Cloning and Characterization of an Alternatively Processed Human Type II Interleukin-1 Receptor mRNA
1996; Elsevier BV; Volume: 271; Issue: 34 Linguagem: Inglês
10.1074/jbc.271.34.20965
ISSN1083-351X
AutoresChanglu Liu, Ronald P. Hart, Xin-Jun Liu, William Clevenger, Richard A. Maki, Errol B. De Souza,
Tópico(s)T-cell and B-cell Immunology
ResumoTwo types of interleukin (IL)-1 receptors with three extracellular immunoglobulin-like domains, limited homology (28%), and different pharmacological characteristics termed type I and type II have been cloned from mouse and human cell lines. Both receptors exist in transmembrane and soluble forms; the soluble IL-1 receptor is thought to be post-translationally derived from cleavage of the extracellular portion of the membrane receptors. In preliminary cross-linking studies with radiolabeled IL-1, we found that monkey kidney COS1 cells express a soluble receptor with molecular mass of ∼55-60 kDa, which is different from previously reported soluble IL-1 receptors. This soluble IL-1 receptor protein from COS1 cells was purified to homogeneity by affinity chromatography using recombinant IL-1β as the ligand and shown to have an affinity for human 125I-IL-1β (KD ∼2-3 nM) comparable to the human type II IL-1 receptor (IL-1RII). The purified protein was microsequenced, and the sequence information was used to design primers to clone the COS1 IL-1RII using reverse transcription-coupled polymerase chain reaction; the DNA comparison with monkey COS1 and human IL-1RII indicate that they are 95% identical at the nucleic acid and amino acid levels. In addition, another cDNA, which represents an alternatively processed mRNA of the IL-1RII gene, was also cloned both from monkey COS1 and human Raji cells and was shown to have ∼95% sequence identity between these species. While the cDNA of the novel alternatively processed gene has a 5′ end identical to the IL-1RII, the 200 base pairs at the 3′ end are different and the sequence predicts a soluble IL-1 receptor protein of 296 amino acids. Radioligand binding studies of the alternatively processed IL-1RII mRNA demonstrated kinetic and pharmacological characteristics similar to the known type II IL-1 receptor. COS7 cells (which lack IL-1 receptor) transfected with the transmembrane form of the human IL-1RII cDNA showed 125I-IL-1β binding in both the membrane fractions and supernatant. In contrast, COS7 cells transfected with the alternatively processed human IL-1RII cDNA showed high affinity 125I-IL-1β binding (Ki ∼ 1.2 nM) predominantly in the supernatant; a very small amount of detectable membrane IL-1 binding activity was also observed presumably due to association of the soluble IL-1 receptor and membrane-integrated proteins. In cross-linking and ligand blot studies, the alternatively processed human IL-1RII cDNA-transfected COS7 cells expressed a soluble IL-1 receptor with molecular masses ranging from 60 to 160 kDa, further indicating the association between this soluble IL-1 receptor and other soluble proteins. In summary, we report the purification and characterization of a soluble IL-1 receptor expressed by COS1 cells and the cloning of an alternatively processed type II IL-1 receptor mRNA from both human and COS1 cells. The alternative splicing of a primary transcript leading to a secreted protein provides a potentially important mechanism by which soluble IL-1RII can be produced. Two types of interleukin (IL)-1 receptors with three extracellular immunoglobulin-like domains, limited homology (28%), and different pharmacological characteristics termed type I and type II have been cloned from mouse and human cell lines. Both receptors exist in transmembrane and soluble forms; the soluble IL-1 receptor is thought to be post-translationally derived from cleavage of the extracellular portion of the membrane receptors. In preliminary cross-linking studies with radiolabeled IL-1, we found that monkey kidney COS1 cells express a soluble receptor with molecular mass of ∼55-60 kDa, which is different from previously reported soluble IL-1 receptors. This soluble IL-1 receptor protein from COS1 cells was purified to homogeneity by affinity chromatography using recombinant IL-1β as the ligand and shown to have an affinity for human 125I-IL-1β (KD ∼2-3 nM) comparable to the human type II IL-1 receptor (IL-1RII). The purified protein was microsequenced, and the sequence information was used to design primers to clone the COS1 IL-1RII using reverse transcription-coupled polymerase chain reaction; the DNA comparison with monkey COS1 and human IL-1RII indicate that they are 95% identical at the nucleic acid and amino acid levels. In addition, another cDNA, which represents an alternatively processed mRNA of the IL-1RII gene, was also cloned both from monkey COS1 and human Raji cells and was shown to have ∼95% sequence identity between these species. While the cDNA of the novel alternatively processed gene has a 5′ end identical to the IL-1RII, the 200 base pairs at the 3′ end are different and the sequence predicts a soluble IL-1 receptor protein of 296 amino acids. Radioligand binding studies of the alternatively processed IL-1RII mRNA demonstrated kinetic and pharmacological characteristics similar to the known type II IL-1 receptor. COS7 cells (which lack IL-1 receptor) transfected with the transmembrane form of the human IL-1RII cDNA showed 125I-IL-1β binding in both the membrane fractions and supernatant. In contrast, COS7 cells transfected with the alternatively processed human IL-1RII cDNA showed high affinity 125I-IL-1β binding (Ki ∼ 1.2 nM) predominantly in the supernatant; a very small amount of detectable membrane IL-1 binding activity was also observed presumably due to association of the soluble IL-1 receptor and membrane-integrated proteins. In cross-linking and ligand blot studies, the alternatively processed human IL-1RII cDNA-transfected COS7 cells expressed a soluble IL-1 receptor with molecular masses ranging from 60 to 160 kDa, further indicating the association between this soluble IL-1 receptor and other soluble proteins. In summary, we report the purification and characterization of a soluble IL-1 receptor expressed by COS1 cells and the cloning of an alternatively processed type II IL-1 receptor mRNA from both human and COS1 cells. The alternative splicing of a primary transcript leading to a secreted protein provides a potentially important mechanism by which soluble IL-1RII can be produced. INTRODUCTIONInterleukin 1 (IL-1) 1The abbreviations used are: IL-1interleukin-1IL-1raIL-1 receptor antagonistIL-1RI and IL-1RIIIL-1 receptor I and II, respectivelyRT-PCRreverse transcription-coupled polymerase chain reactionPAGEpolyacrylamide gel electrophoresisDMEMDulbecco's modified Eagle's mediumPBSphosphate-buffered salineBSAbovine serum albuminRACErapid amplification of cDNA ends. is a hormone-like polypeptide that performs many roles in inflammation and immunity (1Dinarello C.A. Blood. 1991; 77: 1627-1652Crossref PubMed Google Scholar, 2Mizel S.B. FASEB J. 1989; 3: 2379-2388Crossref PubMed Scopus (285) Google Scholar, 3Oppenheim J.J. Kovacs E.J. Matsushima K. Durum S.K. Immunol. Today. 1986; 7: 45-56Abstract Full Text PDF PubMed Scopus (1023) Google Scholar). Currently, two forms of IL-1 (IL-1α and IL-1β) and one IL-1 receptor antagonist (IL-1ra) have been characterized (1Dinarello C.A. Blood. 1991; 77: 1627-1652Crossref PubMed Google Scholar). IL-1α and IL-1β (collectively referred to as "IL-1") and IL-1ra elicit their biological effects by binding to specific receptor molecules on the surface of responsive cells. Two types of IL-1 receptors with three extracellular immunoglobulin-like domains, limited homology (28%), and different pharmacological characteristics termed type I (4Sims J.E. March C.J. Cosman D. Widmer M.B. Macdonald H.R. McMahan C.J. Grubin C.E. Wignall J.M. Jackson J.L. Call S.M. Friend D. Alpert A.R. Gillis S. Urdal D.L. Dower S.K. Science. 1988; 24: 585-589Crossref Scopus (703) Google Scholar, 5Chizzonite R. Truitt T. Kilian P.L. Sterns A.S. Nunes P. Parker K.P. Kaffka K.L. Chua A.O. Lugg D.K. Gubler U. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8029-8033Crossref PubMed Scopus (208) Google Scholar) and type II (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar) have been cloned from mouse and human cell lines. IL-1α, IL-1β, and IL-1ra all bind with comparable affinity to the type I IL-1 receptor (IL-1RI), which is expressed mainly on T cells, fibroblasts, keratinocytes, endothelial cells, synovial lining cells, chondrocytes, hepatocytes, brain, and endocrine tissues (1Dinarello C.A. Blood. 1991; 77: 1627-1652Crossref PubMed Google Scholar, 5Chizzonite R. Truitt T. Kilian P.L. Sterns A.S. Nunes P. Parker K.P. Kaffka K.L. Chua A.O. Lugg D.K. Gubler U. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8029-8033Crossref PubMed Scopus (208) Google Scholar, 7Takao T. Culp S.G. De Souza E.B. Endocrinology. 1993; 132: 1497-1504Crossref PubMed Scopus (90) Google Scholar). On the other hand, IL-1β binds with much higher affinity and selectivity to the type II IL-1 receptor (IL-1RII), found primarily on neutrophils and B cells, including the Raji human B cell lymphoma line (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar, 8Bomsztyk K. Sims J.E. Stanton T.H. Slack J. McMahan C.J. Valentine M.A. Dower S.K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8034-8038Crossref PubMed Scopus (149) Google Scholar, 9Dripps D.J. Verderber E. Ng R.K. Thompson R.C. Eisenberg S.P. J. Biol. Chem. 1991; 266: 20311-20315Abstract Full Text PDF PubMed Google Scholar). Functional characterization studies have indicated that the two receptors exert different effects. While the type I IL-1 receptor is a signal transducing molecule for IL-1 (10Mathias S. Younes A. Kan C.-C. Orlow I. Joseph C. Kolesnick R.N. Science. 1993; 359: 519-522Crossref Scopus (389) Google Scholar, 11Sims J.E. Gayle M.A. Slack J.L. Alderson M.R. Bird T.A. Giri J.G. Colotta F. Re F. Mantovani A. Shanebeck K. Grabstein K.H. Dower S.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6155-6159Crossref PubMed Scopus (544) Google Scholar), the type II IL-1 receptor is thought to be a decoy receptor (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar, 12Colotta F. Re F. Muzio M. Bertini R. Polentarutti N. Sironi M. Giri J.G. Dower S.K. Sims J.E. Mantovani A. Science. 1992; 261: 472-475Crossref Scopus (863) Google Scholar). Very recently, a third member of the IL-1 receptor family (designated as IL-1 receptor accessory protein; IL-1RAcP), which has limited homology to both type I and type II receptors, has been cloned from mouse (13Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite R.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar) and rat (14Liu C. Chalmers D. Maki R. De Souza E.B. J. Neuroimmunol. 1996; (in press)Google Scholar) cells. The IL-1RAcP forms a complex with type I IL-1 receptor and either IL-1α or IL-1β but not with IL-1ra and increases the binding affinity of IL-1β for type I IL-1 receptor when the two proteins are co-expressed (13Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite R.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar).The IL-1RII exists in both membrane and soluble forms (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar). The soluble form of IL-1RII, a glycoprotein with molecular mass ∼ 45 kDa, is thought to be post-translationally derived from cleavage of the membrane form (12Colotta F. Re F. Muzio M. Bertini R. Polentarutti N. Sironi M. Giri J.G. Dower S.K. Sims J.E. Mantovani A. Science. 1992; 261: 472-475Crossref Scopus (863) Google Scholar). In preliminary cross-linking studies with radiolabeled IL-1, we found that monkey kidney COS1 cells, a commonly used cell line for transient gene expression, express a soluble receptor with molecular mass of ∼55-60 kDa, significantly larger than the reported soluble type II IL-1 receptor (12Colotta F. Re F. Muzio M. Bertini R. Polentarutti N. Sironi M. Giri J.G. Dower S.K. Sims J.E. Mantovani A. Science. 1992; 261: 472-475Crossref Scopus (863) Google Scholar). In the present study, we purified the soluble IL-1 receptor expressed in COS1 cells and cloned a novel alternatively processed type II IL-1 receptor mRNA from both COS1 and human cells.EXPERIMENTAL PROCEDURESHuman IL-1β Expression and PurificationThe human IL-1β mature peptide coding sequence with one extra methionine codon at the N terminus was amplified by reverse transcription-coupled polymerase chain reaction (RT-PCR) using primers (P1, 5′-GCC ATG GCA CCT GTA CGA TCA CTG-3′; P2, 5′-TTT CGC CAG CCC TAG GGA TTG AGT-3′) derived from the human IL-1β cDNA sequence (15March C.J. Mosley B. Larsen A. Cerretti D.P. Braedt G. Price V.L. Gillis S. Henney C.S. Kronheim S.R. Grabstein K. Conlon P.J. Hopp T.P. Cosman D. Nature. 1985; 315: 641-647Crossref PubMed Scopus (1197) Google Scholar). The amplified cDNA was sequenced and cloned into a prokaryotic expression vector pET-21-d (Novagen) and transfected into Escherichia coli BL21(DE3) (Stratagene). A single colony was inoculated in 1 liter of LB broth containing 50 µg/ml ampicillin and cultured at 37°C with vigorous shaking. Isopropyl-1-thio-β-D-galactopyranoside was added to the cell culture to a final concentration of 0.5 mM when the cell culture reached an OD of 1.0 (at 600 nm), and the cells were cultured for an additional 2 h. The cell mixture was centrifuged at 4°C, 5000 × g for 30 min. The cell pellet was resuspended in 5 ml of phosphate buffer (40 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) containing 5 mM EDTA. The cell suspension was frozen and thawed twice, then resuspended in 5 ml of pH 7.4 phosphate buffer containing 5 mM EDTA, 0.2% Triton X-100, and 200 µg/ml lysozyme (Sigma). The suspension was mixed gently and incubated at 37°C for 20 min or until the cell lysate became clear. The cell lysate was placed on ice and sonicated for 5 min to break down the bacterial chromosomal DNA and decrease the viscosity of the cell lysate. The mixture was then added to 100 ml of 30 mM sodium citrate buffer at pH 3.5 containing 5 mM EDTA with gentle stirring. Two hundred milliliters of phosphate buffer was added to the mixture, and the pH was adjusted to 5.0. The mixture was centrifuged at 4°C, 10,000 × g for 30 min, and the supernatant was collected. Twenty milliliters of SP-Sepharose medium (Pharmacia Biotech Inc.) pre-equilibrated with phosphate buffer (at pH 5.0) was then added to the supernatant and incubated at room temperature for 30 min with gentle agitation. The mixture was centrifuged 1000 × g at room temperature for 5 min, and the supernatant was discarded. The SP-Sepharose medium was loaded onto a column and washed extensively with phosphate buffer at pH 5.0 and eluted with 30 ml of phosphate buffer adjusted to pH 8.0. The eluant was then directly passed through a DEAE-Sepharose (Pharmacia) column and flow-through fractions, which contain the recombinant human IL-1β purified to homogeneity were collected. In general, 1 liter of cell culture gave a yield of ∼30-50 mg of human recombinant IL-1β.IL-1β Affinity Column PreparationHuman recombinant IL-1β purified as described above was cross-linked to a CNBr-activated Sepharose-4B matrix (Pharmacia) as described by the manufacturer.Ligand Receptor Affinity Cross-linkingMonkey kidney COS1 cells cultured under serum-free conditions (DMEM with 10 mM Hepes, 50 units/ml ampicillin and streptomycin) at 37°C and 5% CO2 were centrifuged at 4°C, 10,000 × g for 1 h, and the supernatant was concentrated 20-fold with Centricon-30 (Amicon). One hundred microliters of concentrated supernatant was incubated with 100 pM 125I-labeled human recombinant IL-1β (125I-IL-1β) (DuPont NEN, specific activity of ∼2200 Ci/mmol), either in the presence or absence of 100 nM unlabeled competitors (recombinant human IL-1β, recombinant human IL-1α, or recombinant human IL-1ra), at 4°C overnight. The cross-linker ethylene glycolbis succinimidylsuccinate (Pierce) was then added to the mixture at a final concentration of 2 mM, and the reaction mixture was incubated at room temperature for an additional 20 min. The reaction mixture was then run on to a 4-20% SDS-PAGE under reducing conditions and the gel was dried and exposed to a x-ray film overnight at −70°C with an intensifying screen (Kodak).IL-1β Affinity Precipitation of IL-1 ReceptorCOS1 cells were lysed with cell lysate buffer: 50 mM Tris HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.02% sodium azide, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin as described by Sambrook et al. (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Fifty microliters of IL-1β-Sepharose-4B affinity bead mixture prepared as described above was added to the cell lysate (1 ml) and incubated at 4°C for 4 h. The IL-1β-Sepharose-4B beads were then spun down and washed three times with ice-cold 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40. The 125I-IL-1β ligand blot was then performed as described below.Soluble IL-1 Receptor PurificationCOS1 cells were cultured in serum-free DMEM supplemented with glutamine, sodium pyruvate, penicillin, streptomycin, and 15 mM Hepes. The cell culture medium was collected, and fresh medium was added every 2 days. In total, 2 liters of medium were collected. The medium was centrifuged at 4°C, 10,000 × g for 1 h and then passed though the human IL-1β affinity column at a rate of 20 ml/h. The column was first washed with PBS plus 0.1% Triton X-100, then washed with PBS alone, and eluted with a 0.5-4 M guanidine HCl gradient, and 1 ml of fractions were collected. Aliquots (5 µl) of eluant from each fraction were directly spotted on to a dry nitrocellulose membrane (Schleicher & Schüll), blocked with 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20 (TBST), 1% BSA, and blotted with human 125I-IL-1β as described below. Fractions with human 125I-IL-1β-binding activity were then pooled and dialyzed first against PBS and then against water overnight at 4°C. The dialyzed sample was then lyophilized and redissolved in 200 µl of water. An aliquot (5 µl) was used for the IL-1β ligand blot in order to determine the recovery of the soluble IL-1 receptor. Aliquots (20 µl) were run on a 4-20% SDS-PAGE under non-reducing conditions. The gel was then stained with Coomassie Brilliant Blue. The band of protein that corresponded to the IL-1β-binding activity was sequenced by the Edman Degradation method (Protein Chemistry Laboratory, UC Davis).Ligand BlotThe sample was first run on a 4-20% SDS-PAGE under non-reducing conditions and then transferred to a nitrocellulose membrane (Schleicher & Schüll). The membrane was blocked with TBST containing 1% BSA at room temperature for 30 min. The blocked membrane was incubated at 4°C overnight with gentle agitation either in the presence or absence of 100 nM human unlabeled IL-1β with TBST containing 1% BSA and 30 pM human 125I-IL-1β. The membrane was then washed four times (5 min each) with ice-cold TBST and exposed to a x-ray film overnight at −70°C with an intensifying screen.Cell TransfectionCOS7 cells were cultured in six-well cell culture dishes (3.5 cm) with DMEM containing 10% fetal calf serum and transfected using LipofectAMINE (Life Technologies, Inc.) as described by the manufacturer.Whole Cell Binding AssayThe cell culture medium was removed, and the cells in 3.5-cm cell culture dishes were directly incubated with 1 ml of DMEM, 15 mM Hepes buffer, plus 1% BSA and 60 pM 125I-IL-1β, either in the presence or absence of 200 nM unlabeled IL-1β at room temperature for 2 h and washed three times with ice-cold PBS. The cells were then lysed with 4 M guanidine HCl, and the cell lysate was counted in a γ counter (Packard).Solid Phase Binding Assay for Soluble IL-1 ReceptorTwenty-fold concentrated serum-free cell culture supernatant (concentrated by Centricon-30) was incubated in a 96-well plate (100 µl/well; high protein-binding, Costar) at 4°C for 2 h, and the medium aspirated. The wells were then blocked with 200 µl of 1% nonfat milk in PBS at 4°C for 2 h followed by two washes with PBS. The plates were then incubated with 100 µl/well of DMEM containing 1% BSA with various concentrations of human 125I-IL-1β or human 125I-IL-1α (1 pM to 20 nM) either in the presence or absence of 500 nM unlabeled IL-1β at 4°C overnight. The binding medium was then aspirated, and the wells were washed three times with 200 µl of ice-cold PBS containing 0.1% Triton X-100. The bound 125I-IL-1 was then eluted from wells by adding 200 µl of 2% SDS, and samples were counted in a γ counter.cDNA Cloning StudiesCloning of COS1 Cell IL-1RII cDNATwo primers (P1, 5′-ATG ACT CTG CTA GGA CGG TCC CAG-3′; P2, 5′-TCA TGG GCA AAT GTC AGG ACA CAG-3′) corresponding to portions of the human IL-1RII cDNA sequence (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar) were synthesized and used to amplify a cDNA pool made from COS1 total RNA using an oligo(dT)-adaptor primer (5′-GAC TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TT-3′) (17Frohman M.A. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego1990: 28Google Scholar) for reverse transcription. The resultant cDNA fragment was cloned, sequenced, and shown to have 95% DNA sequence identity to human IL-1RII cDNA, suggesting that this cDNA fragment is COS1 cell IL-1RII cDNA. One oligonucleotide (P3, 5′-GGA CGG TGC TCT GTG GCT TCT G-3′) designed from the cloned COS1 IL-1RII cDNA fragment sequence was used to amplify the 3′ end of COS1 cell IL-1RII cDNA together with the 3′ end adapter primer (5′-GAC TCG AGT CGA CAT CG-3′) described as 3′ RACE by Frohman (17Frohman M.A. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego1990: 28Google Scholar). Two different clones with identical 5′ end regions (about 600 base pairs) were identified. One clone has 95% overall homology to human IL-1RII cDNA and is designated as COS1 mIL-1RII. The second clone, which has a different 3′ end region, is designated as COS1 sIL-1RII.The complete coding sequence for COS1 mIL-1RII was amplified using two primers; 5′ primer (5′-CTC TGG AAG TTG TCA GGA GCA ATG-3′; P4) was derived from the published human IL-1RII cDNA sequence (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar), and 3′ primer (5′-CAT GTG GTA TGT GGG TCA TAG TG-3′; P5) was designed from the 3′ end of COS1 mIL-1RII cDNA cloned by 3′ RACE. The complete coding sequence for COS1 sIL-1RII was amplified using two primers: 5′ primer (5′-CTC TGG AAG TTG TCA GGA GCA ATG-3′; P4) and 3′ primer (5′-CAT GTG GTA TGT GGG TCA TAG TG-3′; P6), which was designed from the 3′ end sequence of COS1 sIL-1RII cDNA cloned by 3′ RACE.Cloning of the Human Soluble IL-1RII cDNAHuman soluble IL-1RII (hsIL-1RII) cDNA containing the complete coding sequence was amplified using two primers; 5′ end primer (5′-CTC TGG AAG TTG TCA GGA GCA ATG-3′; P4) was designed from the published human IL-1RII cDNA sequence (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar); 3′ end primer (5′-CAT GTG GTA TGT GGG TCA TAG TG-3′; P6) was derived from COS1 sIL-1RII cDNA sequence. The complete coding sequence for human membrane IL-1RII (hmIL-1RII) was amplified using primers: 5′ end primer (5′-CTC TGG AAG TTG TCA GGA GCA ATG-3′; P4) and 3′ end primer (5′-CAT TCC ATT TAT TTC ACT TGG GAT AGG-3′; P7), which was derived from the published human IL-1RII cDNA sequence (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar).RNase Protection AssaysRNase protection assays were performed as described by Sambrook et al. (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) using the following probes.COS1 sIL-1RII ProbeA 460-base riboprobe containing 394-base antisense sequence specific for COS1 sIL-1RII mRNA with 295 bases in common to COS1 mIL-1RII was generated by T7 RNA polymerase (Promega).hsIL-1RII ProbeA 450-base riboprobe containing 394-base antisense sequence specific for hsIL-1RII mRNA with 295 bases in common to hmIL-1RII was generated by T3 RNA polymerase (Promega).DISCUSSIONIn the present study, we have identified and purified to homogeneity a soluble IL-1 receptor from monkey kidney COS1 cell culture supernatant with radioligand binding, molecular weight characteristics, and protein sequence identity comparable to the previously characterized soluble human type II IL-1 receptor (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar). Radioligand binding studies with increasing concentrations of 125I-IL-1β demonstrate saturable high affinity specific binding with a KD value of ∼2-3 nM for the purified COS1 soluble protein. The pharmacological characteristics of the purified protein were determined in cross-linking, ligand blot, and solid phase radioligand binding studies. In contrast to 125I-IL-1β, 125I-IL-1α did not show any appreciable signal in the ligand blot assay and bound only to a very limited extent in the solid phase assay; saturability was not seen even at a 20 nM concentration of radioligand. However, in cross-linking studies, IL-1α at a concentration of 100 nM did compete for 125I-IL-1β binding suggesting that it bound the receptor with low affinity. In contrast, IL-1ra was unable to appreciably compete for 125I-IL-1β binding even at a 100 nM concentration. Taken together, the radioligand binding data demonstrate characteristics of the type II IL-1 receptor with a clear cut preference of the protein for IL-1β and the following rank order of potency: IL-1β > IL-1α> IL-1ra. The molecular mass of the soluble COS1 IL-1 receptor as determined in cross-linking and ligand blot assays is ∼55-60 kDa. This molecular mass is somewhat higher than the 45 kDa reported by Sims et al. (12Colotta F. Re F. Muzio M. Bertini R. Polentarutti N. Sironi M. Giri J.G. Dower S.K. Sims J.E. Mantovani A. Science. 1992; 261: 472-475Crossref Scopus (863) Google Scholar). This may be due to glycosylation differences in the cell types analyzed or the association of the receptor with other proteins. A report by Svenson et al. (18Svenson M. Hansen M.B. Heegaard P. Abell K. Bendtzen K. Cytokine. 1993; 5: 427-435Crossref PubMed Scopus (73) Google Scholar) identified a soluble IL-1 binding activity in human serum that has a size of 70-80 kDa, suggesting that various forms of the soluble IL-1 receptor exist.The purified COS1-soluble IL-1 receptor was microsequenced, and the data indicated that 20 of the 21 amino acids were identical to the human type II IL-1 receptor (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar). The protein sequence information was used to design primers to clone the COS1 mIL-1RII cDNA using RT-PCR. The DNA comparison with COS1 mIL-1RII and human IL-1RII indicates that they are 95% identical at the nucleic acid and amino acid levels. The IL-1RII cDNA cloned from COS1 cells was expressed in COS7 cells and was shown to bind human 125I-IL-1β with an affinity (Ki = 1.2 nM) similar to that of human IL-1RII. Although the human IL-1RII only has a short intracellular domain of 29 amino acids, the deduced amino acid sequence of the COS1 mIL-1RII predicts an even shorter intracellular domain of 24 amino acids. While the intracellular domain of mIL-1RII is believed not to be involved in intracellular signaling, this receptor may be capable of transducing a signal through association of either the membrane or soluble forms of the receptor with other proteins that possess signaling capabilities. A novel member of the IL-1 receptor gene family termed the IL-1 receptor accessory protein associates with the type I IL-1 receptor to bind IL-1 with high affinity (13Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite R.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). Whether a comparable association exists for the type II IL-1 receptor remains to be determined.In addition to the COS1 mIL-1RII cDNA discussed above, another cDNA that represents an alternatively processed mRNA of IL-1RII gene was also cloned both from COS1 cells and Raji cells; there was ∼95% sequence identity between the two species. This cDNA clone is identical to the IL-1RII clone reported previously (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar) except for 200 base pairs at the 3′ end that are different. The sequence of this clone suggested that the protein is a soluble IL-1 receptor. The expression of this alternatively processed cDNA clone in COS7 cells showed that most of the soluble IL-1 receptor was secreted into the supernatant; a very small proportion of the binding was present in the membrane fraction and most likely represents membrane-associated rather than trans-membrane protein. This is in marked contrast to COS7 cells transfected with the well established human mIL-1RII receptor in which equivalent amounts of 125I-IL-1β binding were detected in the membrane and supernatant. The binding assay showed that the novel, alternatively processed soluble IL-1 receptor had similar affinity to the membrane-bound IL-1RII, indicating that the IL-1 binding domain of IL-1RII is coded by the 5′ 0.9 kb of the mRNA coding sequence.125I-IL-1β ligand blot assay of the soluble receptor secreted by human sIL-1RII-transfected COS7 cells demonstrated the presence of multiple bands ranging in molecular mass from 60 to 160 kDa. The higher molecular mass bands probably correspond, in part, to complexes of the soluble receptor with other secretory proteins. This soluble receptor may also form complexes with membrane proteins, since sIL-1RII-transfected COS7 cells show membrane IL-1β binding when compared with background binding of mock-transfected COS7 cells. COS1 cells express relatively high levels of sIL-1RII mRNA, and an IL-1 binding protein with similar binding properties of type II IL-1 receptor but with large molecular weight (>100 kDa) was seen in ligand blot assays. These large IL-1 binding proteins may represent complexes formed between sIL-1RII and other proteins or with each other through the formation of disulfide bonds, since sIL-1RII contains an unpaired cysteine.While both the human mIL-1RII and the alternatively processed sIL-1RII forms of the receptor result in high levels of soluble IL-1RII in the supernatant, they most likely occur by different mechanisms. The mIL-1RII has been postulated to be post-translationally processed to the soluble form of the receptor resulting from cleavage of the extracellular protein. The novel sIL-1RII, on the other hand, is produced intracellularly and secreted as a soluble protein. This is the first demonstration that alternative splicing of the primary transcript can be used to generate a soluble IL-1RII protein. The soluble proteins derived from both forms of the the type II IL-1 receptor most likely serve similar roles as decoy receptors and inhibitors of IL-1 function. The relative contribution of the two receptors to the soluble receptor pool is unknown and is probably dependent on a variety of factors. These include cellular expression patterns, the level of basal expression and most importantly the regulation of the two proteins. A survey of the expression pattern of the two forms of the receptor in various cell lines demonstrates that some cells (e.g. Raji cells) express both forms, while other cells (e.g. 293 cells) only express the membrane form of the receptor.In summary, we have purified and characterized a soluble IL-1 receptor from COS1 cell culture supernatant with comparable pharmacological characteristics and a different molecular mass (55-60 kDa) to type II IL-1 receptor. In addition, we have cloned cDNAs of a novel alternatively processed mRNA from both COS1 cells and human cells, which encodes a protein of 296 amino acids with pharmacological characteristics of the soluble type II IL-1 receptor. The contribution of the newly identified type II IL-1 receptor mRNA to the pool of soluble IL-1 receptors as well as its regulation and physiological role in limiting the actions of IL-1 await future studies. INTRODUCTIONInterleukin 1 (IL-1) 1The abbreviations used are: IL-1interleukin-1IL-1raIL-1 receptor antagonistIL-1RI and IL-1RIIIL-1 receptor I and II, respectivelyRT-PCRreverse transcription-coupled polymerase chain reactionPAGEpolyacrylamide gel electrophoresisDMEMDulbecco's modified Eagle's mediumPBSphosphate-buffered salineBSAbovine serum albuminRACErapid amplification of cDNA ends. is a hormone-like polypeptide that performs many roles in inflammation and immunity (1Dinarello C.A. Blood. 1991; 77: 1627-1652Crossref PubMed Google Scholar, 2Mizel S.B. FASEB J. 1989; 3: 2379-2388Crossref PubMed Scopus (285) Google Scholar, 3Oppenheim J.J. Kovacs E.J. Matsushima K. Durum S.K. Immunol. Today. 1986; 7: 45-56Abstract Full Text PDF PubMed Scopus (1023) Google Scholar). Currently, two forms of IL-1 (IL-1α and IL-1β) and one IL-1 receptor antagonist (IL-1ra) have been characterized (1Dinarello C.A. Blood. 1991; 77: 1627-1652Crossref PubMed Google Scholar). IL-1α and IL-1β (collectively referred to as "IL-1") and IL-1ra elicit their biological effects by binding to specific receptor molecules on the surface of responsive cells. Two types of IL-1 receptors with three extracellular immunoglobulin-like domains, limited homology (28%), and different pharmacological characteristics termed type I (4Sims J.E. March C.J. Cosman D. Widmer M.B. Macdonald H.R. McMahan C.J. Grubin C.E. Wignall J.M. Jackson J.L. Call S.M. Friend D. Alpert A.R. Gillis S. Urdal D.L. Dower S.K. Science. 1988; 24: 585-589Crossref Scopus (703) Google Scholar, 5Chizzonite R. Truitt T. Kilian P.L. Sterns A.S. Nunes P. Parker K.P. Kaffka K.L. Chua A.O. Lugg D.K. Gubler U. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8029-8033Crossref PubMed Scopus (208) Google Scholar) and type II (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar) have been cloned from mouse and human cell lines. IL-1α, IL-1β, and IL-1ra all bind with comparable affinity to the type I IL-1 receptor (IL-1RI), which is expressed mainly on T cells, fibroblasts, keratinocytes, endothelial cells, synovial lining cells, chondrocytes, hepatocytes, brain, and endocrine tissues (1Dinarello C.A. Blood. 1991; 77: 1627-1652Crossref PubMed Google Scholar, 5Chizzonite R. Truitt T. Kilian P.L. Sterns A.S. Nunes P. Parker K.P. Kaffka K.L. Chua A.O. Lugg D.K. Gubler U. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8029-8033Crossref PubMed Scopus (208) Google Scholar, 7Takao T. Culp S.G. De Souza E.B. Endocrinology. 1993; 132: 1497-1504Crossref PubMed Scopus (90) Google Scholar). On the other hand, IL-1β binds with much higher affinity and selectivity to the type II IL-1 receptor (IL-1RII), found primarily on neutrophils and B cells, including the Raji human B cell lymphoma line (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar, 8Bomsztyk K. Sims J.E. Stanton T.H. Slack J. McMahan C.J. Valentine M.A. Dower S.K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8034-8038Crossref PubMed Scopus (149) Google Scholar, 9Dripps D.J. Verderber E. Ng R.K. Thompson R.C. Eisenberg S.P. J. Biol. Chem. 1991; 266: 20311-20315Abstract Full Text PDF PubMed Google Scholar). Functional characterization studies have indicated that the two receptors exert different effects. While the type I IL-1 receptor is a signal transducing molecule for IL-1 (10Mathias S. Younes A. Kan C.-C. Orlow I. Joseph C. Kolesnick R.N. Science. 1993; 359: 519-522Crossref Scopus (389) Google Scholar, 11Sims J.E. Gayle M.A. Slack J.L. Alderson M.R. Bird T.A. Giri J.G. Colotta F. Re F. Mantovani A. Shanebeck K. Grabstein K.H. Dower S.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6155-6159Crossref PubMed Scopus (544) Google Scholar), the type II IL-1 receptor is thought to be a decoy receptor (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar, 12Colotta F. Re F. Muzio M. Bertini R. Polentarutti N. Sironi M. Giri J.G. Dower S.K. Sims J.E. Mantovani A. Science. 1992; 261: 472-475Crossref Scopus (863) Google Scholar). Very recently, a third member of the IL-1 receptor family (designated as IL-1 receptor accessory protein; IL-1RAcP), which has limited homology to both type I and type II receptors, has been cloned from mouse (13Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite R.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar) and rat (14Liu C. Chalmers D. Maki R. De Souza E.B. J. Neuroimmunol. 1996; (in press)Google Scholar) cells. The IL-1RAcP forms a complex with type I IL-1 receptor and either IL-1α or IL-1β but not with IL-1ra and increases the binding affinity of IL-1β for type I IL-1 receptor when the two proteins are co-expressed (13Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite R.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar).The IL-1RII exists in both membrane and soluble forms (6McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (616) Google Scholar). The soluble form of IL-1RII, a glycoprotein with molecular mass ∼ 45 kDa, is thought to be post-translationally derived from cleavage of the membrane form (12Colotta F. Re F. Muzio M. Bertini R. Polentarutti N. Sironi M. Giri J.G. Dower S.K. Sims J.E. Mantovani A. Science. 1992; 261: 472-475Crossref Scopus (863) Google Scholar). In preliminary cross-linking studies with radiolabeled IL-1, we found that monkey kidney COS1 cells, a commonly used cell line for transient gene expression, express a soluble receptor with molecular mass of ∼55-60 kDa, significantly larger than the reported soluble type II IL-1 receptor (12Colotta F. Re F. Muzio M. Bertini R. Polentarutti N. Sironi M. Giri J.G. Dower S.K. Sims J.E. Mantovani A. Science. 1992; 261: 472-475Crossref Scopus (863) Google Scholar). In the present study, we purified the soluble IL-1 receptor expressed in COS1 cells and cloned a novel alternatively processed type II IL-1 receptor mRNA from both COS1 and human cells.
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