©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Low Affinity Binding of Interleukin-1 and Intracellular Signaling via NF-B Identify Fit-1 as a Distant Member of the Interleukin-1 Receptor Family (*)

(Received for publication, February 27, 1995; and in revised form, May 11, 1995)

Arnold Reikerstorfer (1) Herbert Holz (2) Hendrik G. Stunnenberg (2) Meinrad Busslinger (1)(§)

From the  (1)Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria and the (2)European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, 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.


INTRODUCTION

The cytokine interleukin-1 (IL-1)()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) .

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 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) .

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 binding and intracellular signaling capacities.


MATERIALS AND METHODS

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.()

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.()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 nMI-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 nMI-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.


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 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 10M 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) .


RESULTS

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.


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 (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 Specifically but with Low Affinity

To investigate IL-1 binding, the purified proteins were rebound to Ni-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 10M was measured, which compares favorably with a previously determined K value of 0.3 10M 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 (>10M) 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-B

The ligand binding analysis demonstrated that neither IL-1 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.


Figure 3: Signaling via the intracellular domain of Fit-1M activates the NF-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 10M 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.




DISCUSSION

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 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.

The type I IL-1 receptor is known to stimulate gene transcription through activation of NF-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.


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-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) .


FOOTNOTES

*
This work was supported by the Institute of Molecular Pathology, the European Molecular Biology Laboratory, and the Austrian Industrial Research Promotion Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: IL, interleukin; PCR, polymerase chain reaction; bp, base pair(s); NTA, nitriloacetate; PBS, phosphate-buffered saline.

H. G. Stunnenberg, unpublished data.

D. Goeddel, unpublished data.

A. Reikerstorfer, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to G. Smith for a gift of anti-B15R antibodies and to C. Dinarello for providing rat IL-1.


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