(Received for publication, August 30, 1995; and in revised form, October 24, 1995)
From the
Through data base searches, we have discovered new proteins that
share homology with the signaling domain of the type I interleukin-1
receptor (IL-1RI): human ``randomly sequenced cDNA 786''
(rsc786), murine MyD88, and two partial Drosophila open
reading frames, MstProx and STSDm2245. Comparisons between
these new proteins and known IL-1RI homologous proteins such as Toll,
18-Wheeler, and T1/ST2 revealed six clusters of amino acid similarity.
We tested the hypothesis that sequence similarity between the signaling
domain of IL-1RI and the three mammalian family members might indicate
functional similarity. Chimeric IL-1RI receptors expressing the
putative signaling domains of T1/ST2, MyD88, and rsc786 were assayed by
three separate IL-1 responsive assays, NF-B, phosphorylation of an
epidermal growth factor receptor peptide, and an interleukin 8
promoter-controlled reporter construct, for their ability to transduce
an IL-1-stimulated signal. All three assays were positive in response
to the T1/ST2 chimera, while the MyD88 and rsc786 chimeras failed to
respond. These data indicate that the sequence homology between IL-1RI
and T1/ST2 indicates a functional homology as well.
Interleukin-1 (IL-1) ()is an important component of
the mammalian inflammatory response and is produced by many different
types of cells following tissue injury and infection (1) . The
receptors and ligands of the IL-1 pathway have been well defined (for
review see (2) and (3) ). Three ligands, IL-1
,
IL-1
, and IL-1 receptor antagonist (IL-1ra) bind three forms of
IL-1 receptor, an 80-kDa type I IL-1 receptor (IL-1RI)(4) , a
68-kDa type II IL-1 receptor (IL-1RII)(5) , and a soluble form
of the type II IL-1R (sIL-1RII)(6) . The cytoplasmic or
signaling domain of the human IL-1RI consists of 213 amino acids and
has been shown to be essential for cellular responses to IL-1 in
vivo(7) , while the type II receptor has a cytoplasmic
domain of only 29 residues and does not appear to transduce a
signal(8) . The interactions between the ligands and receptors
play an essential role in the stimulation and regulation of the
IL-1-mediated host response to injury and infection. Cells expressing
IL-1RI and treated with IL-1
or IL-1
respond in several
specific ways, including stimulating nuclear localization of the
rel-related transcription factor, NF-
B (for review, see (9) ), activation of protein kinases of the mitogen-activated
protein kinase superfamily that phosphorylate residue threonine 669
(Thr-669) of the epidermal growth factor receptor
(EGFR)(10, 11, 12) , and stimulation of
transcription of the IL-8 gene(13) .
We explore the possibility of another level of complexity in IL-1 signaling, the existence of IL-1R-like proteins and their role in IL-1 and IL-1-like signal transduction pathways.
The IL-1RI cytoplasmic (signaling) domain shares significant homology to the cytoplasmic region of the Drosophila melanogaster transmembrane protein Toll(14) . Toll is a transmembrane protein involved in establishment of dorsal/ventral polarity in the Drosophila embryo. Site-directed mutagenesis and deletion analysis have demonstrated that the cytoplasmic domain of IL-1RI and, in particular, residues conserved between IL-1RI and Toll are essential for transduction of intracellular IL-1-stimulated signals(7, 15, 16) .
In addition to Toll,
four other characterized cDNAs have been recognized as homologous to
the cytoplasmic domain of the type I IL-1 receptor. These include
T1/ST2(17, 18, 19) , the IL-1 receptor
accessory protein (IL-1R AcP)(20) , the tobacco N gene(21) , and the Drosophila protein
18-Wheeler(22) . T1/ST2 is a transmembrane protein that has
been characterized as a novel primary response gene expressed in
BALB/c-3T3 cells. The IL-1R AcP is also a transmembrane protein and was
identified through a monoclonal antibody that blocked the binding of
IL-1 to IL-1RI but recognized a protein distinct from the receptor
itself. Both T1/ST2 and IL-1R AcP share homology to IL-1RI
intracellularly and extracellularly. The tobacco N gene
encodes a protein with an amino-terminal domain that has significant
homology to the cytoplasmic domain of Toll and IL-1RI. Interestingly,
tobacco plants carrying the N gene are resistant to tobacco
mosaic virus by means of a ``hypersensitive response'' that
resembles a localized inflammatory response similar to that induced by
IL-1 in mammalian cells. 18-Wheeler has not been extensively
characterized but is thought to encode a heterophilic cell adhesion
molecule required for morphogenesis.
Recognizing this growing family, we conducted data base searches to identify other cDNAs with sequence homology to IL-1RI and found four. We then asked whether the sequence similarity observed in these novel proteins and in the previously identified T1/ST2 protein indicates a similarity in signaling function as well.
Figure 2: Junction point sequences of chimeric receptor structures. A partial murine IL-1R type I sequence containing the transmembrane/cytoplasmic region is shown at top (underlined). In most constructs, the murine IL-1R portion ends three residues into the cytoplasmic domain (KIF . . . ) (the murine IL-1RI sequence was changed from KVF to KIF to facilitate the chimeric constructions; see ``Experimental Procedures''). In one construct, MyD88-E, the murine IL-1R sequence extends further (SKAS . . . ) as shown in the second line from the top, underlined. The protein sequence at the junction point is shown for each chimeric construct. Bold residues are not present in the native sequence but were inserted into the chimeric receptor at the junction point as shown.
By data base searching, we have discovered two new mammalian members of the IL-1R-like family (see alignment in Fig. 1): a murine protein, MyD88(27) , and a novel human gene, rsc786 (``randomly sequenced cDNA'') ( (28) and GenBank accession no. D13637). MyD88 was originally isolated and characterized as a myeloid differentiation primary response gene, induced upon IL-6-stimulated differentiation of a myeloblastic leukemia cell line, M1, to macrophages (note that while the chimeric receptor transfection experiments described in this paper were in progress, two other groups also noted the sequence similarity between MyD88 and IL-1RI(29, 30) ). The MyD88 gene predicts a protein of 243 residues lacking both a transmembrane region and a signal sequence. The region of homology to the IL-1RI cytoplasmic domain is restricted to the carboxyl-terminal 150 residues of MyD88. The biology of human rsc786 has not yet been investigated, but the nucleotide sequence predicts a transmembrane protein with a cytoplasmic domain of about 175 residues that shows homology to IL-1RI. The extracellular portion of rsc786 does not resemble IL-1RI but contains leucine-rich repeats similar to those of Toll and 18-Wheeler.
Figure 1:
IL-1R family homology line-up.
Alignment of the cytoplasmic domain of the human IL-1 receptor type I
with the corresponding segments of homology found in murine T1/ST2,
murine MyD88, human rsc786, murine IL-1R accessory protein, and the D. melanogaster genes toll, 18-wheeler, and mstProx. The alignment was initially performed using the UWGCG
program Pileup (see program manual for the Wisconsin Package, version
8.0 Genetics Computer Group) and subsequently adjusted subjectively by
the authors. Consensus sequences are indicated for the mammalian
members of the family (consensus = three out of five members for
a specific residue, four out of five members for a functional grouping, top line) and for all eight family members included here
(consensus = five out of eight members for a specific residue,
six out of eight members for a functional grouping, bottom
line). *, charged/polar residues (as shown in single-letter codes)
(D, E, N, Q, K, R, H, T, S); @, aliphatic residues (I, L, M, V);
, aromatic residues (F, Y, W). The dark shaded boxes indicate areas of greatest sequence similarity, and lighter
shaded boxes indicate extended regions of slightly less sequence
conservation. Arrow heads above the sequence indicate the
positions of introns where known (filled arrow heads, human
IL-1 receptor(39) ; open arrow heads, Fit1, the rat
homolog of T1/ST2(40) .
In addition to the mammalian proteins, our data base searches also identified two additional Drosophila IL-1RI homologs. These new IL-1RI family members are the genomic fragment MST84D ( (31) and GenBank accession nos. X67703, S74217, and S74219) and STSDm2245 (``sequence-tagged site,'' GenBank accession no. G01378). Both of these sequences contain short ORFs of unknown function that show homology to the IL-1RI cytoplasmic domain. MST84D also encodes several sperm proteins in addition to the partial IL-1RI-like ORF proximal to the rest of the sequences (we refer to this ORF as MstProx for ``proximal to the Mst locus''). Using a probe derived from this sequence, we screened a Drosophila genomic library and isolated and sequenced clones encoding the MstProx gene. We found that MstProx did indeed contain an ORF encoding a transmembrane receptor protein similar both extracellularly and cytoplasmically to Toll and 18-Wheeler and, of the mammalian family members, most closely related to rsc786. The portion of MstProx homologous to the cytoplasmic domain of IL-1RI is shown in Fig. 1.
An amino acid multiple sequence alignment of the IL-1R-like portion of the IL-1R family members is shown in Fig. 1. Comparison of the homologous portions of these proteins reveals six distinct regions of conservation ranging in size from 8 to 16 residues within the IL-1RI cytoplasmic domain of 213 residues (see Fig. 1and ``Discussion'').
To investigate whether the sequence conservation observed among these genes also reflects a functional conservation with regard to IL-1 signaling, we constructed IL-1RI chimeric receptors for three of the mammalian IL-1R family members and tested the chimeric receptors for their ability to transduce an IL-1-mediated signal in vivo.
The chimeric receptors were
tested for their ability to transduce an IL-1 signal in vivo in a transient transfection system. COS7 cells transfected with
the chimeric constructs were pretreated with a polyclonal antibody (P3)
to the human IL-1RI to block IL-1 binding to the endogenous primate
IL-1R and then assayed for IL-1 response via the transfected chimeric
receptors. We utilized a different species (murine) of IL-1RI for the
extracellular and transmembrane portions of our chimeric receptors,
which is not prevented from binding and responding to IL-1 by the
polyclonal antibody (see data below). Expression levels of the various
chimeras were determined by radioimmunoassay (see ``Experimental
Procedures'' and Table 1). The ability of the various
chimeric receptors to mediate activation of NF-B, Thr-669
phosphorylation, or induction of the IL-8 reporter construct through
IL-1 stimulation is compared in each assay with that of the
non-chimeric native murine IL-1RI transfected into COS7 cells in
parallel.
Figure 3:
Activation of NF-B by IL-1 in COS7
cells transfected with receptor chimeras. A, NF-
B gel
shift assays using nuclear extracts harvested from cells transfected
with empty vector (lanes 1-4), T1/ST2 chimeric receptor (lanes 5 and 6), and murine IL-1RI (lanes 7 and 8) as shown above each lane. Presence of the
polyclonal serum P3 at a 1:100 dilution (raised against human IL-1R
type I) and/or huIL-1
(final concentration = 1.0 ng/ml) is
indicated by a ``+'' above the lane. Lane 9 is
the same as lane 3 except 200 ng of cold double-stranded
synthetic oligonucleotide NF-
B probe was included as competitor. Lane 10 is a binding reaction without nuclear extract (probe
only). B, NF-
B gel shift assays using nuclear extracts
harvested from cells transfected as shown above each lane: empty vector (lanes 1-4), murine IL-1RI (lanes 5 and 6), and five different MyD88 chimeric receptors (lanes 7 and 8, MyD88-A; 9 and 10, MyD88-B; 11 and 12, MyD88-C; 13 and 14,
MyD88-D; 15 and 16, MyD88-E). For details of
constructs, see Fig. 2. Lanes 17 and 19 are
identical to lane 3 except 200 ng of cold double-stranded
synthetic oligonucleotide NF-
B probe was included as competitor in
the sample run in lane 19. Lane 18 is a binding
reaction without nuclear extract (probe
only).
When the T1/ST2 chimeric receptor
was transfected into COS7 cells and stimulated with IL-1 in the
presence of P3 antibody, a clear NF-B response was obtained (see Fig. 3A, lane 6), indicating that the T1/ST2
cytoplasmic domain is capable of activating NF-
B DNA binding
activity in response to IL-1 approximately as well as the full-length
murine IL-1RI (Fig. 3A, compare lanes 6 and 8). Therefore, by functional conservation as well as sequence
homology, T1/ST2 is clearly a member of the IL-1R family.
We also
tested several chimeric constructs for MyD88 and rsc786. Lanes
7-16 of the gel in Fig. 3B are samples
transfected with five different MyD88 chimeric receptors (see Fig. 2for structures). Although some MyD88 chimeras appear to
stimulate NF-B to a small degree in response to IL-1, none of
these stimulations were similar in magnitude to those observed by the
T1/ST2 chimera or native murine IL-1RI nor were they consistently
reproducible. It should be pointed out that the MyD88 chimeric
receptors were expressed at a lower level than the native IL-1RI and
the T1/ST2 chimeras (Table 1). All of the rsc786 chimeric
receptors shown in Fig. 2were tested for their ability to
stimulate NF-
B in response to IL-1, but all failed to activate
NF-
B (data not shown). All expressed as well as or better than the
IL-1R positive control (data not shown).
To investigate the Thr-669 kinase activity stimulated by IL-1 in
transfected cells, we measured the level of phosphorylation of a
synthetic peptide consisting of residues 663-673 of
EGFR(12) . Cytoplasmic extracts from IL-1-stimulated or
unstimulated cells transfected with empty vector, full-length murine
IL-1RI, or chimeric receptors were assayed. Extracts were incubated
with EGFR peptide as substrate for activated Thr-669 kinase in the
presence of [-
P]ATP. Results are expressed
as the ratio of Thr-669 kinase activity detected in cells after IL-1
stimulation to the value observed in unstimulated cells. Fig. 4A shows the results of Thr-669 kinase assays from
cells transfected with empty vector, full-length murine IL-1RI, and the
T1/ST2 chimeric receptor. Open bars indicate samples treated
with the P3 antiserum alone, and shaded bars indicate
pretreatment with P3 followed by a 10-min stimulation with IL-1 (human
IL-1
, 10 ng/ml). Though the presence of P3 alone induces a slight
increase in the Thr-669 kinase activity above the background value of
untreated cells, treatment with IL-1 in the absence of antibody
produces a significantly elevated level of Thr-669 kinase activity.
Pretreatment with P3 followed by stimulation with IL-1 reduces the
Thr-669 kinase activity in vector-transfected cells to a value
comparable with P3 alone (Fig. 4A, sample 1),
indicating that stimulation via endogenous COS7 receptors is blocked. Fig. 4A, sample 2, shows that cells
transfected with full-length murine IL-1 receptor respond to IL-1 via
activated Thr-669 kinase despite pretreatment with the P3 antiserum.
Figure 4:
Activation of Thr-669 kinase by IL-1 in
COS7 cells transfected with receptor chimeras. 2 days
post-transfection, confluent cultures of COS7 cells were incubated in
the presence of the P3 polyclonal anti-human IL-1RI antibody for 10 min (open boxes) or were incubated with P3 antibody followed by
addition of huIL-1 at 10 ng/ml (shaded boxes). Kinase
activity is expressed relative to untreated cells. A, cells
transfected with vector alone (sample 1), murine IL-1RI (sample 2), or the T1/ST2 chimera (sample 3). Each
point represents the mean of three to five independent experiments
assayed in duplicate. B, cells transfected with vector alone (sample 1), with murine IL-1RI (sample 2), or with
four different murine MyD88 chimeras (3, MyD88-A; 4,
MyD88-D; 5, MyD88-B; 6, MyD88-E). For details of
constructs see Fig. 2. Each point is the result of a
representative experiment assayed in
duplicate.
The results with the T1/ST2 chimera are given in Fig. 4A, sample 3. Cells transfected with this chimera, pretreated with P3 antiserum and then stimulated with IL-1, respond to IL-1 with a 3-fold increase in Thr-669 kinase activity (a typical level of response)(26) , clearly indicating that the transfected T1/ST2 chimeric receptor is capable of activating the Thr-669 kinase pathway after IL-1 stimulation. Thus, this second type of assay, Thr-669 kinase activation, also provides functional evidence that the T1/ST2 protein is a member of the IL-1R family.
In Fig. 4B (samples 3 through 6) we show that pretreatment with P3 followed by IL-1 stimulation of cells transfected with our various MyD88 chimeric receptor constructs does not increase Thr-669 kinase activity significantly above the level observed with P3 alone, suggesting that the MyD88-transfected chimeric receptors are unable to respond to IL-1 stimulation by activation of Thr-669 kinase. Similarly, none of the rsc786 chimeric receptors were able to activate the Thr-669 kinase pathway after IL-1 stimulation (data not shown).
Figure 5:
Activation of IL-8 promoter by IL-1 in
COS7 cells transfected with receptor chimeras. 1 day post-transfection,
COS7 cells transfected with the indicated receptor chimera were treated
with the P3 polyclonal anti-human IL-1RI antibody followed by addition
of huIL-1 at 1.0 ng/ml (stippled boxes). Solid boxes indicate identical transfected cell samples similarly treated,
except no P3 or IL-1 was added. Previous experiments indicated that P3
treatment alone had no measurable effect on background signal in this
assay (data not shown). 2 days post-transfection, cells were assayed
for response to IL-1 with the radioimmunoassay as described under
``Experimental Procedures.'' Results are expressed as total
cpm averaged from two duplicate wells.
Many proteins from diverse systems show homology to the
cytoplasmic domain of the IL-1RI. This expanding IL-1RI-like family
includes murine and human proteins, Drosophila proteins, and a
plant (tobacco) protein. We have identified two new mammalian members
and two new Drosophila members. The new mammalian family
members include a murine protein MyD88 (27) and a human gene,
rsc786 (28) . MyD88 was originally isolated and characterized
as a myeloid differentiation primary response gene, involved in
terminal differentiation of M1D+ cells (differentiation inducible
myeloblastic leukemia cell line) to macrophages upon IL-6 stimulation.
rsc786 is essentially uncharacterized. Another murine member, T1/ST2,
was previously characterized as a novel primary response gene expressed
in BALB/c-3T3 cells(17, 18, 19) . The
transmembrane protein muIL-1R AcP (20) has homology to both the
type I and type II IL-1R. IL-1R AcP has recently been shown to increase
the affinity of IL-1RI for IL-1 and may be involved in mediating
the IL-1 response.
The D. melanogaster members of the
IL-1RI family include the transmembrane protein
Toll(32, 33) . The IL-1RI homologous portion of Toll
has been shown to be required for its role in establishing the
dorsal/ventral polarity of the embryo during Drosophila development(14, 33) . Stimulation of Toll by its
presumptive ligand, Spatzle, causes nuclear localization of a Drosophila homolog of NF-B, Dorsal, and activation of the
serine/threonine protein kinase, Pelle(34, 35) . It
has also been shown that the portion of Toll with homology to IL-1RI is
necessary and sufficient for activating the pathway that results in
nuclear localization of Dorsal in Schneider cells(36) .
Clearly, the sequence homology observed between Toll and IL-1RI and the
homologous components of their respective signal transduction pathways
also reflects an evolutionarily conserved functional similarity. Here,
we also present two additional Drosophila family members,
MstProx and STSDm2245, both Drosophila genomic
fragments that contain partial ORFs capable of encoding proteins
homologous to the signaling domain of IL-1RI. Cloning and sequencing
more of the MstProx ORF has confirmed that it is a new member of the
IL-1R family (see Fig. 1).
Besides the mammalian and Drosophila proteins there is also a noteworthy plant member of the IL-1RI family, the tobacco N gene. The N gene encodes a protein with an amino-terminal domain that has significant homology to Toll and the cytoplasmic domain of the IL-1RI(21) . Introduction of the N gene into tobacco mosaic virus-sensitive strains of tobacco confers resistance to tobacco mosaic virus via the ability to mount a hypersensitive response to the virus at the site of infection (21) similar to the acute inflammatory response pathway stimulated by injury and mediated by IL-1 in mammals.
We
have investigated whether the sequence similarities observed between
four of the mammalian members of the family indicate functional
similarities (the muIL-1R AcP sequence was published after these
studies were completed). A homology line-up of selected IL-1R family
members is shown in Fig. 1. Comparison of the homologous
portions of these proteins reveals six distinct regions of conservation
ranging in size from 8 to 16 residues within the IL-1RI cytoplasmic
domain of 213 residues. The most COOH-terminal of the homology domains
has been defined by deletion analysis and site-directed mutagenesis of
the native IL-1RI to be essential for certain IL-1-mediated signals (15) . ()Two of the residues in this domain, Phe-530
and Trp-531, that were defined as essential in the human IL-1RI by
site-directed mutagenesis are also conserved in all members of the
family (see Fig. 1). The significance of these homologies is
unknown, but it is possible that sequence similarities in certain
domains indicate that these domains are conserved because they must
interact with some of the same components of the signal transduction
pathway as the IL-1RI. In addition to activation of signal transduction
pathways, these interactions might alternatively provide regulatory
roles such as amplification or abrogation of the IL-1 signal.
We
have shown that a chimeric receptor consisting of the extracellular and
transmembrane portions of the murine IL-1RI and the cytoplasmic domain
of T1/ST2 is indeed capable of transmitting an IL-1-mediated signal to
the cell as assayed by activation of NF-B, Thr-669 kinase, and an
IL-8 promoter-controlled reporter construct. Activation of NF-
B
and Thr-669 kinase indicates that the T1/ST2 cytoplasmic domain is
capable of activating at least two separate signaling pathways that
respond to IL-1(26) . This demonstrates that the sequence
homology observed between the cytoplasmic domain of the IL-1RI and the
T1/ST2 protein is functionally significant.
It is not clear why
MyD88 and rsc786 were incapable of transmitting a significant
IL-1-mediated signal via their IL-1RI chimeric receptors as assayed by
NF-B, Thr-669 kinase, and IL-8 promoter activation. One
possibility might be that the chimeric receptors, in the context of the
MyD88 and rsc786 proteins, were simply not functional constructs.
Though we did address this possibility by making several versions of
each of these chimeras, none of them signaled in our assays in any
significant way. Another explanation is that the MyD88 and rsc786
chimeric receptors are activating the cell in response to IL-1 but not
through the NF-
B, Thr-669 kinase, or IL-8 promoter pathways. MyD88
and rsc786 may interact with some of the same proteins that the native
IL-1RI and the T1/ST2 protein do but may stimulate these proteins to
signal in subtly different ways not detected in our assays. Perhaps one
or more of the factors responsible for interacting with and signaling
from the IL-1R-like domain of MyD88 and rsc786 are not found in COS7
cells. We find this unlikely since these cells are IL-1 responsive, and
interspecies conservation is sufficient enough to allow signaling
through transfected murine IL-1RI and the murine T1/ST2 chimeric
receptor. A further possibility is suggested by the recent report of an
IL-1 receptor accessory protein(20) . Perhaps a similar
protein, capable of interacting with the extracellular portion of
rsc786 or the amino-terminal domain of MyD88, is required for signaling
by these proteins. Such an accessory protein, even if present in COS
cells, would not be expected to interact with the extracellular portion
of the IL-1R, and it is possible that the endogenous COS cell IL-1R AcP
might not be able to complement the rsc786 and MyD88 signaling domains
for signaling function. It is also possible that MyD88 and rsc786 do
not serve in a signaling capacity. Instead, they may function to
abrogate IL-1 signaling by forming non-productive complexes with
components of the IL-1 signaling pathway, serving to inhibit or
regulate the cell's ability to respond to IL-1. Interestingly,
T1/ST2, the family member most closely related to the IL-1RI in
sequence as well as function, also maps to the same chromosomal
location(37, 38) , while rsc786 is more distantly
related in sequence, does not appear to be functionally conserved as
indicated by our investigation, and also maps to a separate linkage
group than IL-1RI. (
)
Clearly, the IL-1RI homologous
portion of T1/ST2 (when present as part of a chimeric IL-1 receptor) is
capable of transducing an IL-1 response similar to that transduced by
the native IL-1RI. The specificity of this signal could be regulated
through expression patterns of native T1/ST2 receptor and its cognate
ligand that may differ from the expression patterns of IL-1RI and
IL-1 and -
. It remains to be shown how the signals transduced
by T1/ST2 and other possible signals (as yet uncharacterized)
transduced by IL-1RI family members such as MyD88 and rsc786 fit into
the overall scheme of IL-1 signal transduction.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U42425[GenBank].