The International Journal of Biochemistry & Cell Biology 68 (2015) 1520

Contents lists available at ScienceDirect

The International Journal of Biochemistry

& Cell Biology
journal homepage: www.elsevier.com/locate/biocel

Identication of critical regions within the TIR domain

of IL-1 receptor type I
Jrgen Radons a, , Werner Falk a , Stefan Dove b
a
b

Department of Internal Medicine I, University Clinic Regensburg, D-93042 Regensburg, Germany

Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany

a r t i c l e

i n f o

Article history:
Received 3 June 2015
Received in revised form 5 August 2015
Accepted 11 August 2015
Available online 14 August 2015
Keywords:
IL-1
IL-1RI
TIR domain
NF-B
Site-directed mutagenesis
Homology modeling

a b s t r a c t
Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that
play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding
of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology
models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these
residues was investigated by site-directed mutagenesis and a functional luciferase assay reecting NFB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A
and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the C helix (Q469-E473 in
mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
IL-1 is a pleiotropic cytokine playing a crucial role in inammation and regulation of immune responses thereby affecting almost
any cell type (Garlanda et al., 2013). It is also involved in the regulation of other homeostatic functions and the pathogenesis of
different diseases such as chronic inammatory and neurodegenerative diseases, atherosclerosis, and cancer (Lukens et al., 2012;
Multhoff et al., 2012). IL-1 is expressed by tumor, stromal and
endothelial cells and the hosts inltrating immune cells (Multhoff
et al., 2012). IL-1 signal transduction is initiated by binding of
either form of IL-1 to IL-1 receptor type I (IL-1RI), which undergoes
a conformational change allowing association with IL-1 receptor accessory protein (IL-1RAcP). IL-1RAcP does not bind IL-1 but
represents an essential component in the IL-1 signaling pathway

Abbreviations: DEAE, diethylaminoethyl; FACS, uorescence-activated cell sorting; FCS, fetal calf serum; IL, interleukin; IL-1RI, IL-1 receptor type I; IL-1RAcP, IL-1
receptor accessory protein; IL-1RAPL, IL-1RAcP-like; LPS, lipopolysaccharide; Mal,
MyD88 adaptor-like protein; MyD88, myeloid differentiation primary response protein MyD88; NF-B, nuclear factor kappa B; PI3K, phosphatidylinositol 3-kinase;
PBS, phosphate-buffered saline; PKB, protein kinase B; SI, stimulation index; TBS,
Tris-buffered saline; TILRR, Toll-like and IL-1R regulator; TIR, Toll/IL-1 receptor; TLR,
Toll-like receptor; WT, wild type.
Corresponding author at: Mhldorfer Str. 64, D-84503 Alttting, Germany.
Tel.: +49 86714034200.
E-mail address: raj10062@web.de (J. Radons).
http://dx.doi.org/10.1016/j.biocel.2015.08.009
1357-2725/ 2015 Elsevier Ltd. All rights reserved.

(Korherr et al., 1997; Radons et al., 2002; Wesche et al., 1997). The
crystal structure of the ectodomains of an IL-1/IL-1RI/IL-1RAcP
ternary complex has been solved recently (Thomas et al., 2012).
IL-1-mediated receptor heterodimerization leads to recruitment of
the intracellular adapter MyD88 via TIR-TIR domain interactions
culminating in activation of intracellular signaling cascades including NF-B (Boraschi and Tagliabue, 2013; Gay et al., 2011; Watters
et al., 2007). Recently, a novel IL-1RI co-receptor called Toll-like
and IL-1R regulator (TILRR) was identied to associate with IL-RI
thus amplifying IL-1-mediated NF-B activation and inammatory
responses (Zhang et al., 2010; Zhang et al., 2012). IL-1 signaling
also involves recruitment of phosphatidylinositol 3-kinase (PI3K)
to the IL-1 receptor complex (Reddy et al., 1997) and subsequent
activation of NF-B via AKT/PKB (Cahill and Rogers, 2008). IL-1
binding also activates numereous G proteins resulting in an IBindependent NF-B transactivation (Jefferies and ONeill, 2000;
Singh et al., 1999). IL-1 signaling nally regulates gene expression
of a great variety of other genes involved in immune defence, repair
and others (Multhoff et al., 2012; ONeill, 2000). For successful initiation of signaling, oligomerization of TIR domains and association
of adapter molecules was shown to be required (Jain et al., 2014;
Multhoff et al., 2012; Narayanan and Park, 2015; Ve et al., 2015).
Within the TIR domain three typical regions of conservation
can be found: box 1 (D-K-YDAF-SY), box 2 (GYKLCIRDPG), and
box 3 (-FWKx-) (Bowie and ONeill, 2000; ONeill and Dinarello,
2000). Bold amino acids are identical in nearly all members while
conservative substitutions may occur in the other positions. In

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J. Radons et al. / The International Journal of Biochemistry & Cell Biology 68 (2015) 1520

dominant negative TLR-4, the proline in box 2 is substituted by

histidine in C3H/HeJ mice abolishing the host immune response
to LPS (Poltorak et al., 1998). Only IL-1RI and TLR-3 do not harbor
a proline at this position. Results on human IL-1RI mutants indicated the conserved arginine in box 2 of IL-1RI and the FWKxxRY
sequence including box 3 to be crucial for IL-1 signaling (Heguy
et al., 1992). By contrast, in another study single point alanine
mutation of R, W and K (sequence RFWK) did not affect function
(Slack et al., 2000). Boxes 1 and 2 are considered to be involved in
binding of adapter molecules (Bowie and ONeill, 2000; Slack et al.,
2000). Our previous investigations identied certain amino acids
within the loop preceding box 3 in IL-1RAcP as being crucial for
IL-1 responsiveness (Radons et al., 2003). The present study was to
identify amino acids of the IL-1RI TIR domain contributing to receptor signaling. Since the intracellular TIR domains are not part of the
crystal structure of the IL-1/IL-1RI/IL-1RAcP heterotrimer (Wang
et al., 2010), a homology model of the mouse IL-1RI TIR domain
(mIL-1RI-TIR) based on the structure of the human IL-1R accessory protein-like (IL-1RAPL) (Khan et al., 2004) was generated and
used for suggesting promising positions to be mutated. The role of
these residues was identied by site-directed mutagenesis and a
functional luciferase assay reecting NF-B activity in transiently
transfected Jurkat cells.
2. Materials and methods
2.1. Homology modeling of the mIL-1RI TIR domain
An initial model of mIL-1RI-TIR was generated by the program
COMPOSER (Blundell et al., 1988) as part of the molecular modeling
suite SYBYL 7.3 (Certara, L.P., Princeton, NJ). Homology modeling is
based on known crystal structures serving as templates for structurally conserved regions (SCRs). Among structures of TIR domains
present in the Brookhaven Protein Databank (PDB) human TLR-1
(PDB ID 1fyv), TLR-2 (1fyw) (Xu et al., 2000) and IL-1RAPL (1t3g)
(Khan et al., 2004) chain A of the latter was selected as single template since only in this case the degree of homology with mIL-1RI
was sufcient (ca. 30% sequence identity compared to ca. 20% with
TLR-1 and TLR-2). Fig. 1 presents the optimal sequence alignment
and the SCRs determined by COMPOSER.
In the present case of a single template structure, the backbone frameworks of the SCRs of mIL-1RI-TIR and of IL-1RAPL
are identical. Side chains were added to the target SCR backbone

by a knowledge-based approach, taking in account the backbone

secondary structure and the side chain conformation at the corresponding residues of the template. The structurally variable regions
(SVRs) of the model were constructed by the loop search algorithm
within SYBYL. For each SVR, appropriate fragments from a binary
PDB database with the same length as the SVR of the target are
proposed on the base of distances and superpositions of the anchor
residues. After adding hydrogens, the model was energy minimized
using the AMBER-FF99 force eld (Cornell et al., 1995) with AMBERFF99 charges (distance dependent dielectricity constant 4, rst 50
cycles with constrained backbone and steepest descent method)
up to a root mean square gradient of 0.05 kcal/mol (Powell conjugate gradient). Surfaces and electrostatic potentials of the model
were calculated and visualized by the program MOLCAD (MOLCAD
GmbH, Darmstadt, Germany) implied in SYBYL 7.3.
2.2. Construction of the reporter plasmid and mutated forms of
IL-1RI
For determination of transcriptional activity of NF-B, ve
tandemly arranged NF-B binding sites from the HIV long terminal repeat enhancer (5 -GGGACTTTCC-3 ) (Sen and Baltimore,
1986) were cloned via BglII and HindIII into the luciferase
reporter vector pGL3-Basic (pGL35xNF-B). Mutated forms of
mIL-1RI were generated by site-directed mutagenesis using the
QuickChangeTM site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands). The vector pFLAG-IL-1RI, a derivative of
pFLAG-CMV-1 (SigmaAldrich GmbH, Steinheim, Germany) was
used as a template in all mutagenesis experiments. All mutations
were veried by sequence analysis. Expression and protein production by all mutants was analyzed by FACS detection of the Flag
epitope in transfected cells using anti-Flag antibody which was
done with permeated cells.
2.3. Detection of NF-B activity in transiently transfected Jurkat
cells
The human T cell leukaemia cell line Jurkat, lacking a receptor for IL-1 (Kuno et al., 1993), was purchased from the American
Type Culture Collection (ATCC; Manassas, USA) and maintained
in RPMI 1640 medium (PAN Biotech GmbH, Aidenbach, Germany)
containing 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin
and 2 mM glutamine at 37 C in humidied air with 5% CO2 . For

Fig. 1. Sequence alignment of TIR domains of hIL-1RI (UniProtKB P14778), mIL-1RI (P13504) and IL-1RAPL (Q9NZN1, the template for homology modeling). -Helices (blue)
and -sheets (green) are labeled according to Khan et al. (2004). Red frames indicate boxes 1, 2, and 3. mIL-1RI positions subjected to mutation are highlighted in yellow.
SCR1SCR5: structurally conserved regions determined by COMPOSER. 7xseven additional residues not resolved in the IL-1RAPL structure (PDB ID 1t3g).

J. Radons et al. / The International Journal of Biochemistry & Cell Biology 68 (2015) 1520

17

transfection, 100 l of cells (106 /ml) in TBS were mixed with 30 l

of freshly prepared DEAE-dextran solution (10 mg/ml) and 470 l
TBS with 1 g of vector DNA and 0.5 g of the luciferase reporter
plasmid pGL35xNFB harboring ve NF-B binding sites (Radons
et al., 2002). The total amount of DNA in all transfections was kept
constant by adding empty vector DNA. The mixture was incubated
for 10 min with agitation and washed once with RPMI 1640 containing 10% FCS, mercaptoethanol (3 105 M) and gentamycin
(50 g/ml). The pellet was resuspended in 1.5 ml of medium, and
50 l were used for stimulation with either recombinant human
IL-1 (rhIL-1; Pan Biotech, Aidenbach, Germany; 1 or 10 ng/ml)
or recombinant human TNF (rhTNF; 10 ng/ml) for 6 h or remained
unstimulated. NF-B activation was determined by measuring the
luciferase activity in transiently transfected cells using the LucLite
Plus luciferase reporter gene assay kit (Perkin Elmer Life Sciences,
Shelton, USA). Data from corresponding cultures from all experiments were pooled and the highest and the lowest value in all
groups were deleted. Afterwards, a mean cpm was established for
unstimulated cultures which was then used to convert all cpm to
stimulation indices (SI, stimulated/mean unstimulated). A mean SI
was then calculated for the WT transfectants stimulated with TNF
and IL-1. This made it possible to determine an SI in % of WT for all
values; (SI sample/mean SI WT) x 100).
2.4. Statistical analysis
Level, spread, and symmetry of data distribution were conveyed
by a box-and-whisker plot. Statistical differences between medians
were analyzed using the two-tailed non-parametric WilcoxonMann-Whitney test. A P value of less than 0.05 was considered to
reect statistical signicance.
3. Results and discussion
3.1. Structure-based selection of IL-1RI mutants
The homology model of mIL-1RI-TIR based on the crystal
structure of human IL-1RAPL (Khan et al., 2004) comprises all characteristic elements of TIR domains (see sequence alignment, Fig. 1,
and structural overview, Fig. 2A). A ve-strand parallel -sheet is
surrounded by seven -helices. Three typical segments assumed
to be functionally signicant, boxes 1-3, and moreover, the whole
BB-loop belong to the structurally conserved regions (SCRs) and are
probably well approximated by the mIL-1RI-TIR model. Differences
in the structures of IL-1RAPL (Khan et al., 2004) on one hand as
well as TLR-1 and TLR-2 on the other hand (Xu et al., 2000) indicate
variable regions possibly not correctly reproduced by the model. In
particular, (i) the short helices C and C are followed by a wellordered helix C in the case of IL-1RAPL and by a highly disordered
region in the case of TLR-1 and TLR-2, respectively, (ii) C is slightly
shifted in IL-1RAPL vs. TLR-1, and (iii) helix D in TLR-1 and TLR-2
is nearly perpendicular to that in IL-1RAPL (Khan et al., 2004).
The most striking specic feature of both human and mouse
IL-1RI-TIR compared to TIR domains of IL-1RAPL, IL-1RAcP, TLR-1
and TLR-2 is a cluster of acidic amino acids at the C-terminus of
the BB-loop and in helix C (Fig. 2B). Additionally, three serines
before (S465) and within C (S470, S471) are typical. Moreover,
C and the BB-loop belong to the interaction interfaces in homodimers (asymmetric units) of IL-1RAPL (Khan et al., 2004), TLR-1
(Xu et al., 2000) and TLR-2 (Tao et al., 2002) although the crystallographic interfaces of these TIR domains are different and probably
not biologically relevant. In a model based on the crystal structure of the dimeric TLR-10 TIR domain, dimerization of TLR-4 TIR
domains was shown to require formation of a large conserved
platform containing the BB-loop and box 1 motifs and thereby

Fig. 2. Homology model of the mIL-1RI TIR domain. Secondary structure elements
are labeled and colored (-helices blue, -strands green). Specic conserved regions
(boxes 13) are drawn in red. (A) Ribbon and tube representation of the tertiary
structure. Ocher balls indicate mutated positions. (B) Detailed model of the region
with all but one (S519) amino acids subjected to mutation (ocher C atoms). Other
atom colors: O red, N blue, C gray.

forming a potential binding site for intracellular adapters (Bovijn

et al., 2012). Although the involvement of the BB-loop in TLR-4 TIR
dimerization has been described previously, further data from this
group suggest that this region establishes contact with helix E of
TLR-4, rather than helix C as found in the structure of TLR-10 TIR
crystals (Toshchakov et al., 2011). Noteworthy, the crystal structure
of the TIR domain of TLR-6 recently revealed that helix C rather
than the BB-loop plays a crucial role in homodimeric TIR-TIR interactions (Jang and Park, 2014). Accordingly, homodimerization of
Mal-TIR domains lacking the BB-loop involves residues of the C
and D helices of both monomers (Lin et al., 2012), implying that
the functional dimer does not inevitably represent the most stable among possible dimer conformations (Toshchakov et al., 2011).
In Mal-TIR dimers, E190 (D) of one monomer forms a hydrogen
bond with K158 (C ) of the other monomer, while P189 and F193
in D of one monomer form stacking interactions with P155 and

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J. Radons et al. / The International Journal of Biochemistry & Cell Biology 68 (2015) 1520

Fig. 3. Effect of different mutant variants of IL-1RI on NF-B activation. Jurkat cells were transiently co-transfected with 500 ng of luciferase reporter and 1 g of the indicated
IL-1RI constructs. 6 h after transfection, cells were stimulated with different concentrations of rhIL-1 or 10 ng/ml rhTNF. After incubation for an additional 6 h, luciferase
activity was determined in triplicate as a measure of NF-B activation and normalized to the TNF response. Data were visualized by a box-and-whisker plot. The boxes show
the 25th and 75th percentiles together with the medians while the whiskers show the 5th and 95th percentiles, respectively. Data represent the medians of three to six
separate experiments performed in at least triplicates. *, p < 0.05 compared with wild type (WT).

W156 in C of the other monomer, respectively (Lin et al., 2012).

In the case of IL-1RAPL, S488 (C ) of one monomer forms a hydrogen bond with S488 of the other monomer, an interaction that is
reproduced by a disulde bridge of the corresponding cysteines in
the TLR-1 dimer. Thus, it may be suggested that the whole BB-C
face is involved in heterodimerization in the case of IL-1RI, too. Consequently, the specic acidic and polar amino acids in this region
are particularly interesting for in-vitro mutagenesis experiments,
considering also Q469 at the beginning of C , the only amino acid
of the whole BB-C face which differs between mouse and human
IL-1RI (G466).
The conserved proline in box 2 (BB-loop) of nearly all TIR
domains is replaced by valine in IL-1RI species orthologs (mIL-1RI:
V435). Substitution of proline by polar residues histidine in TLR-4
(Poltorak et al., 1998), histidine and aspartate in IL-1RAcP (Radons
et al., 2002) impaired function, whereas an IL-1RAcP P446A mutation was without effect (Radons et al., 2002). Comparison of the
BB-loops in IL-1RAPL and the mIL-1RI model suggests that proline
and valine do not lead to different backbone conformations.
3.2. Effects of IL-1RI mutants on NF-B activation
NF-B activation mediated by several intracellular signaling
pathways was determined by measuring the luciferase activity of

our reporter plasmid pGL35xNFB harboring ve NF-B binding sites (Radons et al., 2002). This NF-B activation assay does
not distinguish between activation via the MyD88-dependent and
AKT/PKB-dependent pathways. Signicant changes in IL-1 responsiveness of transfected Jurkat cells were observed in eight out of 13
tested mutants (Fig. 3). As expected and as control, the mutant with
the complete intracytoplasmatic region missing (Stop380) showed
no response to IL-1. In case of the ve mutants E437K, D438K,
D438A, S519K and Q469P, responses were not signicantly different from that of the wild type. The four mutants E437K/D438K,
E437A, E472A/E473A and 5xA showed decreased IL-1 responsiveness, albeit in case of E437K/D438K and E437A only at the lower IL-1
concentration. Effects of the 5xA mutant were exceptionally strong.
Increased responsiveness to IL-1 was observed for the mutants
E437A/D438A, V435P and Q469A (for V435P and Q469A only at
the higher IL-1 concentration).
It has been shown previously that substitution of the conserved
R within box 2 of IL-1RI by alanine caused a massive reduction
in IL-1-induced NF-B activation (Slack et al., 2000). Moreover,
this box 2 receptor mutant was found to strongly decrease IL-1mediated receptor activation after blockage of TILRR expression
identifying box 2 as being a putative interaction site for this
cytosolic adapter (Zhang et al., 2010). The observed decreased
IL-1 responsiveness of our C-terminal box 2 mutants (E437K,

J. Radons et al. / The International Journal of Biochemistry & Cell Biology 68 (2015) 1520

Fig. 4. Electrostatic potential mapped on a MOLCAD Connolly surface of the mIL-1RI

TIR domain. The potential is based on AMBER-FF99 charges. Colors: most negative
violet, negative blue, neutral cyan, green, positive yellow, most positive
brown. Labeled are amino acids subjected to mutation.

E437K/E438K) might rely, at least in part, on restrictions in TILRR

recruitment.
Since the E472A/E473A mutation reduced IL-1-induced NF-B
activation by 25% and the 5xA variant even by 75%, the specic
C helix in mIL-1RI (and hIL-1RI) is functionally relevant. Fig. 4
presents the electrostatic potential map of the mIL-1RI model. The
acidic nature of C leads to a high negative potential. The region
around helix E including the conserved box 3 is positively charged
due to four basic amino acids (K530, R532, K535, R538). Two of
these basic amino acids (R537 and K540 corresponding to mIL1RI R532 and K535, respectively) are present in IL-1RAcP as well
so that its EE-loop and box 3 are positively charged, too. The TIR
domain of the adaptor protein MyD88 shows a similar charge distribution (Li et al., 2005). Our previous results on IL-1RAcP mutants
indicated that the EE-loop and box 3 are involved in TIR domain
interactions (Radons et al., 2002). Docking of IL-1RAcP and MyD88
based on homology models with human TLR-2 as template and
in-vitro mutagenesis results suggested electrostatic interactions
between the DRD-motif in box 2 (BB-loop) of MyD88 and the lysine
patch in the EE-loop of IL-1RAcP (Li et al., 2005). Heterodimerization of IL-1RAcP and MyD88 precedes homotypic oligomerization
of MyD88 (Akira, 2003; Akira and Sato, 2003; Yamamoto et al.,
2004) again based on box 2-box 3 interactions (Li et al., 2005), and
recruitment of downstream signaling transducers by the MyD88
oligomers. Common for all these TIR-TIR interactions is that a negatively charged pocket in the region of box 2 and C/C may provide
a binding site for protruding positively charged residues in or close
to box 3. The shape of the corresponding regions of IL-1RI shows
this general pattern, too (Fig. 4). Heterodimerization or oligomerization of IL-1RI with IL-1RAcP and/or MyD88 may rely on these
interactions. Thus, IL-1RI is probably involved in transient or stable
TIR-TIR interactions essential for downstream signaling.
Strikingly, mIL-1RI Q469 in the IL-1R specic region (mIL-1RI
amino acids 465, 467-473) may be exchanged by glycine (hIL-1RI
G466). To investigate the signicance of a polar amino acid at this
position, Q469 (C ) was mutated to alanine and proline, respectively. Whereas Q469P was without any effect on NF-B activation,
mutation of Q469 to alanine led to a signicant increase of the IL-1

19

response at the higher IL-1 concentration, suggesting that a small

neutral amino acid like glycine or alanine is more favorable than
the polar glutamine in mIL-1RI.
Due to the IL-1RI-specic amino acids E437 and D438, the Cterminus of the BB-loop contributes to the large negatively charged
region spanned by parts of box 2, C , the DD-loop and D. Lysine
and alanine mutation of these acidic amino acids behaved quite
contradictory. Only E437A and the E437K/D438K double mutant
led to a signicant reduction in IL-1-induced NF-B activation, but
in both cases only at the lower IL-1 concentration. Surprisingly,
the double-A mutant even induced increased effects at both 1 and
10 ng/ml IL-1. Thus, a negative potential at the C-terminal end of
the BB-loop is not necessary for IL-1RI functionality. At least one
basic amino acid is well tolerated.
Among the members of the TIR family, a highly conserved proline can be found in box 2. This residue corresponds to a naturally
occurring missense point mutation resulting in the substitution
of P712 by histidine (the LPSd mutation) in dominant negative
TLR-4 in C3H/HeJ mice selectively leading to LPS hyporesponsiveness (Poltorak et al., 1998). Equivalent substitutions in other TLR
molecules also abrogate inammatory responses (Ozinsky et al.,
2000; Underhill et al., 1999). In contrast to the other TIR family
members, the conserved proline as part of box 2 within the BBloop is substituted by valine in IL-1RI (mIL-RI V435). Exchange of
V435 by proline improved IL-1-mediated NF-B activation, but only
at the higher IL-1 concentration. In accordance with the model in
Fig. 2, the V435P mutation probably stabilizes the BB-loop without major structural effects. Interestingly, TLR-3 bears an alanine
(A795) in place of the conserved BB-loop proline. Mutation of A795
to proline also improved NF-B activation (Verstak et al., 2013)
most likely due to the proposed effects on BB-loop stability thereby
modulating recruitment of downstream adapter molecules.
In nearly all TIR domains, a conserved WX motif can be found
at the E-EE junction with WK in IL-1RAcP, WH in IL-1RAPL and
WS in IL-1RI. To test for specicity, S519 in mIL-1RI was mutated
to lysine thereby reconstituting the IL-1RAcP motif. This mutation
did not affect IL-1-induced NF-B activation. In our previous study
on IL-1RAcP, mutation of the conserved W526 to phenylalanine
and the neighbouring K527 to glycine was without effect on IL1 responsiveness, too (Radons et al., 2002). Taken together, these
ndings argue against a crucial role of this motif within the NF-B
signaling process.
In conclusion, site-directed mutagenesis and a functional
luciferase assay reecting NF-B activity in transiently transfected
Jurkat cells identied the specic C helix of the IL-1RI to be crucial for signaling. Homology modeling of the mouse IL-1RI also
indicated putative TIR-TIR domain interfaces for heterotypic interactions. The negative electrostatic potential and the shape of this
region including the BB-loop (box 2) predestine interactions with
the positively charged area around box 3 of IL-1RAcP or MyD88. The
acidic C-terminal end of the BB-loop is not necessary with respect to
function and dimerization. Back mutation of valine in the BB-loop
into the highly conserved proline of other TIR domains did only
slightly improve NF-B activity, indicating minor stabilization of
the BB-loop.
Acknowledgements
pFLAG-IL-1RI was a generous gift from Michael Martin (Gieen,
Germany). The excellent technical assistance of Daniela Aigner is
gratefully acknowledged.
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