Identification of Two Major Sites in the Type I Interleukin-1 Receptor Cytoplasmic Region Responsible for Coupling to Pro-inflammatory Signaling Pathways
2000; Elsevier BV; Volume: 275; Issue: 7 Linguagem: Inglês
10.1074/jbc.275.7.4670
ISSN1083-351X
AutoresJennifer L. Slack, Kenneth Schooley, Timothy P. Bonnert, Jennifer L. Mitcham, Eva E. Qwarnström, John E. Sims, Steven Dower,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoType I interleukin-1 receptor is the prototype for a family of proteins, which play a central role in early responses to injury and infection. The similarity of function across the family is reflected in similarity in signaling: all members tested couple to activation of NFκB and stress kinases. The coupling to these pathways is mediated by a 200-residue intracellular domain (the Toll/interleukin-1 receptor domain), in which sequence conservation is primarily confined to three short motifs (boxes 1, 2, and 3) located at amino acid residue positions 10 (box 1), 60 (box 2), and 170 (box 3). We have analyzed the contribution of these motifs to function by alanine scanning mutagenesis of the human interleukin-1 receptor type I. Mutant receptors were tested for expression, ligand binding, activation of receptor-associated kinase(s), NFκB, stress kinases, and transcription. Mutations in all three motifs led to low cell surface expression. Mutants in box 3 were, however, wild type for signaling, whereas mutants in boxes 1 and 2 were defective. We conclude that the conserved motifs box 1 and box 2 mediate the coupling of molecules in the family to inflammation signaling pathways. Type I interleukin-1 receptor is the prototype for a family of proteins, which play a central role in early responses to injury and infection. The similarity of function across the family is reflected in similarity in signaling: all members tested couple to activation of NFκB and stress kinases. The coupling to these pathways is mediated by a 200-residue intracellular domain (the Toll/interleukin-1 receptor domain), in which sequence conservation is primarily confined to three short motifs (boxes 1, 2, and 3) located at amino acid residue positions 10 (box 1), 60 (box 2), and 170 (box 3). We have analyzed the contribution of these motifs to function by alanine scanning mutagenesis of the human interleukin-1 receptor type I. Mutant receptors were tested for expression, ligand binding, activation of receptor-associated kinase(s), NFκB, stress kinases, and transcription. Mutations in all three motifs led to low cell surface expression. Mutants in box 3 were, however, wild type for signaling, whereas mutants in boxes 1 and 2 were defective. We conclude that the conserved motifs box 1 and box 2 mediate the coupling of molecules in the family to inflammation signaling pathways. interleukin Toll/interleukin-1 receptor domain IL-1 receptor associated kinase Interleukin-1α and interleukin-1β (IL-1α and IL-1β)1 are two polypeptide cytokines, which play a central role in coordinating immune and inflammatory responses (1.Dinarello C.A. Curr. Top. Microbiol. Immunol. 1996; 216: 133-165Crossref PubMed Scopus (160) Google Scholar). These cytokines and a structurally related antagonist (IL-1ra) bind to a common receptor composed of two type I integral membrane proteins (IL1RI and IL1RAcP) (2.Sims J.E. March C.J. Cosman D. Widmer M.B. MacDonald H.R. McMahan C.J. Grubin C.E. Wignall J.M. Call S.M. Friend D. Alpert A.R. Gillis S.R. Urdal D.L. Dower S.K. Science. 1988; 241: 585-589Crossref PubMed Scopus (818) Google Scholar, 3.Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite P.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar) with which several intracellular signaling molecules have been reported to be associated. The two proteins are part of a family of molecules of diverse structure, which share in common an intracellular 200-residue domain (the Toll/Interleukin-1 receptor domain (TIR)): IL1RRP, ST2, IL18R, and IL18RacP (4.Mitcham J.L. Parnet P. Bonnert T.P. Garka K.E. Gerhart M.J. Slack J.L. Gayle M.A. Dower S.K. Sims J.E. J. Biol. Chem. 1996; 271: 5777-5783Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 5.Parnet P. Garka K.E. Bonnert T.P. Dower S.K. Sims J.E. J. Biol. Chem. 1996; 271: 3967-3970Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 6.Born T.L. Thomassen E. Bird T.A. Sims J.E. J. Biol. Chem. 1998; 273: 29445-29450Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar) and have a similar overall structure to IL-1RI (3 Ig domains (extracellular) and a TIR domain (cytoplasmic)). These receptors couple to two major inflammatory signaling systems, NFκB and stress kinases. The family also contains a second subgroup of receptor molecules, type I membrane proteins with a TIR domain and an extracellular region composed of a series of leucine rich repeats, and the prototypic member is the insect protein Toll (4.Mitcham J.L. Parnet P. Bonnert T.P. Garka K.E. Gerhart M.J. Slack J.L. Gayle M.A. Dower S.K. Sims J.E. J. Biol. Chem. 1996; 271: 5777-5783Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 7.Gay N.J. Keith F.J. Nature. 1991; 351: 355-356Crossref PubMed Scopus (479) Google Scholar, 8.Sims J.E. Dower S.K. Eur. Cytokine Netw. 1994; 5: 539-546PubMed Google Scholar). Toll plays a central role in innate immunity in adult flies, controlling expression of cecropin genes through NFκB-like sites in their promoters (9.Lemaitre B. Nicolas E. Michaut L. Reichhart J.M. Hoffmann J.A. Cell. 1996; 86: 973-983Abstract Full Text Full Text PDF PubMed Scopus (3086) Google Scholar, 10.Levashina E.A. Ohresser S. Lemaitre B. Imler J.L. J. Mol. Biol. 1998; 278: 515-527Crossref PubMed Scopus (101) Google Scholar). Three other TIR insect receptors have been described; mstprox (4.Mitcham J.L. Parnet P. Bonnert T.P. Garka K.E. Gerhart M.J. Slack J.L. Gayle M.A. Dower S.K. Sims J.E. J. Biol. Chem. 1996; 271: 5777-5783Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), 18 wheeler (11.Williams M.J. Rodriguez A. Kimbrell D.A. Eldon E.D. EMBO J. 1997; 16: 6120-6130Crossref PubMed Scopus (223) Google Scholar), and Tehao; 18 wheeler also plays a role in host defense (11.Williams M.J. Rodriguez A. Kimbrell D.A. Eldon E.D. EMBO J. 1997; 16: 6120-6130Crossref PubMed Scopus (223) Google Scholar). Recently, a series of mammalian Tolls have been identified (tlr1 (4.Mitcham J.L. Parnet P. Bonnert T.P. Garka K.E. Gerhart M.J. Slack J.L. Gayle M.A. Dower S.K. Sims J.E. J. Biol. Chem. 1996; 271: 5777-5783Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 12.Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1471) Google Scholar), tlr2 (12.Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1471) Google Scholar), tlr3 (12.Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1471) Google Scholar), tlr4 (12.Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1471) Google Scholar), tlr5 (12.Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1471) Google Scholar), and tlr6 (13.Takeuchi O. Kawai T. Sanjo H. Copeland N.G. Gilbert D.J. Jenkins N.A. Takeda K. Akira S. Gene (Amst.). 1999; 231: 59-65Crossref PubMed Scopus (359) Google Scholar)). Preliminary evidence suggests that these play a role in innate immunity, most compellingly that the lpsd mutation maps to the tlr4 locus (14.Poltorak A. He X.L. Smirnova I. Liu M.Y. VanHuffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6583) Google Scholar). The family also contains several intracellular proteins, in particular a number of plant proteins, which are implicated in host defense (15.Whitman S. Dinesh-Kumar S.P. Choi D. Hehl R. Corr C. Baker B. Cell. 1994; 78: 1101-1115Abstract Full Text PDF PubMed Scopus (1098) Google Scholar). Finally a mammalian family member myD88 (4.Mitcham J.L. Parnet P. Bonnert T.P. Garka K.E. Gerhart M.J. Slack J.L. Gayle M.A. Dower S.K. Sims J.E. J. Biol. Chem. 1996; 271: 5777-5783Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 16.Lord K.A. Hoffman-Liebermann B. Liebermann D.A. Oncogene. 1990; 5: 1095-1097PubMed Google Scholar), composed of an N-terminal death domain and a C-terminal TIR domain, is involved in coupling the IL-1 receptor complex to downstream signaling pathways (17.Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z.D. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar, 18.Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C.Q. Ghosh S. Janeway C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar, 19.Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (997) Google Scholar, 20.Adachi O. Kawai T. Takeda K. Matsumoto M. Tsutsui H. Sakagami M. Nakanishi K. Akira S. Immunity. 1998; 9: 143-150Abstract Full Text Full Text PDF PubMed Scopus (1744) Google Scholar). It appears therefore that this family of molecules and the signal pathways they couple to represents an ancient host defense system present throughout the biological world (21.Rosetto M. Engstrom Y. Baldari C.T. Telford J.L. Hultmark D. Biochem. Biophys. Res. Commun. 1995; 209: 111-116Crossref PubMed Scopus (107) Google Scholar). There are also several viral genes, which show significant homology to this family (8 and Fig. 1), and it has been suggested that like the B15R gene of vaccinia (22.Spriggs M.K. Hruby D.E. Maliszewski C.R. Pickup D.J. Sims J.E. Buller R.M. VanSlyke J. Cell. 1992; 71: 145-152Abstract Full Text PDF PubMed Scopus (293) Google Scholar, 23.Alcami A. Smith G.L. Cell. 1992; 71: 153-167Abstract Full Text PDF PubMed Scopus (430) Google Scholar), a soluble IL-1 receptor, these may play a role in viral subversion of host defenses. IL-1R family receptors couple to downstream signaling pathways via a number of intermediates. Three pelle homologues have been found in mammals (IRAK-1, IRAK-2, and IRAK-M (19.Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (997) Google Scholar, 24.Grosshans J. Bergmann A. Haffter P. Nusslein-Volhard C. Nature. 1994; 372: 563-566Crossref PubMed Scopus (113) Google Scholar, 25.Cao Z.D. Henzel W.J. Gao X.O. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (782) Google Scholar, 27.Wesche H. Gao X. Li X.X. Kirschning C.J. Stark G.R. Cao Z.D. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 32.Yamin T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar)). IRAK-1 associates with AcP and IRAK-2 with IL-1RI (19.Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (997) Google Scholar). The receptor complex also associates with TRAF-6 (28.Cao Z.D. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1135) Google Scholar), myD88 (17.Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z.D. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar), IIP-1 (30.Mitcham J.L. Sims J.E. Cytokine. 1995; 7: 595Google Scholar), and AcPIP (31.Volpe F. Clatworthy J. Kaptein A. Maschera B. Griffin A.M. Ray K. FEB Lett. 1997; 419: 41-44Crossref PubMed Scopus (42) Google Scholar). In vitro kinase assays have shown that IL-1 activates a receptor-associated kinase (33.Martin M. Bol G.F. Eriksson A. Resch K. Brigelius-Flohe R. Eur. J. Immunol. 1994; 24: 1566-1571Crossref PubMed Scopus (86) Google Scholar, 34.Croston G.E. Cao Z. Goeddel D.V. J. Biol. Chem. 1995; 270: 16514-16517Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). However, recent data suggest that the kinase activity of IRAKs is not necessary for IL-1 signaling (27.Wesche H. Gao X. Li X.X. Kirschning C.J. Stark G.R. Cao Z.D. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). TRAF-6 is the upstream regulator of TAK-1 (35.Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z.D. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1034) Google Scholar) and NFκB-inducing kinase (36.Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1174) Google Scholar) coupling IL-1R to activation of NFκB. To dissect the structure-function relationships in signal activation for the TIR family we have analyzed the effects of cytoplasmic domain point mutations on IL-1RI activity. The mutations we have generated have been placed into regions of the domain most conserved across the family. In addition we have mutated several other charged residues predicted to be at the protein surface. Several previous reports have described the results of site-directed mutagenesis studies of the IL-1R domain (34.Croston G.E. Cao Z. Goeddel D.V. J. Biol. Chem. 1995; 270: 16514-16517Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 37.Heguy A. Baldari C.T. Macchia G. Telford J.L. Melli M. J. Biol. Chem. 1992; 267: 2605-2609Abstract Full Text PDF PubMed Google Scholar, 38.Kuno K. Okamoto S. Hirose K. Murakami S. Matsushima K. J. Biol. Chem. 1993; 268: 13510-13518Abstract Full Text PDF PubMed Google Scholar). In general these have focused on the C-terminal conserved region (RFWXN, where X is a basic residue); the region first noted as suggesting a relationship between Drosophila Toll and IL-1RI (7.Gay N.J. Keith F.J. Nature. 1991; 351: 355-356Crossref PubMed Scopus (479) Google Scholar). Our data suggest that this region is mainly involved in controlling receptor trafficking and that signaling interactions are mediated through two other conserved regions. Thus the receptor may interact with intracellular proteins through at least three sites, providing a rationale for the pattern of sequence conservation across this family of signaling proteins. All receptor constructs were cloned into pDC304, a variant of pDC302 (39.Mosley B. Beckmann M.P. March C.J. Idzerda R.L. Gimpel S.D. VandenBos T. Friend D. Alpert A. Anderson D. Jackson J. Wignall J.M. Smith C. Gallis B. Sims J.E. Urdal D. Widmer M.B. Cosman D. Park L.S. Cell. 1989; 59: 335-348Abstract Full Text PDF PubMed Scopus (609) Google Scholar). To construct the wild type chimeric receptor, we first introduced a BglII site into the murine IL-1RI 3′ of the transmembrane domain. This caused a conservative amino acid sequence change from KVF to KIF (KIF is also the human IL-1RI sequence at this position). The cytoplasmic region of the human IL-1RI was then cloned into the expression construct afterBglII digestion of both receptor cDNAs. The result of an alignment of 29 members of the IL-1 receptor family using amino acid sequence fragments spanning the IL-1R homology domain is shown in Fig. 1. The degree of sequence conservation is plotted as the profile score (40.Gribskov M. McLachlan A.D. Eisenberg D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4355-4358Crossref PubMed Scopus (1176) Google Scholar). The analysis reveals three short motifs in the human IL-1 receptor that are most conserved among the family members, YDAYILY (box 1), IYGRDDY (box 2), and TRFWKNV (box 3). In addition there is a region between box 2 and box 3 that shows a low but significant level of sequence homology across the family. We performed alanine substitution, selecting charged residues that, by hydrophobicity analysis, are predicted to lie in regions of the protein that are (a) predicted to be on the surface and (b) are conserved across the family. There were five exceptions K428A, R431A/S, E474A, K492A, and W514A, the first two in a predicted protein kinase C site (KKSRR) the third and fourth in poorly conserved regions, and the fifth a bulky hydrophobic residue, because mutants at these sites have been described previously. In addition we mutated Tyr-374 the C-terminal residue in box 1 to F, because this is more conservative than Y → A. In addition to the point mutants we also generated two triple substitution mutants, in box 1 and box 2 (368AAA: TYDAYILY→ AAAAILY and 411AAA: IYGRDDY → IYAAADY). We reasoned that this set of mutants would alter potential cytoplasmic binding sites with minimal perturbation of overall protein structure. All mutations were made using the Transformer site-directed mutagenesis kit (CLONTECH) and the appropriate mutagenic oligonucleotides in the type I receptor construct HinMext (see above). Mutations were confirmed by sequencing. We have shown previously that HinMext with a wild type cytoplasmic domain is functionally indistinguishable from human IL-1RI. Transfections were done with the DEAE/dextran method, followed by chloroquine treatment, as described, in 293-EBNA cells (human embryonic kidney cells stably expressing the EBNA-1 nuclear antigen (41.McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Huebner K. Croce C.M. Cannizzano L.A. Benjamin D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (635) Google Scholar). 72-h post-transfection, cells were bound with a125I-labeled rat monoclonal antibody (M5) to the murine IL-1RI or with 125I-labeled human IL-1α. Wells were washed, stripped with sodium hydroxide, and counted, and data were presented as total counts averaged over 2 wells. Controls were done in the presence of 100-fold excess unlabeled antibody or IL-1, and these values were subtracted from the data prior to analysis or presentation. Binding dose-response curves for 125I-labeled human IL-1a were done over a 1000-fold range in IL-1 concentration, and values for affinity constants and site numbers were determined by curve fitting with a simple one site binding model using MLAB for the Macintosh (Civilised Software, Silver Spring, MD). Analysis of total receptor expression was done by extracting transfected cells with 1% Triton X-100, spotting 1-ml aliquots onto nitrocellulose filters, blocking with binding medium containing 5% (w/v) non-fat dry milk, and incubating with 125I-labeled rat monoclonal antibody (M5) to the murine IL-1RI or with125I-labeled human IL-1α, as described previously (42.Urdal D.L. Call S.M. Jackson J.J. Dower S.K. J. Biol. Chem. 1988; 263: 2870-2877Abstract Full Text PDF PubMed Google Scholar). Binding of the labeled probes to the receptor blots was analyzed either by γ-counting or by a PhosphorImager. IL-1a and muIL1RM5 were radiolabeled by the chloramine-T method as described elsewhere (42.Urdal D.L. Call S.M. Jackson J.J. Dower S.K. J. Biol. Chem. 1988; 263: 2870-2877Abstract Full Text PDF PubMed Google Scholar). IL-1α was recombinant human protein prepared as described previously from crude material expressed in Escherichia coli(43.Dower S.K. Kronheim S. Hopp T.P. Cantrell M. Deeley M. Henney C.S. Gillis S. Urdal D.L. Nature. 1986; 324: 266-268Crossref PubMed Scopus (373) Google Scholar). 2 days post-transfection, confluent transfected cells were stimulated with 1.0 ng/ml huIL-1α for 30 min for the NF-κB assay. For the Thr-669 kinase assay (44.Bird T.A. Sleath P.R. deRoos P.C. Dower S.K. Virca G.D. J. Biol. Chem. 1991; 266: 22661-22670Abstract Full Text PDF PubMed Google Scholar), stimulation was with 10.0 ng/ml huIL-1 for 15 min. For the IL-8 promoter activation assay, cells were stimulated 1 day post-transfection with 1 ng/ml huIL-1 for 12–16 h. P3 antibody (a polyclonal sheep serum raised against human IL-1R type I) was added 5 min prior to stimulation at a 1:100 dilution to all assays. The antibody does not recognize the mouse extracellular domain of Hin-Mext. For the NFκB gel shift assays, nuclear extracts were prepared as described (4.Mitcham J.L. Parnet P. Bonnert T.P. Garka K.E. Gerhart M.J. Slack J.L. Gayle M.A. Dower S.K. Sims J.E. J. Biol. Chem. 1996; 271: 5777-5783Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) from transfected cell samples, 2 days post-transfection, after appropriate blocking (P3) and IL-1 stimulation. A double-stranded synthetic oligonucleotide probe (5′-TGACAGAGGGGACTTTCCGAGAGGA-3′) containing the kB enhancer element from the immunoglobulin k light chain was 5′-end-labeled by phosphorylation with [γ-32P]ATP. Nuclear extracts (10 mg of total protein) were incubated with radiolabeled probe for 20 min at room temperature, and protein-DNA complexes were resolved by electrophoresis in 0.5 × Tris-borate EDTA, 10% polyacrylamide gels (Novex). The gels were dried and imaged on a PhosphorImager. Quantitative analysis of the images was done using the ImageQuant package (Molecular Dynamics). For the Thr-669 kinase assays (44.Bird T.A. Sleath P.R. deRoos P.C. Dower S.K. Virca G.D. J. Biol. Chem. 1991; 266: 22661-22670Abstract Full Text PDF PubMed Google Scholar), cytoplasmic extracts were prepared from transfected cell samples 2 days post-transfection after appropriate blocking and stimulation. 10 ml of extract was added to 20 ml of a reaction mixture containing 20 mm HEPES, pH 7.4, 15 mm MgCl2, 15 mm ATP (kinase buffer), 75 μmCi/ml [γ-32P]ATP, and 750 mm substrate peptide (residues 663–673 of EGFR). Blanks were incubated with distilled H20 in place of the peptide. After incubation at 30 °C for 20 min, reactions were terminated by the addition of formic acid and cleared by centrifugation, and 30 ml of supernatant were spotted on phosphocellulose paper discs. After washing (three times with 75 mm orthophosphoric acid) and drying, peptide-incorporated counts were determined by Cerenkov counting. Results are expressed as the ratio of Thr-669 kinase activity in cells treated with P3 alone to cells treated with P3 plus IL-1. For IL-1 receptor-associated kinase assays, cells (1 × 105 cells/well in a 12-well tissue culture plate) were transfected with 1000 ng of chimeric wild type or mutant receptors. 2 days post-transfection cells were stimulated with 1 ng/ml IL-1 or left unstimulated, extracted, and immunoprecipitated with the nonblocking rat monoclonal antibody (M5) to the murine IL-1RI. The immunoprecipitates were incubated with [γ-32P]ATP and histone H1, in kinase buffer (see above), washed, and analyzed by SDS-polyacrylamide gel electrophoresis as described previously (33.Martin M. Bol G.F. Eriksson A. Resch K. Brigelius-Flohe R. Eur. J. Immunol. 1994; 24: 1566-1571Crossref PubMed Scopus (86) Google Scholar). For the IL-8 promoter activation assay cells (1 × 105cells/well in a 12-well tissue culture plate) were cotransfected with 500 ng of chimeric wild type or mutant receptor and 1500 ng of the pIL8p reporter plasmid. 1 day post-transfection, the medium was changed, and the cells were blocked with the P3 polyclonal antibody at a 1:100 dilution and either stimulated with 1 ng/ml IL-1 or left unstimulated. 12–16 h post-stimulation, cells were washed twice with binding medium (RPMI 1640, 20 mm HEPES, pH 7.4, 0.1% (w/v) sodium azide) containing 5% (w/v) non-fat dry milk and blocked with 2 ml of the same medium at room temperature for 30 min. Cells were then incubated at room temperature for 60–90 min with 1.5 ml/well of the same medium containing 1 μg/ml of the mouse anti-human IL-2R a antibody 2A3 (45.Dower S.K. Hefeneider S.H. Alpert A.R. Urdal D.L. Mol. Immunol. 1985; 22: 937-947Crossref PubMed Scopus (21) Google Scholar) with gentle rocking. Cells were washed once with the same medium and incubated with 1 ml/well of the same medium containing 1:100 dilution of 125I-goat anti-mouse IgG (Sigma) for 60 min at room temperature. Wells were washed four times with the same medium and twice with phosphate-buffered saline, stripped by the addition of 1 ml of 0.5 m NaOH, and total counts were determined. Results are expressed as total cpm averaged over two wells. 293 EBNA cells (1 × 105 cells/well) cultured in 8-well Lab-Tek Chambered coverglasses were transfected with 1000 ng of plasmid DNA by the DEAE dextran method, cultured for a further 72 h, and fixed with 2% paraformaldehyde (nonpermeabilized) or with 2% paraformaldehyde containing 0.1% Triton X-100 (permeabilized). Autofluorescence was quenched by treatment with 50 mm NH4Cl for 10 min. For the permeabilized cell analysis, cells were then incubated for 1 h in blocking/permeabilization buffer (phosphate-buffered saline containing 5% normal goat serum and 0.1% Triton X-100). Cells were then incubated with rat monoclonal antibody (M5) to the murine IL-1RI (1 μg/ml in blocking or blocking/permeabilization buffer) for 1 h. Cells were washed three times with blocking/permeabilization buffer and incubated with biotinylated goat anti-rat immunoglobulin (Molecular Probes, 2 μg/ml in blocking/permeabilization buffer) for 1 h. Cells were washed three times with buffer and incubated with a streptavidin Texas Red conjugate (Molecular Probes) at 0.2 μg/ml in buffer for 30 min. Cells were washed three times with buffer and twice with phosphate-buffered saline and mounted in 100 mg/ml DABCO (1,4 diaxabicyclo [2.2.2] octane) in 50% glycerol/phosphate-buffered saline. Data were acquired with a Multiprobe 2001 Laser Scanning Confocal Microscope (Molecular Dynamics) interfaced to a Silicon Graphics workstation, using the 568 nm line of a krypton/argon mixed gas laser, a 50-μm pinhole, and a 60x Plan Apo oil immersion objective. After acquisition, data files were exported to a Macintosh, converted to 8 bit TIFF images, and imported into Photoshop for compositing for presentation. All mutants were tested for cell surface expression after transient expression in 293-EBNA cells. Mutations in all three conserved motifs generated a low expression phenotype (Fig. 2). It is not clear from this experiment whether the apparent depression in expression results from a decrease in affinity for ligand or a decrease in receptor expression. However we show below that the affinity of all mutants for ligand is indistinguishable from the chimera with a wild type cytoplasmic domain (see Fig. 6). The effect appears to be clear cut, mutants either fall into a group that are wild type for expression or into a low expression group. Because all three conserved motifs appeared to be involved, subsequent transfection experiments were modified to compensate, for wild type expressers, 50 ng of expression plasmid were used, and for low expressers, 1000 ng, because this is saturating for expression in the system. The expected cell surface expression is indicated by the dotted line in Fig. 2.Figure 6Quantitative analysis of mutant IL-1 receptor function. Upper panel, lack of effect of cytoplasmic domain mutations on IL-1α binding activity of the receptor. 293-EBNA cells were transfected and cultured as described under “Experimental Procedures.” Subsequently cells were incubated with125I-IL-1α spanning a 1000-fold range in concentration (10 points). Unlabeled IL-1 was added to determine nonspecific binding (2 points) as described previously, and data were corrected by subtraction. Counts were determined by γ-counting. Data were converted to molecules ligand bound/cell and free ligand concentration (M) and analyzed by nonlinear least square fitting of a simple one site binding model using MLAB. The affinity constants are shown with the S.E. derived from the curve fit as the error bar. Middle panel, quantitative analysis of the effect of cytoplasmic domain mutations on the capacity of IL-1 receptor to activate NFκB DNA binding activity in response to ligand binding. Data from electrophoretic mobility shift assay experiments were obtained using a PhosphorImager, and the 16 bit digital images were analyzed using ImageQuant software. The NFκB activity in 293-EBNA cells is detected as two bands, a faster moving band, which is constitutive, and a slower band, which is IL-1-inducible; only the slower band was used in the analysis. Data are expressed as the ratio of band intensity after 30 min of IL-1α treatment with that observed in an untreated cultured transfected with the same receptor construct and cultured in parallel. Lower panel, quantitative analysis of the effect of cytoplasmic domain mutations on the capacity of IL-1 receptor to activate stress kinases in response to ligand binding. Transfected cultures were extracted and analyzed for the capacity to phosphorylate the stress kinase substrate peptide (residues 663–673 of EGFR) as described elsewhere (44.Bird T.A. Sleath P.R. deRoos P.C. Dower S.K. Virca G.D. J. Biol. Chem. 1991; 266: 22661-22670Abstract Full Text PDF PubMed Google Scholar). Data are expressed as the ratio of activity after 15 min of IL-1α treatment (10 ng/ml; 6 × 10−10m with that observed in an untreated cultured transfected with the same receptor construct and cultured in parallel. Error bars are from triplicate assays (S.D.).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To analyze this further, the binding of the rat anti-mouse type I IL-1 receptor monoclonal antibody (M5) was compared with that of IL-1. All transfected cells bound both antibody and IL-1 with 50% as many antibody binding sites as IL-1 binding sites (Fig.3 A), irrespective of the level of IL-1 binding sites present. This is consistent with bivalent binding of the antibody, as shown previously. M5 is a nonblocking antibody and recognizes an epitope outside the IL-1 binding site, thus the constant ratio of ligand to antibody bound suggests that none of the mutant receptors have any gross perturbation of ligand binding. Further the data show that at the doses established from Fig. 2, the low expresser group express at the cell surface in the same range as the wild type group. The total level of expression (cell surface and intracellular) of all mutants was determined by M5 and by IL-1 binding in a dot blot assay as described prev
Referência(s)