIRP are AY275836 and AY275837 respectively. B. Alignment of the TIR

domain of human TIRP with that of human TRIF and the smart0025 TIR domain from

the National Center for Biotechnology Information. C. Tissue distribution of human

TIRP mRNA. Human multiple tissue blots were hybridized with a cDNA probe

corresponding to human TIRP coding sequence.

Fig. 2. TIRP interacts with IL-1R1, IL-1RAcP, TRIF and TIRAP. A. TIRP is associated

with IL-1R1 and IL-1RAcP but not TLR2 and TLR4. B. TIRP interacts with TRIF and

TIRAP.

293 cells were transfected with expression plasmids for HA-tagged TIRP and the

indicated FLAG-tagged proteins. Cell lysates were immunoprecipitated with anti-FLAG

antibody or control IgG. The immunoprecipitates were analyzed by Western blots with

anti-HA antibody. The expression levels of all the proteins were comparable as

suggested by Western blot analysis of the lysates (data not shown).

Fig. 3. TIRP interacts with kinase-inactive IRAKs and TRAF6. A. TIRP interacts with

kinase-inactive IRAK and IRAK-4, IRAK-2 and IRAK-M. 293 cells (2x106) were

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transfected with expression plasmids for HA-tagged TIRP and FLAG-tagged IRAK,

IRAK, IRAK-KA, IRAK-4, IRAK-4-KA, IRAK-2 and IRAK-M. Cell lysates were

immunoprecipitated with anti-FLAG (α-F) or control IgG. The immunoprecipitates were

analyzed by Western blots with anti-HA antibody. B. TIRP interacts with TRAF6 and its

TRAF domain, but not with TRAF5. 293 cells (2x106) were transfected with expression

plasmids for HA-tagged TIRP and FLAG-tagged TRAF5, TRAF6 and TRAF6(289-595).

Cell lysates were immunoprecipitated with anti-FLAG (α-F) or control IgG. The

immunoprecipitates were analyzed by Western blots with anti-HA antibody. C. TRAF6

interacts with the TIR domain of TIRP. 293 cells (2x106) were transfected with

expression plasmids for FLAG-TRAF6 and various HA-tagged TIRP mutants. Cell

lysates were immunoprecipitated with anti-HA or control IgG. The immunoprecipitates

were analyzed by Western blots with anti-FLAG antibody.

The expression levels of all the proteins were comparable as suggested by

Western blot analysis of the lysates (data not shown).

Fig. 4. TIRP activates NF-κB and potentiates IL-1 receptor-mediated NF-κB activation.

A. Activation of NF-κB by TIRP and other TIR-containing adapter proteins. 293 cells

(2x105) were transfected with 0.3 µg of NF-κB-luciferase reporter plasmid, 0.2 µg of

RSV-β-gal plasmid, and 1 µg of an expression plasmid for TIRP, MyD88, TRIF or

TIRAP. Sixteen hours after transfection, luciferase and β-gal reporter assays were

performed. Data shown are average luciferase activities normalized by β-gal activities.

B. TIRP does not activate IFN-β promoter. The experiments were done as in A except

the NF-κB-luciferase reporter plasmid was replaced with IFN-β promoter luciferase

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reporter plasmid. C. TIRP potentiates IL-1 receptor-mediated NF-κB activation. 293

cells (2x105) were transfected with 0.3 µg of NF-κB-luciferase reporter plasmid, 0.2 µg

of RSV-β-gal, 1 µg of an expression plasmid for TIRP, and 1 µg of an expression

plasmid for IL-1R1 or IL-1RAcP. Reporter gene assays were performed as in A.

Fig. 5. Functional roles of TIRP in IL-1-induced NF-κB activation pathways. A. Effects

of TIRP dominant negative mutant on NF-κB activation induced by overexpression of

IL-1 receptors, TRAF6, IKKβ, TLR2 and TLR4. 293 cells (2x105) were transfected with

0.3 µg of NF-κB-luciferase reporter plasmid, 0.2 µg of RSV-β-gal plasmid, and 1 µg of

the indicated expression plasmids in the absence (open bars) or presence (solid bars) of 1

µg of expression plasmid for TIRP (78-171). Sixteen hours after transfection, luciferase

and β-gal reporter assays were performed. Data shown are average luciferase activities

normalized by β-gal activities. B. Effects of TIRP dominant negative mutant on NF-κB

activation induced by IL-1 and TNF. 293 cells (2x105) were transfected with 0.3 µg of

NF-κB-luciferase reporter plasmid, 0.2 µg of RSV-β-gal plasmid, 1 µg of expression

plasmid for TIRP (78-171) (solid bars) or empty control plasmid (open bars). Fourteen

hours after transfection, the cells were treated with IL-1 (20 ng/ml), TNF (20 ng/ml) or

left untreated for six hours. Reporter gene assays were performed as in A. C. Effects of

various mutants on TIRP-mediated NF-κB activation. 293 cells (2x105) were transfected

with 0.3 µg of NF-κB-luciferase reporter plasmid, 0.2 µg of RSV-β-gal plasmid,1 µg of

an expression plasmid for TIRP, and 1 µg of the indicated plasmids. Sixteen hours after

transfection, reporter gene assays were performed as in A.

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hTIRPmTIRP

1 40 1 38

M G I G K S K I N S C P L S L S W G K R H S V D T S P G Y H E S D S K K S E D L

M G V G K S K L D K C P - - L S W H K K D S V D A D Q D G H E S D S K N S E E A

hTIRPmTIRP

41 80 39 77

S L C N V A E H S N T T E G P T G K Q E G A Q S V E E M F E E E A E E E V F L K

C L R G F V E Q S S G S E P P T G E Q D - Q P E A K G A G P E E Q D E E E F L K

hTIRPmTIRP

81 120 78 117

F V I L H A E D D T D E A L R V Q N L L Q D D F G I K P G I I F A E M P C G R Q

F V I L H A E D D T D E A L R V Q D L L Q N D F G I R P G I V F A E M P C G R L

hTIRPmTIRP

121 160 118 157

H L Q N L D D A V N G S A W T I L L L T E N F L R D T W C N F Q F Y T S L M N S

H L Q N L D D A V N G S A W T I L L L T E N F L R D T W C N F Q F Y T S L M N S

hTIRPmTIRP

161 200 158 197

V N R Q H K Y N S V I P M R P L N N P L P R E R T P F A L Q T I N A L E E E S R

V S R Q H K Y N S V I P M R P L N S P L P R E R T P L A L Q T I N A L E E E S Q

hTIRPmTIRP

201 235 198 232

G F P T Q V E R I F Q E S V Y K T Q Q T I W K E T R N M V Q R Q F I A

G F S T Q V E R I F R E S V F E R Q Q S I W K E T R S V S Q K Q F I A

sple

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hTIRPhTRIFsmart00255

hTIRPhTRIFsmart00255

hTIRPhTRIFsmart00255

hTIRPhTRIFsmart00255

F L K F V I L H A E D D T D E A L R V Q N L L Q D D F G I K P G I IF Y N F V I L H A R A D E H I A L R V R E K L E A - L G V P D G A TE Y D V F I S Y S G D E D V R N E F L S H L L E Q L R G Y K L C V F

F A E M P - - C G R Q H L Q N L D D A V N G S A W T I L L L T E N FF C E D F Q V P G R G E L S C L Q D A I D H S A F I I L L L T S N FI D D F E - - P G G G D L E N I D E A I E K S R I A I V V L S P N Y

L R D T W C N F Q F Y T S L M N S V N R Q H - - - K Y N S V I P M RD C R - L S L H Q V N Q A M M S N L T R Q G - - - S P D C V I P F LA E S E W C L D E L V A A L E N A L E - Q G G L R V I P I F Y E V I

P L N N - P L P R E - R T P F A L Q T I N A L E E E S R G F P T QP L E S S P A Q L S S D T A S L L S G L V R L D E H S Q I F A R KP S D V R K Q P G S F R K V F K K N Y L K W T E D E K D R F W K K

139478

205523132

A

B

C

Bin et al., Fig. 1

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Bin et al., Fig. 2

IL-1R

IgG α-FIL-1RAcP

IgG α-F

TLR2

IgG α-F

HA-TIRP

IP Ab:

A

WB: αHA

B TRIF TIRAP

IgG α-F IgG α-F

HA-TIRP

IP Ab:WB: αHA

TLR4

IgG α-F

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Bin et al., Fig. 3

IgG α-F IgG α-F IRAK-4 IRAK-4-KA IRAK IRAK-KA

IgG α-F IgG α-F IRAK-2

IgG α-FIRAK-MIgG α-F

HA-TIRP

AIP Ab:

WB: αHA

B IgG α-F IgG α-F IgG α-F

TRAF5 TRAF6 TRAF6(289-596)

HA-TIRP

WB: αHA

aa1-77 aa1-171 aa78-235 aa172-235 aa78-171

IgG α-HA IgG α-HA IgG α-HA IgG α-HA IgG α-HA

FLAG-TRAF6

IP Ab:C

WB: αFLAG

HA-TIRP mutants:

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Bin et al., Fig. 4

TIRAP

vectorTIRP

0

10

15

20

25

30

35

vector TIRP MyD88 TRIF

A

5

Rel

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ucif .

Act

.

0

5

10

15

20

25

vector IL-1R1 IL-1RAcP

C

Rel

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Act

.

0

50

100

150

200

250

vector TIRP MyD88 TRIF TIRAP

B

Rel

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Act

.

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Bin et al., Fig. 5

0

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2

3

4

5

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vector IL-1R1+IL-1RAcP

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10

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TLR2 TLR4

vector

TIRP (78-171)

vector

TIRP (78-171)

50

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Liang-Hua Bin, Liang-Guo Xu and Hong-Bing Shureceptor signaling

TIRP: a novel TIR domain-containing adapter protein involved in Toll/interleukin-1

published online April 28, 2003J. Biol. Chem. 

  10.1074/jbc.M303451200Access the most updated version of this article at doi:

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