(Received for publication, February 27, 1995; and in revised form, May 11, 1995)
From the
The fit-1 gene gives rise to two different mRNA
isoforms, which code for soluble (Fit-1S) and membrane-bound (Fit-1M)
proteins related to the type I interleukin (IL)-1 receptor. To
investigate IL-1 binding, we have synthesized and purified
histidine-tagged polypeptides corresponding to Fit-1S and the
extracellular domain of the type I IL-1 receptor using a vaccinia
expression system. Fit-1S is shown to interact with IL-1 The cytokine interleukin-1 (IL-1) A similar inhibitory
function has been suggested for the B15R protein, a soluble IL-1
receptor encoded by the vaccinia virus (13, 14) . B15R
binds IL-1 We have previously
characterized the Fos-responsive gene fit-1, which codes for
two protein isoforms related to the IL-1
receptor(15, 16) . The membrane-bound protein Fit-1M
is most closely related to the type I IL-1 receptor throughout its
entire coding region, while the secreted Fit-1S protein consists only
of the extracellular domain. The fit-1 gene is transcribed
from two different promoters, and tight coupling of promoter usage to
3` processing is responsible for the generation of the two different
mRNA isoforms(16) . The Fit-1M transcript is predominantly
expressed in hematopoietic cells, while the Fit-1S mRNA is
preferentially expressed in fibroblasts and epithelial
cells(16) . The extracellular domains of Fit-1 and the two
IL-1 receptors (type I and II) share a similar degree of amino acid
sequence identity (26-29%), suggesting that the Fit-1 proteins
may also bind IL-1. Moreover, the mouse homologue (ST2) of the
rat fit-1 gene has been mapped to the same region of mouse
chromosome 1 as the two IL-1 receptor genes(17) , implying that
all three genes have arisen from a common ancestral gene by duplication
events. Here we demonstrate that Fit-1 is indeed a distant member of
the IL-1 receptor family based on its IL-1
The Fit-1M expression
plasmid was obtained by inserting cDNA sequences coding for the
transmembrane and intracellular regions of Fit-1M into a Fit-1S
expression vector consisting of a 1364-bp XbaI fragment of the
Fit-1S cDNA clone C (16) cloned in the XbaI site of
the cytomegalovirus expression vector pRK-7.
Figure 2:
Comparison of the IL-1 binding properties
of the Fit-1S and sIL-1R proteins. A, saturation curve and
Scatchard analysis of IL-1
Figure 1:
Purification of
polyhistidine-tagged soluble receptor proteins. A, schematic
representation of the mouse type I IL-1 receptor (IL-1R) and
of histidine-tagged proteins consisting of the extracellular domain of
the IL-1R (sIL-1R) and Fit-1S. cDNA sequences coding for the
sIL-1R and Fit-1S proteins were inserted into recombinant vaccinia
viruses. TM, transmembrane region; HA, hemagglutinin
epitope tag; His, histidine tag. B, analysis of
purified proteins. Histidine-tagged proteins were purified from
supernatants of vaccinia-infected HeLa cells as described under
``Materials and Methods.'' Purified proteins (
Figure 3:
Signaling via the intracellular domain of
Fit-1M activates the NF-
Previous analyses of the Fos-responsive gene fit-1 revealed that alternative promoter usage coupled with differential
3` processing gives rise to a secreted protein, Fit-1S, and a
membrane-bound isoform, Fit-1M, which is related to the type I IL-1
receptor(16) . In this report we have demonstrated that the
purified Fit-1S protein binds IL-1 The type I IL-1 receptor is known to stimulate
gene transcription through activation of NF-
Figure 4:
Conservation of functionally important
residues in the cytoplasmic domains of the Fit-1M, Toll, and IL-1
receptors. Identical and similar amino acids of the rat type I IL-1
receptor(34) , rat Fit-1M protein(16) , and Drosophila Toll receptor (30) are highlighted by black and grayoverlay, respectively. Filleddots denote amino acid residues that are
essential for IL-1-dependent induction of the IL-2 gene(29) ,
while blacksquares indicate residues critical for
induction of the IL-8 gene(28) . Numbers refer to
amino acid positions of the respective
protein.
Other signal-transducing molecules,
in addition to Fit-1M, are known, which share striking similarities
with the type I IL-1 receptor. The transmembrane protein Toll, which is
involved in establishing the dorsoventral polarity in early embryos of Drosophila, shows significant homology in the intracellular
domain with the type I IL-1 receptor ( (30) and Fig. 4).
Interestingly, signal transduction by Toll results in activation of the
transcription factor Dorsal, which also belongs to the Rel protein
family like NF-
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, but not
with IL-1
. However, Fit-1S binds IL-1
only with low affinity
in contrast to the IL-1 receptor, suggesting that IL-1
is not a
physiological ligand of Fit-1S. Moreover, expression of the
membrane-bound protein Fit-1M in transiently transfected Jurkat cells
did not result in activation of the transcription factor NF-
B
following IL-1
treatment. However, a chimeric protein consisting
of the extracellular domain of the type I IL-1 receptor and of the
transmembrane and intracellular regions of Fit-1M stimulated
NF-
B-dependent transcription as efficiently as the full-length
type I IL-1 receptor. These data indicate that Fit-1M is a signaling
molecule belonging to the IL-1 receptor family.
(
)is
produced in response to tissue injury and infection and regulates a
wide range of immune and inflammatory processes (reviewed in (1) ). The IL-1 family consists of two agonist species,
IL-1
and IL-1
, which share similar biological activities. A
third member, IL-1ra, blocks the biological response of the two
agonists both in vitro and in vivo(2) . All
three forms bind with comparable affinities to two different cell
surface receptors, an 80-kDa (type I) receptor mainly expressed on
T-cells and fibroblasts (3) and a 68-kDa (type II) receptor
predominantly found on B-cells and macrophages(4) . While both
receptors are related in their extracellular ligand-binding regions,
only the type I IL-1 receptor contains a long cytoplasmic domain (213
amino acids) capable of transmitting an intracellular
signal(5, 6) . At the nuclear level, activation of the
transcription factor NF-
B is one of the major mechanisms by which
IL-1 modulates transcription of genes involved in immune and acute
phase responses(7) . The type II receptor, which contains only
a short cytoplasmic sequence of 29 amino acids lacks any signaling
function (6) and is thought to inhibit IL-1 activity by acting
as a decoy target for IL-1(8) . The membrane-bound form of the
type II receptor also serves as a precursor for a shed, soluble
receptor protein, which further antagonizes IL-1 action. This soluble
receptor was detected in supernatants of B-lymphoid and myeloid cells (9, 10, 11) as well as in human plasma and
synovial fluid(10, 12) .
with high affinity and is not essential for virus
replication in tissue culture cells. In vivo, B15R functions,
however, as an attenuation factor by binding and neutralizing IL-1, as
deletion of its gene from the virus genome increases the pathogenicity
of vaccinia in infected mice(13) .
binding and
intracellular signaling capacities.
Cytokines
Mouse IL-1 and IL-6 were obtained
from Sigma, mouse IL-1
from Genzyme, and
I-labeled
mouse IL-1
(100 µCi/µg) from DuPont NEN.
PCR Olgonucleotides
The oligonucleotides are as
follows: a, GCGAAGCTTCGACCATGGGGCTTTGGGCTTTGGCA; b,
CGCGTCGACAATTTGTGAGAGACACTCCTT; c, GCGAAGCTTCCGTGAACAACACAAATGGAG; d,
CGCGTCGACGTCAGGGACTGGGTATATTAAC; e, GGGGATCCAGTTCGTTGCTGTCCTGTGGCAG; f,
GCGAAGCTTCAAAAGTGTTTCAGGTCCAAGCA; g, CAGGTACCGGGTATATTAACTGCACATGC; h,
CCGGTACCTGACTTCAAGAATTACCTC; i, TTCCCGGGCTAGCCGAGTGGTAAGTGTG; k,
AAGGTACCAATTGACCACCAAAGCAC; l, GTCCCGGGCTCAAAAGTGTTTCAGGTCCA.Expression Plasmids
The coding region of Fit-1S
(from amino acids 7 to 335) was isolated as a 987-bp HindIII-SalI fragment from the Fit-1S cDNA
clone C (16) by PCR amplification with the oligonucleotide pair
a/b. The sequences coding for the extracellular domain of the mouse
IL-1 receptor (amino acids 1-336; (3) ) were isolated as
a 1023-bp HindIII-SalI fragment by PCR
amplification using cDNA from Balb/c 3T3 cells and the primer pair c/d.
Each of these fragments was ligated together with a 160-bp SalI-SmaI fragment, encoding three copies of a
hemagglutinin epitope tag followed by 10 histidine codons and an
in-frame stop codon(18) , into the HindIII
and SmaI sites of the vaccinia expression vector
p-gpt-6-2.
(
)
(
)A
780-bp MscI-HindIII fragment of Fit-1M cDNA,
which was obtained by PCR amplification using rat spleen cDNA and
primers e and f, was cloned into the MscI and SmaI
sites of the Fit-1S expression vector. The coding region of the mouse
type I IL-1 receptor was isolated by PCR amplification from Balb/c 3T3
cDNA as two separate DNA fragments linked via an Asp718 site,
which was newly created at codons 333 and 334 (3) without
affecting the coding capacity. The 1020-bp HindIII-Asp718 fragment containing the
extracellular domain was obtained with primers c and g, and the 733-bp Asp718-XmaI fragment comprising the transmembrane and
intracellular regions with primer pair h/i. The two cDNA fragments were
cloned into the HindIII and XmaI sites of
the expression vector pRK-7. The chimeric IR-FM gene was generated by
replacing the Asp718-XmaI fragment of the IL-1
receptor expression vector by a 728-bp Asp718-XmaI
fragment obtained from the Fit-1M cDNA clone F (16) by PCR
amplification with primers k and l. All DNA constructs were verified by
DNA sequencing.
Expression and Purification of Polyhistidine-tagged
Proteins
Recombinant vaccinia viruses were generated and
amplified as described(19) . Vaccinia-infected HeLa cells were
kept in Joklik medium in the absence of fetal calf serum, and the
medium was changed 6 h after infection to remove most of the
vaccinia-encoded B15R protein, which is predominantly synthesized
during the early phase of infection. The supernatant of the infected
cells was collected 24 h later, adjusted to 20 mM Hepes pH
7.9, and incubated overnight at 4 °C with 1 ml of
Ni-NTA-agarose/100 ml of supernatant. The
Ni
-NTA-agarose beads were washed with 3 volumes of
PBS and then with 2 volumes of PBS containing 40 mM imidazole.
The histidine-tagged proteins were eluted with 200 mM imidazole in PBS containing 10% glycerol. Insulin (Sigma) was
added as carrier protein to a final concentration of 0.1%. Purification
of the histidine-tagged proteins was followed by Western blot analysis
with the monoclonal antibody 12CA5 directed against the hemagglutinin
epitope tag.
IL-1 Binding Assays
Histidine-tagged proteins
(20 µg) were rebound to Ni
-NTA-agarose beads
(0.5 ml). All binding assays were performed in a total volume of 25
µl containing PBS and 0.1% bovine serum albumin. The experiment
shown in Fig. 2A was carried out by incubating
Ni
-NTA-agarose beads containing 1 ng of sIL-1R (mixed
with empty carrier beads) with 0.5-10 nM
I-labeled mouse IL-1
for 15 h at 4 °C with
gentle rocking. The beads were washed twice with 250 µl of PBS
containing 0.1% bovine serum albumin. Input and bound radioactivity was
measured in a
-counter. An excess (60 nM) of unlabeled
mouse IL-1
competitor was added to a duplicate binding reaction to
determine the nonspecifically bound radioactivity. For the competition
experiment shown in Fig. 2B,
Ni
-NTA-agarose beads containing
200 ng of Fit-1S
protein were incubated with 2.5 nM
I-labeled
mouse IL-1
plus increasing concentrations of unlabeled mouse IL-1
or IL-6 for 3 h at room temperature followed by two wash steps and
measurement of the bound radioactivity.
binding to sIL-1R. Direct binding of
I-labeled mouse IL-1
to purified sIL-1R was measured
as described under ``Materials and Methods.'' A K value of 0.14
10
M
was determined by replotting the binding data in the Scatchard
coordinate system (on the right). B, competition of
binding of radiolabeled IL-1
to Fit-1S by unlabeled IL-1 and IL-6.
Binding of 1 ng of
I-labeled mouse IL-1
(100
µCi/µg) to purified Fit-1S (200 ng) in the presence of
increasing amounts of unlabeled interleukins was determined as
described under ``Materials and Methods.'' Average values of
two independent experiments are shown.
Transient Transfection and Luciferase Assay
Jurkat
cells were grown in RPMI 1640 medium supplemented with 10% fetal calf
serum (Life Technologies, Inc.). Cells (10) were
transfected by the DEAE-dextran method with 4 µg of expression
plasmid and 2 µg of a luciferase reporter gene containing three
NF-
B sites upstream of the herpes simplex virus thymidine kinase
promoter(20) . Transfected cells were split onto 6-cm dishes,
and IL-1 was added 24 h after transfection. Following an additional
24-h incubation, cells were harvested, washed once with PBS, and lysed
in lysis buffer (Boehringer Mannheim; kit 1363727). Luciferase
activities were determined according to standard protocols and
normalized to the protein content of the cell lysate measured by the
method of Bradford(21) .
Purification of Fit-1S and sIL-1R
IL-1 binding
studies are usually performed with membrane-bound IL-1 receptors
present on intact cells. An extensive search of hematopoietic cells
did, however, not reveal any cell line that was suitable for ligand
binding assay, since all Fit-1M-positive cell lines also expressed at
least one of the two IL-1 receptors. An alternative approach was
suggested by the fact that polypeptides consisting of the extracellular
domain of the type I IL-1 receptor bind IL-1 with similar or at most
10-fold lower affinity than the full-length
protein(22, 23) . We therefore decided to perform IL-1
binding studies with purified Fit-1S protein, which was synthesized in
a vaccinia expression system to guarantee correct protein modification.
A polypeptide consisting of the extracellular domain of the type I IL-1
receptor (sIL-1R) was used as a control for the binding assay. Both
proteins were tagged at their C terminus by inserting a stretch of 10
histidines and 3 hemagglutinin epitopes (Fig. 1A) to
facilitate biochemical purification and detection by Western blot
analysis, respectively. cDNAs coding for these tagged proteins were
inserted downstream of the strong 11K late promoter of vaccinia virus,
and the corresponding proteins were isolated from supernatants of
vaccinia-infected HeLa cells by chromatography on
Ni-NTA-agarose. As shown in Fig. 1B,
both proteins, Fit-1S and sIL-1R, were purified to near homogeneity and
appeared to be glycosylated as indicated by their retarded migration on
SDS-polyacrylamide gels in relation to the molecular mass of the
unmodified polypeptides (41 and 43 kDa, respectively). A minor protein
species with a similar molecular mass (
37 kDa) as the vaccinia
protein B15R (13, 14) was co-purified with Fit-1S.
However, this protein proved to be different from B15R, as it did not
react with anti-B15R antibodies on Western blots in contrast to the
starting material (supernatant of vaccinia-infected HeLa cells, Fig. 1C). According to its size, this minor polypeptide
could correspond to the unmodified Fit-1S protein.
400 ng)
were analyzed by electrophoresis on a 10% SDS-polyacrylamide gel and
visualized by silver staining. The sizes (in kDa) of marker proteins (M) are shown to the left.C,
Western blot analysis of the starting material (supernatant (sup.) of vaccinia-infected HeLa cells) and purified proteins
with polyclonal rabbit antibodies directed against
B15R.
Fit-1S Binds IL-1
To investigate IL-1 binding, the purified proteins were
rebound to Ni Specifically but with Low
Affinity
-NTA-agarose beads and incubated with
increasing concentrations of
I-labeled IL-1, and the
radioactivity that remained bound to the beads after two washing steps
was measured. To validate this ligand binding assay, we determined the
affinity of mouse IL-1
for the control sIL-1R protein. As shown by
the Scatchard analysis in Fig. 2A, an association
constant (K
) of 0.14
10
M
was measured, which compares
favorably with a previously determined K
value of 0.3
10
M
for binding of IL-1
to a soluble type I IL-1
receptor(23) . This evidence clearly demonstrates that the
vaccinia expression system, purification protocol, and binding assay
used are appropriate for studying ligand binding of soluble IL-1
receptors. In contrast to sIL-1R, the Fit-1S protein had to be used at
high concentrations (>10
M) to detect
any binding of IL-1
. As a consequence, saturation of Fit-1S with
ligand could not be achieved, thus precluding the determination of an
affinity constant by Scatchard analysis. Instead, competition assays
were performed to determine the specificity of ligand binding. Fit-1S
was incubated with
I-labeled IL-1
(1 ng) in the
presence of increasing amounts of unlabeled mouse IL-1
, IL-1
,
and IL-6 (Fig. 2B). IL-1
was clearly able to
compete, although with low efficiency, as a 100-fold excess of
unlabeled IL-1
competed only 50% of the radiolabeled ligand away
from the Fit-1S protein. In contrast, IL-1
and the unrelated IL-6
entirely failed to compete even at high concentrations. Moreover,
radiolabeled rat and human IL-1
did also not interact with
Fit-1S.
(
)We conclude therefore that IL-1
binds specifically to Fit-1S, even though this binding is of low
affinity.
Signaling via the Intracellular Domain of Fit-1M Results
in Activation of NF-
The ligand binding analysis
demonstrated that neither IL-1B
nor IL-1
is a physiological
ligand of Fit-1M. However, the sequence similarity of Fit-1M and the
type I IL-1 receptor in their cytoplasmic domains (16) strongly
suggests that Fit-1M is a signal-transducing molecule that activates
similar intracellular pathways as the type I IL-1 receptor. To test
this possibility, we have generated a chimeric receptor (IR-FM)
consisting of the extracellular domain of the type I IL-1 receptor and
the transmembrane and intracellular regions of the Fit-1M protein (Fig. 3A). Stimulation of the type I IL-1 receptor is
known to result in activation of the transcription factor NF-
B (7) . Moreover, the human T cell line Jurkat has been reported
to lack endogenous IL-1 receptors(24) . We therefore
transiently transfected Jurkat cells with an NF-
B-dependent
luciferase gene and expression vectors directing the synthesis of
Fit-1M, the type I IL-1 receptor, and the chimeric protein IR-FM (Fig. 3B). Cells transfected with the empty expression
vector pRK-7 or the Fit-1M expression vector failed to activate
transcription of the luciferase gene in response to treatment with
either mouse IL-1
or IL-1
. In contrast, the chimeric receptor
IR-FM induced luciferase expression as strongly as the type I IL-1
receptor (Fig. 3B). We next investigated the
possibility that stimulation of Fit-1M may require higher
concentrations of IL-1. However, Fit-1M was unable to induce luciferase
expression even in cells that were treated with a 1000-fold higher dose
of IL-1
than is required for half-maximal stimulation of the IL-1
receptor (Fig. 3C). The transfection efficiency of
Jurkat cells was not high enough to directly demonstrate Fit-1M
expression by Western blot analysis. For this purpose we used the
easily transfectable COP-8 fibroblasts where Fit-1M synthesis could be
readily detected.
The transient transfection data therefore
confirmed that IL-1
is not a physiological ligand of Fit-1M.
However, these data also demonstrated that the intracellular domain of
Fit-1M has a similar signaling potential as the type I IL-1 receptor.
B pathway. A, schematic
representation of wild-type and chimeric receptor proteins. Amino acid
residues are numbered according to the published sequences of the mouse
type I IL-1 receptor (3) and rat Fit-1M protein (16) . TM, transmembrane domain. B, transient transfection
assay. Jurkat cells were transfected with the indicated expression
plasmids or the empty expression vector pRK-7 together with an
NF-
B-dependent luciferase gene as described under ``Materials
and Methods.'' Cells were left untreated or incubated with 6
10
M mouse IL-1
or IL-1
for 24 h. The luciferase activities of three experiments were
normalized to the protein content of the cell lysates and are shown as
average values relative to the expression level observed in untreated
control (pRK-7) cells. C, dose-response curves of wild-type
and chimeric receptors. Transient transfection assays were performed as
described in panel B with the exception that the transfected
cells were treated with increasing concentrations of mouse
IL-1
.
but not IL-1
. However,
IL-1
interacts only with low affinity, indicating that it cannot
be a natural ligand of Fit-1S. The cytokines used in our study were of
mouse origin, while the fit-1 gene was isolated from the rat
genome(16) . However, species-specific differences are unlikely
to account for the lack of IL-1
binding and low affinity binding
of IL-1
, as the two cytokines share 94% (IL-1
) and 98%
(IL-1
) of sequence similarity between mouse and rat(25) .
Moreover, rat IL-1
also failed to interact with
Fit-1S.
B(7) .
Although signaling from the IL-1 receptor to the transcription factor
NF-
B is not well understood, IL-1 has recently been shown to
activate a novel protein kinase cascade related to the
mitogen-activated protein kinase pathway(27) . Two lines of
evidence suggested to us that Fit-1M may be able to activate a
signaling cascade similar to that of the type I IL-1 receptor. First,
the intracellular domains of both proteins are of similar lengths and
share 34% of sequence identity(16) . Second, amino acid
residues of the type I IL-1 receptor, which are essential for
IL-1-inducible transcription of the IL-2 and IL-8
genes(28, 29) , have been conserved in the cytoplasmic
domain of Fit-1M (Fig. 4). We have now provided direct evidence
by transient transfection assay that the intracellular domain of Fit-1M
as part of the chimeric protein IR-FM is capable of signaling to
NF-
B like the type I IL-1 receptor. Fit-1M and the IL-1 receptor
therefore share similar intracellular signaling pathways. The wild-type
Fit-1M protein could, however, not be activated by IL-1, confirming
that a different ligand must interact in vivo with this
receptor. It is thus conceivable that Fit-1M and the IL-1 receptor lead
to activation of a similar set of genes in response to stimulation by
different extracellular signals.
B(31) . Other members of the conserved
Toll/IL-1 receptor family are the myeloid differentiation marker
MyD88(32) , which is encoded by an IL-6-inducible gene, and the
product of the N gene of tobacco, which mediates resistance to
tobacco mosaic virus(33) .
We are grateful to G. Smith for a gift of anti-B15R
antibodies and to C. Dinarello for providing rat IL-1.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.