(Received for publication, October 24, 1995; and in revised form, December 12, 1995)
From the
T1/ST2 is a receptor-like molecule homologous to the type I interleukin-1 receptor. Despite this sequence similarity, we have been unable to demonstrate binding of T1/ST2 to any of the three interleukin-1 species. In searching for a ligand for T1/ST2, we have cloned a cell surface protein to which it binds. This protein is unable to initiate signal transduction by the T1/ST2 receptor in several in vitro assays.
The cytokine interleukin-1 exerts profound effects on a wide
variety of cell types and tissues. These effects are mediated through a
single receptor, the type I IL-1 ()receptor, which is
comprised of an extracellular portion formed by three immunoglobulin
domains, a single membrane-spanning segment, and a 215-amino acid
cytoplasmic domain responsible for the signaling functions(1) .
In recent years, a number of homologs of the IL-1 receptor have been
discovered. One of the most intriguing of these is a molecule called
T1, ST2, or fit-1 (referred to herein as T1/ST2), which has
now been cloned independently three times on the basis of its strong
induction by proliferative signals(2, 3, 4) .
These clones encode a secreted molecule possessing considerable
sequence similarity to the extracellular, ligand-binding portion of
both the type I and type II IL-1 receptors. Subsequently, a second form
of T1/ST2 mRNA has been found, which encodes a transmembrane version of
the same molecule, the cytoplasmic domain of which is also very similar
to that of the type I IL-1 receptor(5) . The T1/ST2 gene is
tightly linked to the genes encoding the type I and type II IL-1
receptors in both mouse and human, and the intron/exon structures of
murine T1/ST2 and human IL-1RI are virtually identical, providing
further evidence that the two genes derive from a common
ancestor(6, 7) .
In adult mice and rats, the soluble and transmembrane versions of the T1/ST2 receptor are expressed predominantly in different tissues, a consequence of transcription from different promoters leading to the use of different polyadenylation sites and thereby to the production of different forms of mRNA(4, 8, 9) . The soluble version is produced primarily by fibroblasts, whereas the transmembrane version is found predominantly in hematopoietic tissues and in lung. During embryonic development, transmembrane receptor is again expressed predominantly in hematopoietic organs, whereas the soluble receptor is made in a limited set of tissues (skin, bone, eye) during a short period in the mid to late stages of gestation and then turned off again(8) . Receptor protein is found in defined locations near to but not at the same place as receptor mRNA, suggesting that the soluble receptor diffuses away from the cells that make it until it encounters a ligand to which it can bind. Expression of soluble T1/ST2 receptor expression is also turned on in certain mammary tumor cells(4, 9, 10) , consistent with its initial cloning as a gene induced upon stimulation of proliferation.
One attractive hypothesis, consistent with the above data, would postulate the existence of a ligand whose actions on the membrane-bound form of the T1/ST2 receptor are in some way incompatible with unrestrained cell proliferation. Soluble T1/ST2 receptor, induced upon stimulation of cell proliferation, could bind this ligand and prevent it from signaling through the membrane-bound receptor, thus freeing the way for cell growth.
We have attempted to identify a ligand for the T1/ST2 receptor. As part of this study, we have also asked whether T1/ST2 binds any of the IL-1 species, which seemed possible given the sequence similarity between T1/ST2 and IL-1RI. However, consistent with the absence in the literature of any evidence for the existence of IL-1 binding proteins other than the type I and type II receptors, we find no evidence for T1/ST2 binding to any of the IL-1 ligands. We have identified and cloned a protein that binds the T1/ST2 receptor but have not yet been able to demonstrate a functional response to this interaction.
Human and murine T1/ST2 extracellular regions were amplified by polymerase chain reaction from KG-1 and 3T3 cell RNA, respectively, and fused to human IgG1 sequences as described(11) . The Ig portion was mutated to reduce binding to Fc receptors(12) . The fusion protein in the vector pDC409 (13) was expressed in COS cells and purified on a protein A column.
A BIAcore biosensor (Pharmacia Biosensor) was used to examine binding of IL-1 ligands to the human T1/ST2 Fc fusion protein, essentially as described in detail in Arend et al.(14) . Briefly, a goat anti-human IgG serum covalently coupled to the dextran matrix of a hydrogel chip was used to capture the human T1/ST2 Fc protein from concentrated COS cell supernatant. The appropriate IL-1 ligand, at 1 µM concentration, was reacted with the captured protein, and the change of mass per unit area over time was measured.
Screening of cell lines for expression of T1/ST2 binding protein, determination of the affinity of binding of T1/ST2 Fc protein to A172 cells, and expression cloning of the binding protein from an A172 library were carried out as described(15, 16) . The human binding protein cDNA was used as a probe to isolate murine binding protein clones from a 70Z/3 pre-B cell library by cross-hybridization. Two cDNA clones each were isolated for mouse and human binding protein; they differed in length by four (human) or five (mouse) nucleotides at the 5`-end, but those from a given species were otherwise identical throughout the coding region. The longer mouse clone appears to be missing three N-terminal amino acids by comparison with the human clones.
For chromosome mapping, metaphase spreads from normal human lymphocytes were prepared according to the method of Fan et al.(17) . The human T1/ST2 binding protein cDNA was labeled with biotin-16-dUTP (Boehringer Mannheim) by nick translation. Fluorescence in situ hybridization and detection of immunofluorescence were performed according to the technique of Pinkel et al.(18) with minor modifications(19) . The chromosome preparations were stained with both diamidino-2-phenylindole and propidium iodide (Oncor) (17) and observed with a Zeiss Axiophot fluorescence microscope. Hybridization of the 1.3-kilobase cDNA probe to human chromosomes was performed on three separate occasions and revealed specific labeling on chromosome 19. Fluorescent signals were detected on chromosome 19 in 62 of 82 metaphase spreads examined. Nonspecific hybridization was moderately low, and 153 of 284 signals (53.9%) were located on chromosome 19. All signals on chromosome 19 were located at 19p13, with most appearing to reside at or beside sub-band 19p13.2.
To analyze expression
patterns, RNA samples (5 µg) were electrophoresed, transferred to
nylon membranes, and hybridized as described (16) to antisense
riboprobes made from human (Fig. 4A) or mouse (Fig. 4B) T1/ST2 binding protein cDNA. Evenness of
loading was monitored by staining the filters with methylene blue
following transfer. In Fig. 4C, a Clontech blot
(catalogue no. 7760-1) containing 2 µg of poly(A) RNA in each lane was hybridized to the human riboprobe.
Figure 4:
Northern blots using antisense riboprobes
from human (A and C) or mouse (B) T1/ST2
binding protein cDNA clones on the corresponding RNA. In A and B, poly(A) RNA was used for the A172, EL4,
and 70Z/3 samples; the other lanes contained total RNA. The arrows represent the positions of 28 and 18 S rRNA. All RNAs in C are poly(A)
.
Soluble human T1/ST2 binding protein expression constructs were made in pDC409 by fusing the first 194 amino acids (Met. . . . Glu-Arg-Val-Asn) at their C terminus to either the human IgG1 Fc mutein (see above) or to the epitope tag FLAG(20) . The equivalent murine versions were made by including the first three amino acids of the human binding protein to substitute for the missing murine N terminus. Proteins were expressed in CV1/EBNA cells (16) and either used directly as cell supernatant, with or without concentration, or purified on a protein G column (Fc construct) or an M2 anti-FLAG antibody column (FLAG construct) followed by elution with 12.5 mM sodium citrate, pH 2.8, and subsequent neutralization.
Construction of the reporter plasmid containing nucleotides
-130 to +44 of the human IL-8 promoter (21) fused to
the IL-2R chain coding sequence, transfection and stimulation of
COS cells, and measurement of IL-2R
chain expression by use of the
2A3 anti-IL2R
antibody (22) followed by
I-goat anti-mouse Ig serum will be described in detail
elsewhere. (
)The basic cassette (MuIL1R/-), containing
the murine IL-1RI extracellular and transmembrane regions with no
cytoplasmic portion, and the MuIL1R/ST2 chimera (mouse IL-1RI
extracellular and transmembrane regions fused to the mouse T1/ST2
cytoplasmic domain) are described in Mitcham et
al.(23) . For the chimera containing the murine T1/ST2
extracellular portion, amino acids 1-358 of the full-length
murine ST2 protein (5) were fused to amino acids 363-576
of the mouse type I IL-1 receptor (numbering is from the initiating
methionine). The equivalent human chimera contained amino acids
1-334 of the human T1/ST2 protein, the transmembrane region
(amino acids 330-358) of the full-length murine ST2 protein
(since no sequence is available for the transmembrane version of the
human receptor), and the amino acids 363-576 (the cytoplasmic
portion) of the mouse type I IL-1 receptor. All chimeras were
constructed in the expression plasmid pDC304, a variant of
pDC302(24) . Stimulations of transfected COS cells were
performed with 1 ng/ml of human IL-1
or with either 50 ng/ml or 1
µg/ml of human T1/ST2 binding protein Fc with equivalent results.
Similarly, the presence or absence of the sheep anti-human IL-1RI
antiserum P3 had no effect on stimulations using human T1/ST2 binding
protein Fc.
The only measurable binding in the
experiment was observed with IL-1ra as a ligand (Fig. 1) and
represented less than 10% occupancy of the T1/ST2 Fc fusion protein at
1 µM ligand. The apparent rate of dissociation was too
fast to measure. These data correspond to a K in
the range of 10
-10
M
, which is too low to be
physiologically significant and which may simply represent nonspecific
interaction of the ligand with the chip surface, with the goat
anti-human IgG used for immobilization, or with the Fc portion of the
fusion protein. With IL-1
and IL-1
, the BIAcore traces showed
no evidence of binding whatsoever. We conclude that the T1/ST2 receptor
does not bind in a significant way to any of the known IL-1 family
members.
Figure 1:
Lack of binding of IL-1,
IL-1
, and IL-1ra to the T1/ST2 receptor, measured by
BIAcore.
Figure 2:
A,
flow cytometry profiles showing that human T1/ST2 Fc protein binds
specifically to A172 cells. SA-PE, streptavidin-phycoerythrin
detection reagent. B, binding curve (top) and
Scatchard plot (bottom) of human T1/ST2 Fc protein binding to
A172 cells. Cells were incubated with various concentrations of T1/ST2
Fc protein, and binding was subsequently detected using a saturating
concentration of I-mouse anti-human IgG(15) . The
deduced site numbers and affinities are R
, 1945
sites/cell; K
, >1
10
M
; R
,
26358 sites/cell; K
, 2.6
10
M
.
To
isolate cDNA clones of the T1/ST2 binding protein, we used an
expression cloning strategy described
previously(15, 16) . A cDNA library from the A172 cell
line was generated in the mammalian expression vector
pDC410(25) . The library was transfected in pools of 2000 into
CV1/EBNA cells growing on slides, and after 2 days the cells were
incubated with the T1/ST2 receptor Fc fusion protein followed by a
second step of I-rabbit anti-human IgG. The slides were
coated with photographic emulsion, exposed, and screened under a
low-power microscope for the presence of silver grains superimposed on
cells, indicating expression of a cDNA capable of binding to the T1/ST2
Fc fusion protein.
Two positive clones were found after screening 5
10
clones of an oligo(dT) primed library. The cDNA
sequence predicts a type I transmembrane protein of 227 amino acids,
with a 170-amino acid extracellular portion lying between a potential
signal peptide and the transmembrane domain (Fig. 3). The
protein also contains a 12-amino acid cytoplasmic tail. No similarity
to any of the IL-1 ligands is apparent from the sequence of this clone
nor is there any resemblance between the hydrophobicity plots, as is
found for example between IL-1
and -
and acidic fibroblast
growth factor(26) . However, there are a number of open reading
frames as well as expressed sequence tags in the DNA sequence data
bases that show significant homology to the T1/ST2 binding protein (see
legend to Fig. 3). One of these has been established to be an
endosomal protein in yeast and another a microsomal protein in dog
pancreas; the others are uncharacterized. Northern blots reveal that
the
1.5-kilobase T1/ST2 binding protein mRNA is expressed widely (Fig. 4). The gene encoding the human T1/ST2 binding protein was
mapped to chromosome 19p13.2 (data not shown; see ``Materials and
Methods'').
Figure 3: The amino acid sequences of the human (top) and murine (bottom) T1/ST2 binding proteins, as deduced from the cDNA clones. Identities are indicated by vertical lines. The murine clones appear to be missing the N-terminal portion of the signal sequence. The predicted site of signal peptide cleavage is indicated by an arrow head. The predicted transmembrane segment is underlined. There are a number of homologous cDNA sequences in the NCBI data bases. Four of these are human expressed sequence tags (three of them represented multiple times), which can be retrieved using accession numbers T17481, T48838, and T48839; T27390 and F06012; R54717, H03613, H27167, and H27168; and R54718. The others are a dog microsomal membrane protein (X53592)(29) , a Chinese hamster coated vesicle protein (U26264), which is a homolog of the second group of human expressed sequence tags, a yeast endosomal protein (X67317) (30) , and four open reading frames found by yeast genomic sequencing of chromosome I (L22015, translation of complement 4145-4792), chromosome IV (Z48432, translation of complement 18667-19344), chromosome VIII (U00059, translation of 18834-19472), and chromosome XV (X87331, translation of complement 9721-10344). An open reading frame on yeast chromosome XIII (Z49810, translation of 10178-10813) may also be a family member.
The human T1/ST2 binding protein cDNA clone was used as a probe to isolate a murine counterpart from the 70Z/3 pre-B cell line. The mouse sequence is very similar to the human one, showing 95% amino acid identity overall and 97% identity within the extracellular portion of the protein. Both human and mouse T1/ST2 receptor Fc fusion proteins bound to both human and mouse binding proteins when the latter were expressed transiently in COS cells (data not shown).
To facilitate further studies, tagged versions of the binding proteins were made by fusing either the human IgG1 Fc moiety, described above, or the FLAG sequence (DYKDDDDK) (20) to the C terminus of their extracellular portions. It seemed likely that these tags would not interfere with function, since normally the extracellular portion is joined to its own transmembrane region at the same point. Both varieties of fusion construct, for both human and mouse binding proteins, showed good binding to mouse 3T3 cells in fluorescence-activated cell sorter experiments (Fig. 5; data not shown).
Figure 5: Flow cytometry profiles of murine 3T3 fibroblasts following staining with human or mouse T1/ST2 binding protein Fc. Human IgG and murine IL-4 receptor Fc protein were used as negative controls.
Figure 6:
Failure to activate DNA binding by
NFB upon stimulation of 3T3 cells with T1/ST2 binding protein Fc
fusion. 3T3 cells were stimulated with the following: nothing (lane
4); the negative control, murine IL-4 receptor Fc (lane
1, 250 ng/ml; lane 2, 10 ng/ml); the positive control,
human IL-1
(lanes 3, 6, 7, 10 ng/ml; lanes 8, 9, 250 ng/ml); human T1/ST2 binding protein
Fc (lane 10, 10 ng/ml; lane 11, 250 ng/ml); or murine
T1/ST2 binding protein Fc (lane 12, 10 ng/ml; lane
13, 250 ng/ml). No extract was added to the probe in lane
5. In lanes 7 and 9, a 100-fold excess of
unlabeled NF
B oligonucleotide was added as competitor to
demonstrate specificity. NF
B electrophoretic mobility shift assays
were conducted as described(31) .
A
second assay used to assess functional activity of the T1/ST2 binding
protein is the ability to stimulate transcription from the IL-8
promoter. In this assay, the human IL-8 promoter has been
fused to the coding region of the human IL-2 receptor
chain. The
reporter construct is transfected into COS cells along with an
appropriate receptor chimera, the cells are stimulated with ligand, and
expression of IL-2R
on the cell surface is measured using
I-labeled anti-IL-2R
antibody. Transcription of the
transfected IL-8 promoter/IL-2R
chain reporter can be stimulated
by IL-1 through either the endogenous COS cell IL-1 receptors (not
shown) or, as can be seen in Fig. 7, through a transfected
murine IL-1 receptor after antibody blocking of the endogenous
receptors. Moreover, reporter expression can be stimulated by IL-1
through a transfected chimeric receptor containing the extracellular
and transmembrane segments of the murine IL-1 receptor fused to the
cytoplasmic portion of the murine T1/ST2 receptor (Fig. 7; (23) ). However, neither IL-1 nor the T1/ST2 binding protein
was able to induce transcription of the reporter construct through the
reverse chimera containing the extracellular and transmembrane segments
of the murine T1/ST2 receptor fused to the cytoplasmic portion of the
murine IL-1 receptor. Human IL-1
also failed to stimulate the IL-8
promoter when tested on a comparable chimera containing the
extracellular portion of human T1/ST2, confirming once again that
IL-1
is not a ligand for T1/ST2. Cell surface expression of the
chimeric receptors was confirmed by flow cytometry after staining with
either antibody or T1/ST2 binding protein Fc (data not shown).
Figure 7:
Failure of T1/ST2 binding protein to
activate transcription from the IL-8 promoter. COS cells were
transfected with the indicated receptor plasmids together with a
reporter plasmid containing the IL-8 promoter driving expression of the
IL-2 receptor chain cDNA. 24 h later, the cells were stimulated
overnight with medium (solid bars), 1 ng/ml IL-1
(hatched bars), or 1 µg/ml human T1/ST2 binding protein Fc (stippled bars). They were then incubated with mouse
monoclonal antibody 2A3 against IL-2R
followed by
I-goat anti-mouse Ig serum and counted. Stimulations were
done in the presence of a 1/100 dilution of sheep anti-human IL-1RI
serum P3(23) , which at this concentration blocks binding of
IL-1 to the endogenous COS cell IL-1 receptors but has no effect on
binding to the transfected mouse IL-1RI extracellular region. The P3
serum does not bind to human or mouse T1/ST2 (data not shown). The
designations (X/Y) of the transfected receptor
plasmids refer to the origin of the extracellular and transmembrane
regions (X) and cytoplasmic domain (Y) of the chimera
(see ``Materials and Methods''); for example, MuIL1R/ST2
contains the mouse IL-1RI extracellular and transmembrane regions fused
to the cytoplasmic portion of mouse T1/ST2. In other experiments, human
and mouse IL-1RI cytoplasmic domains were found to function equally
well in this assay.
Despite its considerable homology to type I and type II IL-1
receptors, we find that the T1/ST2 protein does not have any measurable
affinity for any of the three known IL-1 species (IL-1, IL-1
,
IL-1ra). Other researchers have also failed to find binding to
IL-1
or IL-1
(8) . One group (27) has reported
a low binding affinity for IL-1
; we have not been able to confirm
this. Recently another IL-1 receptor homolog, called IL-1 receptor
accessory protein (AcP), has been reported(28) . The AcP
associates with the type I IL-1 receptor and increases its affinity for
IL-1; it may also play a role in IL-1 signal transduction. It is
conceivable that the T1/ST2 protein functions similarly, although its
restricted tissue distribution would suggest that it cannot be required
for IL-1 signaling.
We have attempted to test another hypothesis,
namely, that the T1/ST2 receptor has a ligand(s) of its own, capable of
eliciting biological responses in receptor-bearing cells. Making use of
a receptor/Fc fusion protein, we have cloned a cell surface molecule
capable of binding to T1/ST2. The binding protein shows no sequence
similarity to any of the IL-1 species. It does appear to be widely
expressed and to possess homology to a number of other mostly
uncharacterized open reading frames or cDNAs. We have tested the
binding protein for biological activity in two assays, activation of
DNA binding by NFB and induction of transcription from the IL-8
promoter. Both of these assays respond well to IL-1, acting either
through the type I IL-1 receptor or through a chimeric receptor
consisting of the extracellular and transmembrane portions of the IL-1R
fused to the cytoplasmic domain of T1/ST2. Neither responded to the
T1/ST2 binding protein. In the NF
B assay, the endogenously
expressed T1/ST2 receptor was used, whereas the IL-8 assay used a
chimeric receptor in which the T1/ST2 extracellular portion was fused
to the IL-1R cytoplasmic part. We have also asked whether the T1/ST2
binding protein has any activity in a number of assays of immune and
hematopoietic function with consistently negative results (data not
shown).
The assays used to measure biological function were chosen
because we had good reason to believe that a ligand for T1/ST2 should
read out in them. The T1/ST2 cytoplasmic domain has been demonstrated
to be capable of leading to NFB activation in the context of a
chimeric receptor containing the IL-1R extracellular portion. It is
reasonable then to assume that natural T1/ST2 receptor, responding to
its true ligand, would also lead to NF
B activation. 3T3 cells, in
which this assay was done, express natural T1/ST2 receptors and are
capable of activating NF
B in response to other stimuli. Similarly,
in the COS cell assay, the IL-1R cytoplasmic domain is capable of
activating the IL-8 promoter in the context of the endogenous monkey
IL-1RI, a transfected mouse IL-1RI, or a chimeric receptor fusing the
mouse IL-1RI extracellular and transmembrane portions to the human
IL-1RI cytoplasmic domain. It is reasonable to assume that it will also
be capable of eliciting IL-8 promoter activity when present in a
properly activated chimera, which fuses it to the T1/ST2 extracellular
and transmembrane portions, especially since the reverse chimera (IL-1R
extracellular and transmembrane domains, T1/ST2 cytoplasmic domain)
functions well in the assay upon IL-1 stimulation.
The lack of biological activity shown by the cDNA reported here suggests that it may not be a true ligand for the T1/ST2 receptor and that the interaction between the two proteins may be of no physiological relevance. Many other explanations exist, however. Although we believe that our assays should have responded to a T1/ST2 ligand, it remains possible that the binding protein will display activity in other settings. Alternatively, perhaps the binding protein serves as an antagonist, similar to the IL-1ra, to regulate the activity of an undiscovered primary ligand. Yet another possibility is that the T1/ST2 receptor does not function on its own but requires an accessory molecule, similar to the IL-1R AcP(28) , to respond to the binding protein and that this accessory protein is not expressed in 3T3 or COS cells. Finally, the functional interaction was examined in only one direction, i.e. the effect of the binding protein on T1/ST2 receptor-bearing cells. Instead, induction of a biological signal may occur in cells bearing the membrane-bound binding protein. At the moment, we are unable to distinguish among these hypotheses.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U41804[GenBank], U41805[GenBank].