Recruitment Controls Toll-like
Jonathan C. Kagan1and Ruslan Medzhitov1,2,*
1Section of Immunobiology
2Howard Hughes Medical Institute
Yale University School of Medicine, New Haven, CT 06520, USA
Toll-like receptors (TLRs) play a critical role in
the immune system as sensors of microbial
infection. Signaling downstream from TLRs is
initiated by the recruitment of adaptor proteins,
including MyD88 and TIRAP. These adaptors
play essential roles in TLR signaling, but the
mechanism of their function is currently un-
known. Here we demonstrate that TIRAP and
MyD88 have distinct functions and describe a
mechanism of recruitment of TIRAP and MyD88
to TLR4. We find that TIRAP contains a phos-
phatidylinositol 4,5-bisphosphate (PIP2) bind-
ing domain, which mediates TIRAP recruitment
to the plasma membrane. TIRAP then functions
to facilitate MyD88 delivery to activated TLR4
to initiate signal transduction. These results es-
tablish that phosphoinositide-mediated adap-
tor recruitment initiates a specific signal-trans-
response to infectious microorganisms (Iwasaki and
Medzhitov, 2004). TLRs recognize microbial molecules
ranging from bacterial cell-surface components to viral
genomes (Akira and Takeda, 2004). TLR signaling initiates
with the recruitment of TIR-domain-containing adaptor
proteins to the cytoplasmic TIR domain of an activated
TLR. Recruitment of one or more of these adaptors
(MyD88, TIRAP/Mal, TRIF/TICAM-1, and TRAM/TICAM-
2) to a TLR initiates signaling events that activate the
NF-kB, AP-1, and IRF families of transcription factors
(Akira and Takeda, 2004). These transcription factors in-
duce the expression of genes involved in host defense
TLR4 is the best characterized member of the TLR fam-
ily and is the only receptor that uses all four TIR-domain-
containing adaptors. Activation of TLR4 occurs in re-
sponse to bacterial lipopolysaccharide (LPS) and induces
two signaling pathways that are termed the MyD88-de-
pendent and MyD88-independent pathways (Akira and
Takeda, 2004). The MyD88-dependent pathway requires
both MyD88 and TIRAP to activate NF-kB and proinflam-
matory cytokine production (Horng et al., 2002; Yama-
moto et al., 2003a, 2003b). MyD88-independent signaling
events arecontrolled byTRIFand TRAM and induce IRF3-
dependent type I interferon production (Fitzgerald et al.,
2003; Hoebe et al., 2003; Oshiumi et al., 2003a, 2003b;
Yamamoto et al., 2003a, 2003b). Other TLRs utilize a sub-
set ofadaptors. Forexample, TLR2 signaling requires only
MyD88 and TIRAP (Horng et al., 2002; Yamamoto et al.,
2003a, 2003b). TLR2 signaling activates NF-kB but not
IRF3, indicating that adaptor recruitment determines the
specificity of the signaling pathways induced by TLRs.
However, the functional differences between the four
adaptors and the mechanisms of differential adaptor en-
gagement by TLRs are currently unknown.
TLRs can be divided into two groups based on their
subcellular location. The first group (TLRs 1, 2, 4, 5, and
6) are found at the plasma membrane. The second group
(TLRs 3, 7, and 9) are intracellular and likely signal from
acidic endosomes. Interestingly, all of the intracellular
TLRs signal in a TIRAP-independent fashion (Horng
et al., 2002; Yamamoto et al., 2002), suggesting a link be-
tween TLR localization and adaptor use.
The differential localization of TLRs indicates that the
MyD88-dependent signaling pathway can be initiated
from several cellular compartments. The mechanisms
that regulate the delivery of TLR adaptors to activated re-
ceptors are unknown, and their elucidation is essential for
understanding the design of TLR signaling pathways in
general. In this study, we investigate the mechanisms
that control TLR adaptor recruitment to distinct subcellu-
lar locations. TIRAP represents a good model to address
this question because it is utilized by a subset of TLRs
that signal from the plasma membrane but not by TLRs
that signal from endosomes. We found that TIRAP resides
on membranes that shuttle between the plasma mem-
brane and endosomes by an ADP ribosylation factor 6
Cell 125, 943–955, June 2, 2006 ª2006 Elsevier Inc. 943
(ARF6) dependent process. We demonstrate that TIRAP
contains a phosphatidylinositol 4,5-bisphosphate (PIP2)
binding domain that mediates TIRAP recruitment to mem-
onstrate that the primary function of TIRAP in TLR signal-
ing is to control the recruitment of MyD88 to TLR4. Thus,
branes dictates differential adaptor recruitment to TLRs
residing in different compartments.
TIRAP Is Localized to the Plasma Membrane
and Endocytic Vesicles
centrated at the leading edge of the MEFs (data not
shown). Macrophages do not typically have a leading
edge, but instead have membrane ruffles, which are dis-
crete regions of the plasma membrane that are biochemi-
cally similar to the leading edge of fibroblasts (Allen et al.,
1998; Araki et al., 1996). Accordingly, TIRAP was enriched
rophages (Figure 1A). In addition to being concentrated at
throughout the cell (see Figure S1A in the Supplemental
Data available with this article online). Identical results
were obtained when examining the localization of TIRAP
tagged with GFP, FLAG, or HA (Figure 1A; see also below
and Figure S1B), indicating that the localization did not re-
sult from the tag and represents a property of the TIRAP
protein itself. These data indicate that TIRAP resides at
Figure 1. A Putative PIP2 Binding Do-
main Controls TIRAP Localization to the
TLR2xTLR4 KO (B) macrophages expressing
TIRAP-GFP and stained for F-actin. TIRAP lo-
calization is concentrated at membrane ruffles,
as evidenced by the costaining with F-actin in
the merged images (right column). All images
experiments where over 500 cells were exam-
ined per condition and, unless otherwise indi-
cated in the text, >95% of the cells displayed
(C) Deletion of all but 40 residues retains the
localization of wt TIRAP. Residues 15–35 of
TIRAP phenocopy wt TIRAP.
The PIP2 binding motif (residues 15–35) is
shown with the lysines mutated in TIRAP 43
underlined. Note that TIRAP mutants lacking
an intact PIP2 binding motif are mislocalized
(TIRAP 43, TIRAP 1–20, and TIRAP 1–20TIR).
944 Cell 125, 943–955, June 2, 2006 ª2006 Elsevier Inc.
These findings were corroborated by biochemical frac-
tionation of macrophages expressing TIRAP-GFP, which
demonstrated that TIRAP cofractionated with membranes
but was absent from cytosolic fractions (Figure S1C).
Upon detergent extraction, TIRAP was redistributed into
the cytosolic fraction. Since TIRAP lacks any obvious
membrane-anchoring domains, we next investigated the
mechanism of TIRAP localization to membranes and its
role in TLR signaling.
TIRAP Localization Is TIR Independent and Sensitive
to Cytochalasin D
TIRAP regulates signaling by TLR2 and TLR4 (Horng et al.,
2002; Yamamoto et al., 2002). To determine whether
ies were performed in macrophages deficient in both TLR2
and TLR4. No differences in TIRAP localization were
observed when comparing wild-type (wt) or TLR2xTLR4
knockout (KO) macrophages (Figure 1B).
The localization of TIRAP P125H, which is unable to in-
teract with TLR4 (Horng et al., 2001), was examined as an
independent approach to determine the role of TLRs in
TIRAP localization. The distribution of TIRAP P125H was
ization to membranes is not controlled by TIR-dependent
interactions between TIRAP and TLRs and therefore in-
volves a previously unrecognized property of this adaptor.
The colocalization between TIRAP and actin at mem-
brane ruffles led us to examine the role of actin in TIRAP
localization. Transfected cells were treated with cytocha-
lasin D, an inhibitor of actin polymerization, and TIRAP lo-
calization was examined. Within 30 min of cytochalasin D
treatment, TIRAP was redistributed from being localized
primarilyattheplasma membranetobeing enrichedonin-
tracellular tubules and vesicles (Figures S1D and S1E).
Prominent redistribution was observed in 55% ± 6% of
the cells examined, with the remaining cells simply losing
TIRAP concentration at the cell surface. TIRAP-positive
tubules and vesicles were also observed in untreated cells
not create a new location for this protein but shifted the
equilibrium to favor an intracellular distribution. These
results indicate that TIRAP cycles between the plasma
membrane and an intracellular location by an actin-de-
A Putative Phosphoinositide Binding Domain
Regulates TIRAP Localization
To identify the region that controls TIRAP localization, de-
letion mutants were generated. Each mutant was GFP
tagged, and the resulting proteins were examined for their
ability to phenocopy the localization of wt TIRAP. TIRAP
mutants lacking the TIR domain (TIRAP 1–85) retained
the ability to localize to the cell surface (Figure 1C;
Figure S2A). Similarly, the first 40 amino acids of TIRAP
(TIRAP 1–40) were sufficient to phenocopy wt TIRAP lo-
calization (Figure 1C; Figure S2A). These results provide
further evidence that membrane localization of TIRAP is
TIRAP 1–40 contains a region (residues 15–35) with
a high isoelectric point (10.46) compared to the entire pro-
tein (7.55) that is enriched in basic and aromatic residues
(Figure 1D). These properties are often found in domains
that bind PIP2 (McLaughlin et al., 2002). The localization
of this putative PI binding motif was identical to wt TIRAP
(Figure 1C; Figure S2A). In contrast, residues 1–20 contain
no membrane-targeting activity (Figure 1C; Figure S2A).
The putative PI binding domain contains six conserved
lysines. Since mutation of lysine residues in PI binding
domains can abolish lipid binding and disrupt subcellular
localization (Stauffer et al., 1998), we generated mutant
versions of TIRAP where lysines were substituted with
alanines. Membrane localization of TIRAP was abolished
by mutation of four of the lysines (TIRAP 43) (Figure 1C;
Figure S2A). These data indicate that the N-terminal re-
gion with properties of a PI binding motif is necessary
and sufficient to direct TIRAP localization.
TIRAP Is a Phosphoinositide-Interacting Protein
To determine whether TIRAP binds to lipids, we per-
formed several in vitro protein/lipid interaction assays.
First, a GST-TIRAP fusion protein was overlaid onto nitro-
cellulose membranes containing a variety of lipid species
(PIP strips). GST-TIRAP, but not GST, bound to all PIs and
phosphatidylserine (PS), but not to phosphatidylethanol-
amine (PE), phosphatidylcholine (PC), or any other lipid
examined (Figure 2A). As an independent approach, we
taining known concentrations of lipids. GST-TIRAP, but
not GST, cosedimented with liposomes containing PIP2
or PI(4)P, but not liposomes containing PC and PE (Fig-
ure S3B). To determine which lipid is bound preferentially,
quantitative analysis was performed on TIRAP’s ability to
bind liposomes containing PIP2, PI(4)P, PI, or PS. We fo-
cused our attention on these lipids because each is en-
riched at the plasma membrane at steady state, with the
exception of PI. Under all conditions tested, GST-TIRAP
interacted preferentially with PIP2-containing liposomes
(Figure 2B). Thus, the recruitment of TIRAP to the plasma
membrane is likely to be mediated primarily by interac-
tions with PIP2.
We next analyzed the requirement for the putative PI
binding motif of TIRAP for lipid binding. Mutations of two
conserved lysines (TIRAP 23) reduced, and mutation of
ing ability of TIRAP (Figure 2A; Figure S3A). Thus, TIRAP
43 is defective in both lipid binding in vitro and cellular
localization in vivo, suggesting that PI binding (most likely
to PIP2) is required for TIRAP localization.
We next examined the functional significance of
TIRAP’s PI binding activity and cellular localization. TIRAP
KO macrophages were retrovirally transduced with either
wt TIRAP, TIRAP 43, or TIRAP constructs lacking the TIR
domain (TIRAP 1–85). Only macrophages reconstituted
with wt TIRAP regained the ability to produce IL-6 in
Cell 125, 943–955, June 2, 2006 ª2006 Elsevier Inc. 945
response to LPS treatment (Figure 2C). The minimal activ-
ity of TIRAP 43 is likely due to residual ability of TIRAP 43
to bind lipids, as these mutations severely diminish but
consisting of the minimal localization motif (residues 15–
35) fused to the TIR domain (15–35-TIR) also comple-
mented TIRAP deficiency (Figure 2C). In contrast, a con-
struct lacking the PI binding domain, where the first
20 amino acids were fused to the TIR domain (1–20TIR),
was mislocalized (Figure 1C) and nonfunctional (Fig-
ure 2C). These data indicate that TIRAP’s function is
tein therefore consists of two functionally distinct do-
mains, a C-terminal TIR domain responsible for the inter-
action with TLRs and MyD88 and an N-terminal PI
binding domain that is responsible for subcellular localiza-
tion. Both domains are necessary for TLR4 signaling, indi-
cating a requirement of membrane targeting for TIRAP
function as a TLR adaptor.
TIRAP Binds Preferentially to PIP2 In Vivo
To determine which PIs are required for TIRAP localiza-
tion, different classes of PIs were depleted from cells.
Treatment of cells with the PI-3 kinase inhibitor wortman-
nin disrupted the localization of a PI(3)P-specific PX do-
main (Figures S4A, S4B, and S4E). However, wortmannin
did not affect TIRAP localization (Figures S4A, S4B, and
S4E), indicating that 30-PIs are not required for TIRAP
Salmonella enterica serovar Typhimurium (S. typhimu-
rium) encodes a PI phosphatase, SopB (also known as
phosphorylates 40- and 50-PIs in vivo, and overexpression
of SopB is an effective means of depleting cellular levels
of PIP2 (Terebiznik et al., 2002). We examined the effect
of SopB on TIRAP localization in CHO cells, which tolerate
high levels of SopB expression. Expression of SopB, but
not the catalytically inactive mutant SopBC460S, disrupted
the plasma-membrane localization of TIRAP (Figure 2D;
Figure 2. TIRAP Is a Phosphoinositide
(A) PIP strips reveal GST-fusion protein binding
selectivity. The left panel indicates the identity
of each lipid spot. GST-TIRAP, but not GST,
interacts with all PIs tested and PS. TIRAP 43
is defective for lipid binding.
demonstrating that TIRAP binds preferentially
to PIP2 when compared with plasma-mem-
(C) TIRAP KO macrophages expressing the
genes indicated on the x axis. The transduction
efficiency of each macrophage population was
determined to be 30%–40% as assessed by
FACS and direct examination by fluorescence
microscopy. Macrophages were challenged
with the indicated concentrations of LPS and
processed for IL-6 production. All mutants
that lack PIP2 binding ability are defective for
signaling. Data presented in (C) and (E) are
the mean ± SD of an experiment performed at
least two times.
(D) CHO cells were transfected with plasmids
encoding TIRAP-HA and the indicated SopB
allele. Note that the prominent plasma-mem-
brane staining by TIRAP is lost upon expres-
sion of SopB.
(E) SopB expression disrupts TLR4-mediated
NF-kB activation in 293T cells.
946 Cell 125, 943–955, June 2, 2006 ª2006 Elsevier Inc.
Figure S4E), suggesting that 40- and/or 50-PIs are required
for TIRAP localization. Furthermore, expression of SopB,
but not SopBC460S, blocked TLR4-induced NF-kB acti-
vation (Figure 2E), demonstrating a role of 40- and/or 50
PIs in TLR4 signaling.
To corroborate these findings, the localization of TIRAP
was examined in cells expressing a phosphatase that acts
specifically on 50-PIs, INP54p (Raucher et al., 2000). Like
SopB, INP54p disrupted cell-surface localization ofTIRAP
(Figure S4C and S4E). In contrast, a construct that con-
tained the same membrane localization motif as the
INP54p, but no phosphatase domain, did not effect TIRAP
distribution (Figures S4D and S4E). These data indicate
a requirement of 50-PIs for TIRAP localization.
To determine more specifically which lipid (or lipids) is
the primary mediator of TIRAP localization in vivo, we
examined the ability of individual PIs to direct localization
of this adaptor. This was accomplished by generating chi-
meric TIRAP constructs where the N terminus (containing
the PI binding domain) was replaced by a PI binding
domainof a well-defined and restricted specificity. There-
sulting chimeras contain a heterologous N-terminal PI
binding domain and a C-terminal TIR domain. Since PIs
(and their interacting proteins) are enriched in distinct
subcellular locations (De Matteis and Godi, 2004), only
a subset of these chimeras should phenocopy wt TIRAP
localization. Thus, using localization as a readout, we
eliminated a variety of PIs as mediators of TIRAP localiza-
tion. TIRAP chimeras whose subcellular distributions are
directed by interactions with PI(3)P (PX domain from
gp91-phox; PX-TIR), PI(4)P (PH domain from Fapp1;
FAPP1-TIR) or PI(3,4,5)P3 (PH domain from GRP1;
GRP1-TIR) displayed a distribution that was distinct
from that of wt TIRAP (Figures 3B–3E). PX-TIR was found
mainly on endosomes, FAPP1-TIR was found on the
Golgi, and GRP1-TIR was scattered throughout the cell
with minimal membrane staining. These subcellular distri-
whose transport is dictated by interactions with these
respective lipids (De Matteis and Godi, 2004). Thus, the
primary mediator (or mediators) of TIRAP localization is
not PI(3)P, PI(4)P, or PI(3,4,5)P3. In contrast, the TIRAP
chimera whose localization is directed by interactions
with PIP2 (PH domain of PLCd1; PLC-TIR) displayed
Figure 3. An Amino-Terminal PIP2 Bind-
ing Domain Is Necessary and Sufficient
for TIRAP Function
(A–F) Micrographs of macrophages expressing
the indicated TIRAP chimeras. The localization
of wt TIRAP is phenocopied only by the PIP2-
specific (PLC-TIR) (A)andtheSH4-TIRchimera
(G) TIRAP KO macrophages expressing the
genes indicated on the x axis. The transduction
efficiency of each macrophage population was
determined to be 30%–40% as assessed by
FACS and direct examination by fluorescence
microscopy. Macrophages were treated with
the indicated concentrations of LPS and pro-
cessed for IL-6 production. Wild-type TIRAP
and PLC-TIR complemented TLR4 signaling,
whereas all other TIRAP chimeras did not.
Data presented are the mean ± SD of an exper-
iment performed at least two times.
Cell 125, 943–955, June 2, 2006 ª2006 Elsevier Inc. 947
subcellular distribution identical to that of wt TIRAP; both
wt TIRAP and PLC-TIR were enriched at the cell surface
and membrane ruffles (Figure 3A).
We next examined the effect of PI binding specificity on
TIRAP function as a TLR4 adaptor. We tested the ability of
the chimeras to complement the signaling defect of TIRAP
KO macrophages in response to LPS. PX-TIR, GRP-TIR,
and FAPP1-TIR were each unable to complement the sig-
naling defect of TIRAP KO macrophages (Figure 3G). In
contrast, the PLC-TIR chimera restored LPS responsive-
ness (Figure 3G).
Collectively, these data indicate that PIP2 is the primary
mediator of TIRAP localization and that PIP2 binding
specificity is essential for TIRAP function.
Plasma-Membrane Localization Is Not Sufficient
for TIRAP Functional Activity
Intracellular proteins can be localized to the plasma mem-
brane by several different mechanisms, and we therefore
for TIRAP functional activity or whether PIP2-mediated
targeting was specifically required. To address this ques-
tion, wegenerated aTIRAP mutant thatretained the ability
to target to the plasma membrane by a PIP2-independent
mechanism. The SH4 domain of the Src-family tyrosine
kinase Fyn is sufficient to direct heterologous proteins to
the plasma membrane (van’t Hof and Resh, 1999). Mem-
brane localization requires that the domain be both myris-
toylated and farnesylated. We substituted either the entire
N-terminal portion of TIRAP (up to the TIR domain) or the
PIP2 binding region of TIRAP with the Fyn SH4 domain
(generating SH4-TIR and SH4-link-TIR chimeras, respec-
Fyn chimeras localized to the plasma membrane of mac-
rophages, where they costained with F-actin (Figure 3F
and data not shown). Interestingly, while both SH4-TIRAP
constitute TIRAP function in TIRAP KO macrophages
(Figure 3G). These data indicate that TIRAP localization
to the plasma membrane is not in itself sufficient for
functional activity. Rather, PIP2-mediated recruitment of
TIRAP is required.
by Crosstalk with Integrin Signaling Pathways
PIP2 is a critical hub in many cellular processes, and local
concentrations of PIP2 are affected by a variety of signal-
ing pathways (McLaughlin et al., 2002). We hypothesized
that PIP2-dependent control of TIRAP function could pro-
vide a mechanism for coupling TLR4 signaling with the
signaling pathways that regulate PIP2 metabolism. A po-
tential connection with integrin signaling is particularly in-
teresting in this regard because b2 integrins play multiple
essential roles in macrophage biology (Hynes, 2002). Sig-
nalingbyintegrinspromotes PIP2synthesis andfacilitates
the formation of focal adhesions, cell migration, and
phagocytosis (Hynes, 2002). CD11b is the only b2 integrin
expressed on macrophages (Bhat et al., 1999). Interest-
ingly, CD11b was shown to be involved in LPS signaling,
but the mechanism of its role is unknown (Flo et al.,
2000; Perera et al., 2001). We reasoned that one function
of CD11b may be to promote TIRAP concentration at the
plasma membrane by its ability to stimulate PIP2 synthe-
sis. To test this, the localization of TIRAP was examined in
CD11b KO macrophages. TIRAP was found primarily in
membrane ruffles in 94% ± 5% of wt macrophages
(Figure 4A). In contrast, TIRAP localization was evenly dis-
persed throughout the plasma membrane of CD11b KO
macrophages; only 9% ± 3% of cells exhibited a ruffling
phenotype (Figure 4A). Time-lapse microscopy confirmed
that CD11b KO macrophages were defective for mem-
brane ruffling that is typically exhibited by wt macro-
phages (Movie S1, Movie S2, Movie S3, and Movie S4).
Whereas TIRAP-GFP and PLCd1-PH-GFP cycled through
membrane ruffles in wt macrophages (Movie S1 and
namic changes in TIRAP and PLCd1-PH localization were
not observed in CD11b KO macrophages (Movie S2 and
Movie S4). These results suggested that CD11b promotes
TIRAP enrichment at the plasma membrane by regulating
We next examined the role of CD11b in TIRAP-depen-
dent TLR4 signaling. CD11b KO macrophages exhibited
a defect in LPS-induced IL-6 production similar to TIRAP
KO macrophages (Figure 4B). In contrast, LPS-induced
expression of RANTES (a TIRAP-independent gene) did
not require CD11b (Figure 4B). Thus, of the two pathways
induced by TLR4, only TIRAP-dependent signaling is reg-
ulated by CD11b. Moreover, CD11b was not required
for IL-6 production induced by CpG DNA, which signals
through TLR9 via the TIRAP-independent pathway
Collectively, these results support a model whereby
CD11b, by triggering local PIP2 production, facilitates
the recruitment of TIRAP to the plasma membrane and
positively regulates TLR4-TIRAP-MyD88 signaling.
The ARF6 GTPase Regulates TIRAP Localization
and TLR4 Signaling
Integrins stimulate PIP2 production in part by activation of
ARF6. ARF6 is a GTPase that regulates the activity of PI 4-
phosphate 5-kinase (PI5K), which generates PIP2 at the
plasma membrane and endosomes (Honda et al., 1999).
on ARF6-positive endosomes (Brown et al., 2001). Treat-
ment of cells with cytochalasin D, or expression of ARF6
proteins from the plasma membrane to internal compart-
ments (Brown et al., 2001). TIRAP localization is sensitive
to cytochalasin D (Figures S1D and S1E), suggesting that
itiscontrolled byARF6. To examine more directly whether
calization in cells expressing wt ARF6 or an ARF6 mutant
that is unable to bind GTP (ARF6T27N) (Radhakrishna
and Donaldson, 1997). Wild-type ARF6 colocalized with
TIRAP at the plasma membrane in both MEFs and
948 Cell 125, 943–955, June 2, 2006 ª2006 Elsevier Inc.
macrophages (Figure 5A and data not shown). Expression
of ARF6T27N caused the redistribution of TIRAP into ves-
icles that colocalize with ARF6 (Figure 5B). In agreement
with previous studies (Radhakrishna and Donaldson,
1997), the clathrin adaptor a-adaptin did not colocalize
with ARF6T27N (Figure 5C). Additionally, TIRAP localiza-
tion was not affected by expression of Rab5Q79L, a regu-
(Figure5D).Thus,TIRAPtransport isregulated specifically
Peptides composed of residues 2–17 of ARF6 interfere
with processes regulated by this GTPase (Galas et al.,
1997). We designed a peptide containing these residues
fused to the cell-permeating domain of the Drosophila an-
tennapedia protein (Derossi et al., 1998) to determine the
role of ARF6 in TLR4 signaling. Treatment of MEFs with
ARF6 peptides prevented LPS-induced production of
the chemokine KC (Figure S5A). LPS-induced KC produc-
tion was unaffected by control peptides that also contain
an antennapedia sequence. The effect of ARF6 peptides
was specific in that it did not affect IL-1 receptor (IL-1R)
mediated KC production (Figure S5B). This finding is of
particular interest because MyD88-dependent signaling
pathways downstream of TLR4 and IL-1R are identical,
with the exception of TIRAP. Collectively, these results in-
dicate that ARF6 regulates TIRAP localization and thereby
controls TLR4 signaling.
TIRAP Functions to Facilitate MyD88 Recruitment
to the Plasma Membrane
The role of TIRAP in TLR signaling is enigmatic. It is not
clear why TLR2 and TLR4 require TIRAP to induce the
MyD88-dependent signaling, while other TLRs and IL-1R
trigger the same pathway independently of TIRAP. One
possibility is that TIRAP activates signaling pathways dis-
tinct from MyD88 (Mansell et al., 2006). Another possibility
is that TIRAP functions to recruit MyD88 to TLR2 and
TLR4, while other TLRs can recruit MyD88 by a mecha-
nism not dependent on TIRAP. This latter possibility is
consistent with the fact that TIRAP-dependent signaling
is always MyD88 dependent, while MyD88-dependent
stream of TLR9 or IL-1R.
To address the possible role of TIRAP in MyD88 recruit-
ment, we examined the subcellular distribution of MyD88.
GFP-tagged MyD88 was found in discrete foci scattered
throughout the cytosol (Figure 6A). Identical results were
obtained with HA- or FLAG-tagged MyD88 (data not
shown), indicating that the localization is not a conse-
quence of the tag but represents a property of MyD88 it-
self. When coexpressed with TIRAP, MyD88 was relocal-
ized to the cell periphery, where it became enriched at the
plasma membrane (Figure 6B). TIRAP therefore facilitates
MyD88 delivery to the plasma membrane. In contrast,
MyD88 was not required for TIRAP delivery to the cell sur-
face, as MyD88 KO macrophages exhibited normal TIRAP
distribution (data not shown).
To examine the mechanism by which TIRAP recruits
MyD88, we examined the requirements for the TIR and
the PIP2 binding domains of TIRAP. A TIRAP point mutant
with a nonfunctional TIR domain (TIRAP P125H) was
unable to alter MyD88 localization (Figure 6C), indicating
that TIRAP recruits MyD88 by a TIR-dependent process.
To address the role of the PIP2 binding domain in
MyD88 recruitment, we employed the SH4-TIR chimera.
Whereas both wt TIRAP and SH4-TIR were found at the
Figure 4. Crosstalk between Integrin and TLR4 Signaling by
the TLR-Independent Recruitment of TIRAP to Membrane
(A) Micrographs of macrophages of the genotype noted expressing
either TIRAP-GFP (bottom panels) or PH PLC-GFP (top panels). Both
TIRAP and PLC are concentrated at membrane ruffles in wt cells.
TIRAP and PLC membrane staining is diffuse in CD11b KO cells,
indicating a defect in the ability to synthesize large amounts of PIP2.
(B and C) Macrophages from wt, TIRAP KO, or CD11b KO mice were
treated with LPS followed by ELISA to detect the cytokine indicated.
CD11b and TIRAP KO macrophages have defects in IL-6 production,
but not RANTES production (B). Neither TIRAP nor CD11b is required
for TLR9-induced IL-6 production (C). Data presented are the mean ±
SD of an experiment performed at least two times.
Cell 125, 943–955, June 2, 2006 ª2006 Elsevier Inc. 949
plasma membrane, SH4-TIR could not relocalize MyD88
to the plasma membrane (Figure 6D), indicating that
PIP2 binding is necessary for MyD88 recruitment. The in-
ability of the SH4-TIR to recruit MyD88 provides a plausi-
ble explanation for the inability of this chimera to reconsti-
tute LPS responsiveness to TIRAP KO cells (Figure 3G). In
contrast, the PLC-TIR chimera efficiently relocalized
MyD88 to the cell surface (Figure 6E), indicating that
PIP2 binding and TIR domains of TIRAP are sufficient for
MyD88 recruitment and signal transduction. These data
indicate that TIRAP functions to control MyD88 delivery
to PIP2-containing membranes to initiate TLR4 signaling.
Targeting MyD88 to PIP2-Containing Membranes
Bypasses the Requirement for TIRAP in TLR4
The data presented above indicate that TIRAP can recruit
MyD88 to PIP2-containing membranes. If this is the only
function of TIRAP, then targeting MyD88 to PIP2-contain-
ing membranes by other means should bypass the re-
quirement for TIRAP in TLR4 signaling. However, if TIRAP
is required for the recruitment of some additional signaling
components, then targeting MyD88 to PIP2 membranes
would not bypass the requirement for TIRAP in TLR4
signaling. To address these possibilities, we endowed
MyD88 with PIP2 binding specificity by the addition of
the PIP2-specific PH domain from PLC to the C terminus
of MyD88 (MyD88-PLC). As described above, wt MyD88
is not detectable at the plasma membrane and is found
in foci scattered throughout the cell (Figure 6A). In con-
trast, MyD88-PLC was found at the plasma membrane
We next tested whether MyD88-PLC can substitute for
wt MyD88 in TLR signal transduction. Wild-type MyD88
and MyD88-PLC, but not TIRAP, restored TLR4 signaling
in MyD88 KO macrophages (Figure 6G). Interestingly,
MyD88 KO cells reconstituted with MyD88-PLC had en-
hanced sensitivity to LPS, suggesting that MyD88 recruit-
menttoPIP2-containing membranesisarate-limiting step
in the induction of TLR4 signaling (Figure 6G).
We next performed complementation experiments in
TIRAP KO macrophages. As expected, TIRAP, but not
MyD88, rescued the signaling defect of TIRAP KO cells
(Figure 6G). Strikingly, MyD88-PLC rescued the TLR4
Figure 5. ARF6 Regulates TIRAP Locali-
Micrographs of MEFs expressing TIRAP-HA (A
and B) and the indicated allele of ARF6-GFP.
Cells were stained for TIRAP and a-adaptin
(C) to identify clathrin-coated vesicles.
(A) TIRAP localizes with wt ARF6 at the cell sur-
(B) TIRAP is redistributed into ARF6+ endo-
somes by ARF6T27N.
(C) Minimal costaining is seen between a-
adaptin and ARF6T27N.
(D) MEFs expressing Rab5Q79L and TIRAP-
HA. Minimal costaining is observed between
Rab5Q79L and TIRAP, indicating that TIRAP
is selectively localized to ARF6+ endosomes.
All images are representative micrographs
from at least three independent experiments
where over 500 cells were examined per condi-
tion and >95% of the cells displayed similar
950 Cell 125, 943–955, June 2, 2006 ª2006 Elsevier Inc.
signaling defect in these cells (Figure 6G). Moreover,
MyD88-PLC rescued the signaling defect in MyD88x-
TIRAP KO MEFs (Figure 6H). Wild-type MyD88 rescued
to IL-1b treatment (a TIRAP-independent response), but
not in response to LPS treatment (Figure 6H). MyD88x-
TIRAP KO cells reconstituted with MyD88-PLC regained
the ability to respond to LPS but, surprisingly, did not re-
gain the ability to respond to IL-1b (Figure 6H), suggesting
that TLR4 and IL-1R initiate MyD88-dependent signaling
from distinct membrane subdomains, which may explain
their differential requirement for TIRAP.
These data establish that the requirement for TIRAP in
TLR4 signaling is bypassed by endowing MyD88 with
Figure 6. The Primary Function of TIRAP
Is to Recruit MyD88 to the Plasma Mem-
MyD88-HA (A), MyD88-HA and TIRAP (B),
TIRAP P125H (C), SH4-TIR (D), or PLC-TIR (E).
MyD88 is found in foci that do not label the
cell surface (identified by FITC-cholera toxin
B) (A). MyD88 is recruited to the cell surface
upon wt TIRAP (B) or PLC-TIR (E) expression.
(F) MyD88-PLC is found at the cell surface of
(G) MyD88 KO or TIRAP KO macrophages
expressing the genes indicated on the x axis.
The transfection efficiency was 30%–40% as
assessed by FACS and direct examination by
fluorescence microscopy. Cells were treated
with LPS, and the production of IL-6 was mea-
sured. Both MyD88 and MyD88-PLC restored
signaling to MyD88 KO cells; only MyD88-
PLC restored LPS responsiveness to TIRAP
KO cells. Data presented in (G) and (H) are
the mean ± SD of an experiment performed at
least two times.
(H) MyD88xTIRAP KO MEFs expressing the
genes indicated on the x axis were stimulated
with LPS (10 ng/ml to 1 ng/ml) or IL-1b (20 ng/
ml to 7 ng/ml) and analyzed for KC. MyD88-
PLC uniquely restored signaling to these cells.
Cell 125, 943–955, June 2, 2006 ª2006 Elsevier Inc. 951
a PIP2 binding domain. This indicates that TIRAP’s func-
tion is to recruit MyD88 to PIP2-containing membranes
for the initiation of TLR signaling triggered by TLR2 and
TLR4. Collectively, these data provide evidence for a cas-
cade of events that is initiated by TIRAP recruitment to the
PIP2-containing plasma-membrane subdomains followed
byMyD88recruitmentto TLR4via TIR-dependent interac-
tions with TIRAP.
TLRs use four TIR-containing adaptors, and their differen-
tial functions havebeen enigmatic. While MyD88 and TRIF
control activation of distinct signaling pathways, it has
been unclear why TIRAP and TRAM are used by some
TLRs but not by others. In addition, while MyD88 and
TRIF can be used without TIRAP or TRAM in the case of
some TLRs, TIRAP and TRAM are never used without
MyD88 or TRIF, respectively. This suggests that TIRAP
and TRAM may have related functions distinct from that
of MyD88 and TRIF. Here we used TIRAP and MyD88 to
demonstrate distinct functions of these adaptors and the
mechanism of TIRAP function.
We demonstrated that TIRAP is a peripheral-membrane
protein and that it contains an N-terminal PIP2 binding re-
gion, which is required for its function and localization to
the plasma membrane. The PIP2 binding specificity of
TIRAP may explain why this adaptor is used by TLR2 and
TLR4, both of which are localized to the plasma mem-
brane (where PIP2 is concentrated), but not by TLR3,
TLR7, and TLR9, which are localized in the intracellular
compartments devoid of PIP2. However, TIRAP is not uti-
lized by TLR5 or IL-1R, which are found on the plasma
membrane. Interestingly in this regard, MyD88-PLC could
recover TLR4 but not IL-1R signaling in MyD88xTIRAP KO
cells, suggesting that TLR4 and IL-1R signal from distinct
subdomains of the plasma membrane. Collectively, these
data demonstrate that TIRAP functions to recruit MyD88
to PIP2-rich plasma-membrane subdomains to initiate
TLR2 and TLR4 signaling. Recruitment of MyD88 to other
compartments, including endosomal compartments de-
void of PIP2, is mediated by a TIRAP-independent mech-
anism. It would be interesting to determine whether
MyD88 recruitment to these other compartments is facili-
lipids found in late endosomes where TLR9 signaling is
PIP2 has multiple functions in cellular physiology, and
its local concentrations are controlled by many signaling
tial mechanism for control of TIRAP-dependent TLR sig-
naling by other signaling pathways that control PIP2 turn-
over. In support of this notion, we found that in CD11b KO
macrophages, the localization of PIP2 binding proteins to
the cell surface is diminished and membrane ruffling is
ablated. As a result, TIRAP recruitment to the plasma
membrane is compromised. Consequently, TLR4 signal-
ing events that require TIRAP are defective in CD11b KO
cells. These results are consistent with previous studies
showing a selective defect of MyD88-dependent gene ex-
pression in response to LPS compared to TRIF-depen-
dent gene expression (Perera et al., 2001). It is possible
thatother regulatorsof PIP2 metabolism,including certain
growth-factor receptors and GPCRs, may similarly regu-
late TIRAP-dependent TLR signaling.
ARF6 acts downstream of integrins and is an estab-
lished positive regulator of PIP2 production. We found
that ARF6 controls TIRAP localization and TLR4 signaling,
its role in TLR signaling is to control TIRAP localization to
distinct membrane subdomains through the local control
of PIP2 concentration. The fact that IL-1R signaling is
not controlled by ARF6 suggests specificity to the role of
ARF6 in regulating TLR4 signaling. However, we find it un-
likely that ARF6 uniquely controls TLR4 signaling. Rather,
we suggest that the regulation of adaptor recruitment by
ARF6 may be a general strategy used to coordinate the
delivery of signaling adaptors to discrete locations of the
A number of microbial pathogens are known to manip-
ulate PI metabolism, in particular PIP2 (Pizarro-Cerda and
Cossart, 2004). The SopB phosphatase used in this study
is a substrate of the inv/spa-encoded type III secretion
system from S. typhimurium. The primary function of the
phosphatase activity of SopB is to manipulate phagocyto-
signaling by TLR4, suggesting that microbial manipulation
of PIP2 levels may also serve the purpose of altering the
TLR-dependent signaling pathways that are triggered
upon pathogen recognition.
Since the identification of TIRAP, it has been unclear
why this adaptor is required for TLR4 signaling. TIRAP
doesnotcontain adiscernable effectordomainthat would
link receptor activation with a downstream signaling cas-
cade. Unlike TIRAP, MyD88 is not membrane associated
and has to be recruited to several distinct cellular com-
partments where different TLRs reside. We found that
of TIRAP to recruit MyD88 is TIR dependent and requires
PIP2 binding activity. Interestingly, the SH4-TIR chimera
cannot recruit MyD88, despite the fact that it is localized
at the plasma membrane. This is in contrast to the PLC-
TIR chimera, which, like wt TIRAP, can relocalize MyD88
and complement LPS responsiveness to TIRAP KO cells.
Thus, PIP2 binding activity of TIRAP is required to initiate
MyD88-dependent signaling by TLR4. These data support
a model whereby PIP2 regulates TIRAP recruitment to the
plasma membrane, where TIRAP functions primarily to
control the recruitment of MyD88 to activated TLR4, by
a TIR-dependent mechanism (Figure 7).
This model helps distinguish the functions of MyD88
and TIRAP. MyD88 can be defined as a bona fide ‘‘signal-
ing adaptor’’ that bridges an activated receptor to a
kinase-dependent signaling cascade. TIRAP, in contrast,
functions primarily as a ‘‘sorting adaptor’’ that is required
952 Cell 125, 943–955, June 2, 2006 ª2006 Elsevier Inc.
for the efficient recruitment of MyD88 to a subset of TLRs.
TIRAP therefore facilitates the assembly of TLR signaling
complexes but does not participate directly in triggering
ing adaptors helps explain why no TLRs have been found
to utilize TIRAP without MyD88, since in the absence of
a signaling adaptor, the sorting adaptor has no function.
Data in support of this model come from the experiments
demonstrating that the requirement for TIRAP can be by-
passed by MyD88-PLC, which is recruited to the plasma
membrane directly by a PIP2-dependent process rather
than indirectly by a TIRAP-dependent process. We spec-
ulate that by analogy to TIRAP and MyD88, TRAM and
TRIF function as sorting and signaling adaptors, respec-
tively. TRAM may function to recruit TRIF to TLR4, but
not to TLR3, consistent with the differential requirement
for these adaptors for TLR3 and TLR4 signaling (Oshiumi
et al., 2003b; Yamamoto et al., 2003b). MyD88 partici-
pates in signal transduction by TLRs localized to distinct
subcellular compartments. While TIRAP functions to re-
cruit MyD88 to TLR4 on the plasma membrane, a distinct
mechanism is likely responsible for the recruitment of
MyD88 to TLRs on endosomes.
In summary, this study reveals a functional difference
between TLR adaptor proteins and identifies the mecha-
nism of TIRAP function in the TLR4 signaling pathway.
function allows for signal integration between TLR4 and
other receptors that control PIP2 metabolism.
Cell Culture, Immunofluorescence, and Transfections
MEFs, CHO cells, and 293T cells were transfected using Fugene-6
(Roche) according to the manufacturer’s instructions and incubated
for 24 hr at 37ºC. Where indicated, cytochalasin D (1 mM; Sigma) or
wortmannin (1 mM; Sigma) was added to the cells 30 min prior to fixa-
tion. Cells were fixed in 2% paraformaldehyde for 20 min at 25ºC and
permeabilized with 50 mM digitonin for 10 min. Samples were treated
with block buffer (1% bovine serum albumin, 50 mM ammonium chlo-
ride in PBS) for 30 min and the appropriate antibodies diluted into
block buffer. Where indicated, AlexaFluor 594-phalloidin (Molecular
Probes) was included in the block buffer to identify F-actin. Antibody
binding was detected using AlexaFluor secondary antibodies from
Molecular Probes and visualized with an Axioplan 2 epifluorescence
microscope (Carl Zeiss). 0.1 micron sections were captured with an
Axiocam HRm digital camera, and images were processed using
Adobe Photoshop. Luciferase production from transfected 293T cells
was measured by the Luciferase Assay System (Promega) according
to the manufacturer’s instructions. Bone-marrow-derived macro-
phages were prepared as described previously (Celada et al., 1984).
C57BL6 mice were purchased from Jackson Laboratory. MyD88 KO
mice were provided by S. Akira. TIRAP KO mice were described pre-
viously (Horng et al., 2002). Bone marrow from CD11b KO mice was
provided by E. Harvill and B. Kelsall. Macrophage transfections were
performed either by nucleofection (AMAXA) using the mouse macro-
phage transfection reagent or by retroviral transduction (Kagan and
Roy, 2002). Staining of macrophages was performed as described
above for MEFs except that the cells were processed 4 hr after trans-
Lipid Binding Assays
PIP strips and PIP arrays (Echelon Biosciences) were immersed in
block buffer (10 mM Tris [pH 8.0], 150 mM NaCl, 0.1% Tween 20,
0.1% ovalbumin) for 1 hr. Strips were probed for 2 hr at 25ºC with
the indicated GST fusion protein (50 ng/ml) in the presence of an
anti-GST antibody (Sigma). Blots were then washed in block buffer
three times for 10 min each and probed with an HRP-conjugated
anti-mouse IgG (Amersham) for 30 min in block buffer. Bound protein
was detected using ECL (Amersham). Lipids used to create liposomes
were purchased from Avanti. PC/PE liposomes contained a 3:1 molar
ratio of PC:PE. Liposomes containing PI species retained the 3:1 ratio
of PC:PE but also contained the indicated % of PI. The lipid species
were dissolved in a 2:1 chloroform/methanol solution in borosilicate
tubes and dried by exposure to nitrogen gas to generate a lipid bilayer.
Figure 7. Model to Describe the Regula-
tion of Adaptor Recruitment to Activated
The b2 integrin CD11b regulates the steady-
state levels of PIP2 at the plasma membrane
and the enrichment of TIRAP at membrane ruf-
fles. This is a TLR-independent process. Integ-
rin interactions with CD14 upon exposure to
LPS may lead to the activation of ARF6 and
subsequent induction of PIP2 synthesis by
PI5K. These events concentrate TIRAP at the
cell surface to facilitate the interactions be-
tween TIRAP and TLR4. The primary function
cilitates the TIR-dependent recruitment of the
‘‘signaling adaptor’’ MyD88. MyD88 recruit-
ment triggers assembly of a signaling complex
at the TLR4 TIR domain. The brackets sur-
rounding the signaling complex indicate the
possibility that induction of TLR4 signaling
requires the delivery of TLR4 to PIP2-rich do-
mains of the plasma membrane.
Cell 125, 943–955, June 2, 2006 ª2006 Elsevier Inc. 953
Dried lipids were resuspended in 300 mM sucrose to yield a final lipid
concentration of 1 mg/ml. Sedimentation assays were performed by
tein in cytosol buffer (25 mM HEPES [pH 7.2], 25 mM potassium chlo-
ride, 2.5 mM magnesium acetate, 150 mM potassium glutamate).
Binding proceeded for 15 min at 37ºC, and the samples were centri-
fuged at 100,000 3 g for 10 min. The supernatant was then aspirated,
and the pellets were examined after SDS-PAGE by silver staining. For
quantitative analysis of protein/liposome interactions, liposomes were
prepared as described above except that NBD-PC was used. Fifty mi-
crograms of liposomes was mixed with one hundred micrograms of
the appropriate GST protein in cytosol buffer and 30 ml of glutathione
Sepharose beads. Binding proceeded for 30 min at 25ºC, and then
the samples were centrifuged at 2000 3 g for 2 min. The sedimented
material was washed twice with cytosol buffer and then with 100 ml
of 10% SDS to release the bound lipids. Fluorescence recovered in
the pellet was assessed by spectrofluorimetry. Fluorescence recov-
ered by beads coated with GST represented ?150 fluorescence units
ing of liposomes to the beads.
Plasmids and Protein Purifications
Plasmids containing HA-tagged TIRAP, FLAG-tagged TIRAP P125H,
GST-TIRAP, CD4-TLR4, and the NF-kB-dependent reporter pBIIX
were described previously (Horng et al., 2001). The HA-TIRAP vector
was used as a template to clone TIRAP into pEGFP-N1 (Clontech). All
mutants of TIRAP and MyD88 were generated by PCR using HA-TIRAP
or HA-MyD88 as a template and cloning the product into pEGFP-
N1. GST fusions with mutant TIRAP were generated by cloning the
cDNA from pEGFP-N1 into pGEX-4Ti (Amersham). Retroviral vectors
encoding various TIRAP cDNAs were generated by subcloning the
GFP-tagged cDNA from pEGFP-N1 into pMSCV2.2. ARF6 and Rab5
plasmids were provided by C. Roy (Yale). PLCd1, GRP1, Fapp1, and
the PX-domain-containing plasmids were provided by W. Mothes
(Yale). INP54p and Lyn plasmids were provided by R. Isberg (Tufts).
SopB plasmids were provided by J. Galan (Yale). GST-fusion proteins
were purified from BL21 E. coli using glutathione Sepharose 4B(Amer-
sham) according to the manufacturer’s instructions. Purity of each
GST preparation was confirmed by SDS-PAGE/silver staining.
Supplemental Data include Supplemental Experimental Procedures,
five figures, and four movies and can be found with this article online
and E. Harvill for providing reagents used in this study. We would like
to thank J. Morgan for helpful discussions and assistance with lipo-
some assays. Thanks to J. Blander, G. Barton, T. Horng, D. Stetson,
L.R. Marek, and A. Neild for helpful discussions. This work is sup-
ported by fellowships from the Arthritis Foundation and the NIH
(AI07019) to J.C.K. and NIH grants AI-46688 and UO1A161360-01 to
R.M. R.M. is an Investigator of the Howard Hughes Medical Institute.
Received: April 7, 2005
Revised: December 8, 2005
Accepted: March 21, 2006
Published: June 1, 2006
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