(Received for publication, May 12, 1995; and in revised form, August 18, 1995)
From the
The ST2/T1 receptor, a homologue of the interleukin 1 receptor (IL-1R), was expressed in COS and Drosophila S2 cells as a human IgG-Fc fusion protein. While a type I IL-1RFc fusion protein bound human IL-1 in vitro, the ST2Fc fusion protein did not. Furthermore, IL-1 stimulated a synthetic interleukin-8 promoter reporter gene that was cotransfected into Jurkat cells with a full-length IL-1R type I (IL-1R1) or a chimeric receptor composed of the IL-1RI extracellular domain and ST2 intracellular domain. In contrast, IL-1 did not stimulate the interleukin-8 promoter when cotransfected with a full-length ST2 or an ST2 extracellular/IL-1R intracellular domain fusion protein. Both IL-1RI and the IL-1R/ST2R chimeric receptor also activated a receptor-associated kinase and CSBP/p38 MAP kinase. Using ST2Fc receptor, we have identified, through receptor precipitation, receptor-dot blot and surface plasmon resonance, a putative ligand of ST2 secreted from Balb/c 3T3 and human umbilical vein endothelial cells. The putative ligand was also able to stimulate CSBP/p38 MAP kinase through the ST2 receptor. These results suggest that the ST2 is not an IL-1 receptor but rather has its own cognate ligand.
ST2/T1 was identified as a late response gene induced by either serum or overexpression of v-mos or Ha-ras oncogenes in Balb/c 3T3 or NIH 3T3 cells(1, 2) . The ST2/T1 (designated ST2 hereafter) gene encodes a 38.5-kDa peptide that is secreted from 3T3 cells as a heavily glycosylated protein of 50-60 kDa(3) . Subsequently, an alternatively spliced form of murine ST2 and rat Fit-1 were cloned that encoded a single transmembrane-spanning protein retaining the extracellular domain found in the soluble ST2 receptor(4, 5) .
ST2 belongs to the immunoglobulin
superfamily and bears significant amino acid identity (25%) to the
extracellular portion of both type I and type II interleukin 1
receptors (IL-1R)(
)(2) . Some of the intracellular
residues that are required for signal transduction through the IL-1R
and are conserved in the Drosophila Toll protein are also
found in the membrane form of ST2(6, 7) . Furthermore,
the gene for ST2 was mapped to mouse chromosome 1 closely
linked to the il-1r locus containing both type I and type II
receptor genes in support of their common ancestry(8) .
Both
soluble and membrane bound ST2 receptors are predominantly expressed in
hematopoietic tissues in vivo and in established
hematopoietic, epithelial, and fibroblast cell lines in
vitro(5, 9) . This expression pattern partially
overlaps with that of the type I and type II
IL-1Rs(10, 11) . Soluble IL-1Rs have also been
identified from various sources (12, 13, 14) including vaccinia virus. The
vaccinia virus coded protein, B15R, binds to IL-1 and has been
shown to be involved in viral pathogenesis by attenuating host response
elicited due to IL-1 production(15, 16) . Thus,
soluble IL-1Rs may modulate IL-1-mediated responses by sequestering it
and inhibiting its proinflammatory responses(17) . The ST2
receptor may play a similar role for its ligand.
We wished to determine if ST2 is a receptor for IL-1 or some other ligand in order to further understand its function. In the present work we have expressed a soluble and membrane form of ST2 and show that it is not a receptor for IL-1. Instead, we show for the first time that the ST2 receptor binds a previously uncharacterized ligand and signals in a manner similar but not identical to IL-1.
The sequence for the intracellular portion of MST2 was also amplified by reverse transcriptase-mediated PCR using the forward primer 5`-AAGTTCCAGCAATGACATGGATTG-3` corresponding to codons 280-287 of membrane MST2 containing a 5` XcmI restriction site. The reverse primer, 5`-GTCTCTAGATCACAAGTCCTCTTCAGAAATGAGCTTTTGCTCAAAGTGTTTCAGGTCTAAGCATGCCTTG-3`, corresponding to codons 559-567, contained a sequence for the myc epitope (20) 9E10 (EQKLISEEDL) and a stop codon followed by an XbaI restriction site. After confirmation of sequence, the PCR product was cloned in place of the 3` end of the soluble ST2 and Fc sequence in the COSFc vector between the XcmI and XbaI sites, yielding the 567-amino acid full-length COSMST2R. The MST2/IL-1R chimera was constructed by amplifying the intracellular portion of IL-1R from amino acids 330-567 with the following primers: 5`-CCAATTGATCACACTAATTTCCAGAAGCACATGATT-3` (codons 330-337 with in frame BclI restriction site) and 5`-CTTTCTAGATCACAAGTCCTCTTCAGAAATGAGCTTTTGCTCCCCGAGAGGCACGTGAGCCTCTCTTTGCAGTTT-3` (codons 559-567 followed by the myc epitope, stop codon, and a XbaI site). The PCR product was used to replace the intracellular portion of the ST2 receptor in COSMST2R from BclI to XbaI. The final product MST2/IL-1R contained amino acids 1-331 of the ST2 receptor fused to amino acids 330-568 of the IL-1R.
The full-length IL-1R construct and the
truncated version, IL-1R360, which lacks all but five amino acids
after the transmembrane domain, was a kind gift from Dr. R. Einstein.
For the IL-1R/MST2R chimera, the intracellular portion of ST2 receptor
was amplified using the forward 5`-AAAGCTTCAGATGGCAAGCTCTACGATGCGTAC-3`
(codons 378-385 with a HindIII site) and the reverse
primer
5`-CAGGTGACCTCACAAGTCCTCTTCAGAAATGAGCTTTTGCTCAAAGTGTTTCAGGTCTAAGCATGCCTTGCCACT-3`
(codons 557-567 with myc epitope, stop codon, and a BstEII site).
The intracellular portion of IL-1R from the HindIII site (amino acid 378 onward) was then replaced with
the PCR-amplified intracellular portion of MST2 (from amino acid 378 to
567). The expression vector for these receptors contain a
cytomegalovirus promoter and a bovine growth hormone polyadenylation
sequence. The authenticity of each construct was confirmed by transient
expression of the corresponding proteins in COS cells as analyzed by
immunoprecipitation from S metabolically labeled cells.
The IL-8 promoter from -185 to +44 (21, 22) was made by PCR-mediated gene synthesis containing a 5` HindIII and a 3` XbaI site. The PCR product was first cloned into PCRII to confirm the sequence. The insert was then removed by digestion with HindIII and XbaI and subcloned into corresponding sites in the PCATE vector (Promega, Madison, WI), which contains a bacterial chloramphenicol acetyl transferase (CAT) gene cassette, to generate the reporter plasmid IL-8P/CAT.
For receptor precipitation, 1-2.5 µg of various
purified Fc fusion proteins were mixed with S-labeled 3T3
conditioned medium and 20 µl of protein A-agarose (Life
Technologies, Inc.) and incubated overnight at 4 °C. Protein
A-agarose pellets were collected by centrifugation and washed 3 times
with PBSTDS buffer (PBS containing 1% Triton X-100, 0.1% SDS, and 0.01%
sodium deoxycholate). Pellets were solubilized in sample buffer and
resolved through SDS-PAGE, fixed, dried, and visualized by
autoradiography. In some experiments the pH of 3T3 conditioned medium
was lowered to 3.0 by 0.1 M HCl and then immediately
neutralized before the receptor precipitation assay. For
immunoprecipitation, polyclonal rabbit antiserum (preimmune or immune)
generated against Drosophila-expressed ST2 was used instead of
Fc fusions. Cytoplasmic extracts from 3T3 cells were made by washing
the cells in ice-cold PBS and lysis in PBSTDS containing 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 5 units/ml
aprotinin for 20 min on ice followed by centrifugation at 15,000
g for 20 min to remove nuclei and cell debris. For
cross-linking experiments, the homobifunctional cross-linker
disuccinimidyl suberate (Pierce) was added to preformed ligand-receptor
complexes in binding buffer at a final concentration of 1 mM for 30 min, followed by the addition of Tris-HCl, pH 7.4 to 10
mM. Sample buffer was then added, and cross-linked product was
resolved by SDS-PAGE. Endogenous soluble ST2 protein was partially
purified by immunoprecipitation from concentrated 3T3 cell conditioned
medium using anti-ST2 polyclonal antibody. ST2 was eluted from
antibody-agarose beads using 10 mM sodium carbonate buffer, pH
11, neutralized to pH 7.4 and used for cross-linking studies.
For the CSBP/p38 kinase assay, 2 10
cells were transfected with different receptor constructs, and 48
h later one-half of cells were stimulated with IL-1
or -
or
concentrated 3T3 conditioned medium for 5-20 min and lysed, and
immune complex kinase assays were carried out for 30 min at 30 °C
as in Raingeaud et al.(25) using anti-CSBP polyclonal
antibodies(26) . The kinase reaction was stopped by the
addition of SDS-PAGE buffer, boiled for 2 min, and resolved by
SDS-PAGE. The bottom half of the gel containing the myelin basic
protein (MBP) was prepared for autoradiography, and the top half of the
gel containing CSBP was prepared for immunoblotting. The radioactivity
in each band was quantitated in a Betascope. For the
receptor-associated kinase assay, cells were lysed and
immunoprecipitated with a monoclonal anti-IL-1RI antibody (Genzyme,
Cambridge, MA), and the kinase assay was performed as in Croston et
al.(27) with the exception that MBP (5 µg) was also
added as an exogenous substrate in the kinase assay. These experiments
were performed with cells stimulated with both IL-1
and -
,
and comparable results were obtained. Therefore, the data only for
IL-1
are presented.
Figure 1: Immunoblot of various purified recombinant Fc fusion proteins. 200 ng of HST2Fc, MST2Fc, and human IL-1RFc proteins from COS (lane 1-3) and Drosophila cells (lane 4-6) were resolved by SDS-PAGE, transferred to nitrocellulose membrane, probed with horseradish peroxidase-conjugated goat anti-human Fc antibody, and developed by ECL. The position of the molecular weight markers is indicated on the left.
Figure 2:
Binding of Fc fusion proteins to IL-1.
Binding of increasing concentrations of I-IL-1
(specific activity, 25,000 cpm/ng) to human IL-1RFc, HST2Fc, and
IgG.
Figure 3:
Receptor-mediated precipitation of I-IL-1
by human IL-1RFc and HST2Fc fusions. A, 100 ng of Fc fusion proteins immobilized on 20 µl of
protein A-Sepharose were incubated for 3 h with 2 ng of
I-IL-1
(specific activity, 60,000 cpm/ng) without
any competitor or with 2 µg each of various unlabeled competitors
as indicated. After incubation the beads were collected by
centrifugation, washed 3 times to remove unbound ligand, counted in a
counter (A), and then resuspended in sample buffer and
resolved by SDS-PAGE (B).
Figure 4:
Cross-linking of iodinated IL-1 and
IL-1
to ST2 and IL-1R. ST2 immunopurified from 3T3 condition
medium (3T3-ST2, lanes 1 and 2 and lanes
5 and 6) or 10 ng of purified sIL-1R (lanes 3 and 4 and lanes 7 and 8) or 100 ng of
HST2Fc (lanes 9-12) or 10 ng of human IL-1RFc (lanes
13 and 14) were incubated with 2 ng of iodinated IL-1s
(specific activity, 60,000 cpm/ng) as indicated. After 3 h at room
temperature, the homobifunctional cross-linker disuccinimidyl suberate
was added to a 1 mM final concentration and incubated for an
additional 30 min. Cross-linked products were analyzed by SDS-PAGE and
autoradiography. Even numbered lanes show cross-linking in the
presence of 1000-fold molar excess of unlabeled
ligands.
Figure 5: Binding of ST2 and IL-1R Fc fusion proteins to various IL-1 s in BIAcore. Binding (response units) of various IL-1 ligands and anti-ST2 immune (IM-ST2-Ab) or preimmune (PI-Ab) serum to IL-1RFc (black brick bar) or HST2Fc (white brick bar) immobilized on Biacore chip surface through protein A.
Figure 6:
Schematic representation of various
receptor constructs and the results of CAT and kinase assays. A, diagram of full-length MST2R and human IL-1R
(IL-1R) and various chimeras: IL-1R/360, IL-1R with all but 5
amino acids deleted from the intracellular domain; IL-1R/MST2R, the
extracellular and the transmembrane portion of IL-1R fused to the
intracellular domain of ST2 receptor; and MST2R/IL-1R, the
extracellular domain of the ST2 receptor fused to the transmembrane and
the intracellular domain of IL-1R. Results of CAT assays (a
representative experiment of three done in duplicates) from Jurkat
cells transfected with expression vectors for the different constructs
together with the reporter plasmid IL-8P/CAT and treated with or
without 10 ng of IL-1
are shown. CM, chloramphenicol; ACM, acetylated chloramphenicol. The extent of acetylation was
also calculated and represented graphically. B, result of
immune complex kinase assay with CSBP isolated from Jurkat cells
transfected with IL-1R
360, IL-1R, or IL-1R/MST2R and treated with (white bar) or without (black bar) 10 ng/ml of
IL-1
for 20 min.
P-phosphorylated MBP used as a
substrate is indicated. The radioactivity in each band was quantitated
and presented in a graphical format. The basal kinase activity in
unstimulated cells in each case is considered as 1. From the kinase
reaction, an immunoblot of CSBP is also presented. IgG is the heavy
chain of anti-CSBP antibody used for immunoprecipitation. C,
same as B except that the cells were treated with IL-1
for 5 min, and anti-IL-1R antibody was used for immunoprecipitation
followed by immune complex kinase assay with MBP as substrate. The
basal level of kinase activity in unstimulated cells is considered as
1.
The observation that the intracellular
portion of the ST2 receptor can substitute for the intracellular
portion of IL-1R suggests that the signal transduction pathways for the
intracellular portion of the two receptors are similar. IL-1 is known
to activate a recently discovered stress-activated MAP kinase known as
CSBP/p38(25, 26, 37, 38) . We next
investigated whether the chimeric receptor also activated this MAP
kinase. As shown in Fig. 6B, CSBP/p38 was activated in
response to IL-1 in cells expressing the IL-1R and the IL-1R/MST2R
chimera but not in cells expressing the truncated IL-1R
360
receptor. It has also been reported that an
80-kDa
IL-1R-associated protein kinase is required for IL-1-mediated
activation of NF-
B (27) and that another protein kinase
that phosphorylates MBP co-immunoprecipitates with type I IL-1R in
response to IL-1 in T cells (39) . We therefore examined if
either kinase was activated by the chimeric receptor. While we were
unable to detect the
80-kDa autophosphorylating kinase, we did
detect an MBP-phosphorylating protein kinase activity that was induced
within 5 min following IL-1
stimulation in cells transfected with
the IL-1R and the IL-1R/MST2R chimera but not with the truncated
IL-1R
360 receptor (Fig. 6c). These data suggest
that at least part of the signal transduction pathway between the IL-1
and the ST2 receptors are common. We could not detect activation of
these kinases by either MST2R or the MST2R/IL-1R chimera in response to
IL-1 (data not shown).
Figure 7: Immunoprecipitation of soluble and membrane-bound ST2 receptor and receptor-mediated precipitation of putative ST2 ligand from metabolically labeled 3T3 and HUVEC conditioned media and cell extracts. A, immunoprecipitation of soluble ST2 with preimmune (PI) and immune (IM) anti-MST2 serum (lanes 1 and 2, arrow) and full-length membrane-bound ST2 receptor (lanes 3 and 4, open and filled arrowheads) from 3T3 cell conditioned medium and cell extract, respectively. B, precipitation of soluble ST2 from HUVEC (lanes 1 and 2, filled arrowhead) with preimmune (PI) and immune (IM) anti-HST2 serum. Precipitation of putative ST2 ligand (lanes 3 and 4, arrow and open arrowhead) with HST2Fc and IgG is shown. C, precipitation of putative ST2 ligand (arrow and open arrowhead) using MST2Fc, control IgG, and protein A-agarose beads from 3T3 cells (lanes 1-3). The conditioned media were made from exponentially growing cells for immunoprecipitation and serum-starved cells for receptor-precipitation, respectively.
We used mouse and human ST2Fc fusion
proteins to identify ST2 binding proteins in metabolically labeled
media from HUVEC and 3T3 cells made quiescent by serum starvation. As
shown in Fig. 7B, an 18-kDa (arrow) and
an
32-kDa protein (open arrowhead) were precipitated from
HUVEC medium by HST2Fc (lane 3) but not by control IgG (lane 4). There are additional proteins also precipitated by
HST2Fc. However, only the
18- and the
32-kDa proteins were
precipitated by the mouse ST2Fc (MST2Fc) from quiescent 3T3 cell medium (Fig. 7C, lane 2, arrow and open
arrowhead) but not by protein A-agarose beads alone (lane
1) or by control IgG (lane 3). Preincubation of labeled
conditioned medium with soluble ST2 protein inhibited the precipitation
of both the
18- and the
32-kDa proteins by ST2Fc (data not
shown). These two proteins were not precipitated by ST2Fc from either
HUVEC or 3T3 cell lysates (data not shown).
The experiment was
repeated with metabolically labeled conditioned media from
exponentially growing 3T3 cells in the presence of serum. Both human
and murine ST2Fc fusion proteins precipitated an 18-kDa protein (Fig. 8, lanes 1 and 2, arrow).
However, the intensity of this band was very faint. Since exponentially
growing cells secrete a large amount of soluble ST2 protein, whereas
quiescent cells do not (32) ,
we suspected that
most of the ligand may be bound to the secreted endogenous ST2. To
release this potential pool of ligand, labeled conditioned medium from
these cells was briefly treated with acid and neutralized before the
addition of various Fc fusion proteins. As shown in Fig. 8, lanes 6 and 7, the intensity of the
18-kDa band
increased dramatically following this brief acid treatment. All control
Fc fusion proteins were negative in this assay (Fig. 8, lanes 3-5 and lanes 8-10). Acid treatment
also led to an increase in the signal of other proteins in the high
molecular weight range which was not reproducible and varied among
different experiments. These high molecular weight proteins probably
result from aggregation of ST2Fc fusion protein alone or with other
labeled proteins in the conditioned medium, perhaps due to denaturation
of serum proteins and/or ST2 following acid treatment. Alternatively
some of these proteins may represent other accessory proteins
coprecipitated with the ST2 ligand-receptor complex.
Figure 8: Precipitation of ST2 ligand with various Fc fusion proteins. ST2 ligand (arrow) was precipitated with HST2Fc, MST2Fc, human IL-5RFc, EPORFc, and human IgG before (lanes 1-5) and after acid (lanes 6-10) treatment from metabolically labeled 3T3 cell medium obtained from exponentially growing cells as described under ``Materials and Methods.''
To confirm the
size of ST2 ligand, we passed the concentrated serum-free 3T3
conditioned medium over a Superdex 75 gel filtration column and assayed
the resulting fractions by a dot blot assay using ST2Fc. As shown in Fig. 9, fractions corresponding to <47 and >15 kDa were
positive in this assay, with maximum signal obtained with fraction
corresponding to 20 kDa. These data are consistent with our
earlier results from receptor-precipitation studies.
Figure 9:
Dot blot assay on fractions obtained from
gel filtration chromatography of concentrated 3T3 conditioned media.
3T3 conditioned medium was concentrated 10-fold and passed over a
Superdex 75 gel filtration column at pH 3.0. Fractions after void
volume were collected and analyzed by dot blot assay with MST2Fc. The
spots were quantitated by densitometric scanning and are represented as
arbitrary intensity units. The approximate molecular masses of
fractions 2, 4, and 6 are indicated. The molecular masses of fractions
1, 3, and 5 are >80, 32, and
14 kDa,
respectively.
As further
evidence for the existence of the ST2 binding proteins, we used BIAcore
analysis. A similar assay has been successfully used to identify the
ligand for the ECK receptor protein-tyrosine kinase(28) . As
shown in Fig. 10, both unconcentrated (3T3 1) and a
10-fold concentrated (3T3 10
) 3T3 cell conditioned medium showed
significant binding (white brick bars) to ST2Fc protein
captured through immobilized protein A. Soluble ST2 competed for this
binding, thus showing its specificity (+MST2). Similarly, a
10-fold concentrated conditioned medium from HUVEC (HUVEC 10
)
also showed specific binding. The 20-50-kDa fraction, obtained
from concentrated 3T3 conditioned medium after passage through a
Superdex 75 gel filtration column (see Fig. 9) was also positive
in this assay (data not shown). No binding was observed with
concentrated control media (DMEM 10
) to ST2Fc or with various
conditioned media to IL-1RFc (black brick bars). Conditioned
media from either 3T3 or HUVEC did not show any binding to unactivated
chip surface, protein A, or unrelated immobilized Fc fusions. We
screened conditioned media from several other cell lines including
Jurkat cells for this binding activity, but we were unable to find any
other cell lines positive in this assay.
Figure 10:
ST2 binding activity in 3T3 and HUVEC
conditioned media in BIAcore assay. Binding of unconcentrated
(1) or 10-fold concentrated (10
) cell conditioned media
to human or mouse ST2Fc (white brick bars) or IL-1RFc (black brick bars) immobilized on BIAcore sensor chip surface
via protein A. Control (DMEM), 3T3 or HUVEC media were applied with or
without preincubation with soluble ST2 (+MST2 or
+HST2).
To look for signal
transduction by the putative ST2 ligand, we examined CSBP/p38
activation and IL-8 promoter/CAT stimulation in Jurkat cells that have
endogenous ST2 receptor. The concentrated 3T3 conditioned
medium was able to activate CSBP/p38 MAP kinase (Fig. 11)
similar to IL-1
. The activation of CSBP could be blocked >80%
by preincubation of medium with MST2Fc protein, suggesting that the ST2
ligand is functional. However, the same 3T3-concentrated medium failed
to induce transfected IL-1 promoter/CAT or HIV1LTR/CAT reporter genes
(data not shown). Since Jurkat cells have endogenous ST2 receptor we
could not test the chimeric ST2/IL-1 receptor.
Figure 11:
Activation of CSBP MAP kinase by 3T3
conditioned medium. 2 10
Jurkat cells were treated
either with 1 ml of 10
concentrated DMEM or 3T3 conditioned
medium or with concentrated 3T3 conditioned medium preincubated with 10
µg/ml MST2Fc. After 20 min cells were lysed, CSBP was
immunoprecipitated, and a kinase assay was performed using MBP as a
substrate (upper panel). CSBP was also immunoblotted from the
kinase reaction (middle panel). IgG is the heavy chain of
anti-CSBP antibody used for immunoprecipitation. The quantitated
radioactivity is represented graphically (lower panel). The
basal level of kinase activity in unstimulated cells is considered as
1.
To identify the ligand(s) of ST2, we have used an ST2Fc
fusion protein to assess binding to purified IL-1s and crude cell
lysates and media. Our data establish very clearly that none of the
IL-1s are ligands for ST2. We did not detect binding via receptor
precipitation, cross-linking, BIAcore, or signal transduction assays.
This is in contrast to a recent report published while this paper was
under review, which detected weak binding of rat ST2/Fit-1 to murine
IL-1 (40) . While we occasionally did detect weak,
competable binding of human IL-1
and IL-1
to high
concentrations of ligands and human ST2Fc in receptor precipitation
assays, the binding was not saturable. Furthermore, we could not detect
any binding of these proteins (including ST2 and IL-1
from mouse)
by the more sensitive BIAcore, which can detect affinities in the
µM range, so we concluded that ST2 does not bind IL-1. We
agree with these authors, however, that IL-1 does not signal through
ST2. A second preliminary report is in agreement with our
data(41) .
In contrast to the negative data with IL-1, we
were able to identify a ligand in 3T3 and HUVEC conditioned media using
some of the same assays. In both cell media, two proteins of 18
and 32 kDa were specifically precipitated by ST2Fc. Size exclusion data
also indicated a ligand with a molecular mass of
20 kDa, which,
along with the variable appearance of the 32-kDa protein, suggests that
the ligand binds as a monomer rather than a heterodimer. Although we do
not know the relationship of the 32- and 18-kDa proteins, it is
possible that the 32-kDa protein is a precursor of the 18-kDa protein,
reminiscent of IL-1. However, we did not detect these proteins in cell
extracts. The intensity of the 18-kDa protein also varied, depending
upon the presence of endogenous ST2 as evidenced by an increase in
signal after acid treatment of conditioned media made from
exponentially growing cells. In quiescent cells, where no ST2 is made,
there was no effect of acid treatment. These data suggest that ST2
ligand is continuously made, whereas the expression of soluble ST2 is
modulated by serum and growth conditions.
Although we have not been able to define a biological activity for the ST2 ligand(s) we have discovered, the ability of this ligand to activate the stress-activated MAP kinase CSBP/p38 in cells expressing the receptor argues that it is functional. ST2 shares this signal transduction pathway with IL-1RI. Indeed, a chimeric receptor consisting of the extracellular domain of IL-1RI and the intracellular domain of ST2 functions like IL-1RI, inducing a receptor-associated protein kinase, CSBP/p38, and IL-8 promoter-dependent transcription in response to IL-1 binding. This contrasts with the response of ST2 receptor to the ligand we have discovered, where only CSBP/p38 activation was observed.
Several key residues of the IL-1RI required for signal transduction have been defined. Three basic residues (Arg-431, Lys-515, and Arg-518) and three aromatic residues (Phe-513, Trp-514, and Tyr-519) that are conserved in human, murine, and chicken IL-1 receptors are required for IL-1 signal transduction(6, 7) . All six are conserved in murine ST2, and all but Tyr-519 are also conserved in the Drosophila toll protein(6, 7) . The region 435-484 of IL-1R is also similar in sequence to the box 1- and box 2-like elements present in gp130, the signal-transducing subunit of the IL-6 receptor family(42) , and deletion of this region in IL-1RI abolishes its capacity to induce IL-8 gene expression(22) . The experiments with the chimeric IL-1R/ST2 receptor suggest that these regions are functionally conserved in ST2, so that the differences observed between IL-1 and the putative ST2 ligand must be due to other components of ST2. One possibility is that association with a second subunit is required for signaling, as has been suggested for IL-1R(43) . Differences might then be due to the association of ST2 with a different second subunit after ligand binding, which does not trigger the IL-8 promoter.
To conclude, our experiments provide the first evidence for a unique ligand for ST2 that is distinct from IL-1. To further characterize the putative ST2 ligand, we are purifying it in sufficient quantities for sequencing and cloning. The availability of cloned material should then allow further evaluation of the biological role of ST2 and its potential intracellular signaling pathways.