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• W1972508857 abstract "The interferon-α (IFNα) receptor consists of two subunits, the IFNα receptor 1 (IFNaR1) and 2 (IFNaR2) chains. Following ligand binding, IFNaR1 is phosphorylated on tyrosine 466, and this site recruits Stat2 via its SH2 domain. In contrast, IFNaR2 binds Stat2 constitutively. In this study we have characterized the Stat2-IFNaR2 interaction and examined its role in IFNα signaling. Stat2 binds the major IFNaR2 protein but not a variant containing a shorter cytoplasmic domain. The interaction does not require a STAT SH2 domain. Both tyrosine-phosphorylated and non-phosphorylated Stat2 bind IFNaR2 in vitro; however, relatively little phosphorylated Stat2 associates with IFNaR2 in vivo. In vitro binding assays defined IFNaR2 residues 418–444 as the minimal interaction domain and site-specific mutation of conserved acidic residues within this domain disrupted in vitro and in vivobinding. An IFNaR2 construct carrying these mutations was either (i) overexpressed in 293T cells or (ii) used to complement IFNaR2-deficient U5A cells. Unexpectedly, the activity of an IFNα-dependent reporter gene was not reduced but, instead, was enhanced up to 2-fold. This suggests that this particular IFNaR2-Stat2 interaction is not required for IFNα signaling, but might act to negatively inhibit signaling. Finally, a doubly truncated recombinant fragment of Stat2, spanning residues 136–702, associated with IFNaR2 in vitro, indicating that the interaction with IFNaR2 is direct and occurs in a central region of Stat2 marked by a hydrophobic core. The interferon-α (IFNα) receptor consists of two subunits, the IFNα receptor 1 (IFNaR1) and 2 (IFNaR2) chains. Following ligand binding, IFNaR1 is phosphorylated on tyrosine 466, and this site recruits Stat2 via its SH2 domain. In contrast, IFNaR2 binds Stat2 constitutively. In this study we have characterized the Stat2-IFNaR2 interaction and examined its role in IFNα signaling. Stat2 binds the major IFNaR2 protein but not a variant containing a shorter cytoplasmic domain. The interaction does not require a STAT SH2 domain. Both tyrosine-phosphorylated and non-phosphorylated Stat2 bind IFNaR2 in vitro; however, relatively little phosphorylated Stat2 associates with IFNaR2 in vivo. In vitro binding assays defined IFNaR2 residues 418–444 as the minimal interaction domain and site-specific mutation of conserved acidic residues within this domain disrupted in vitro and in vivobinding. An IFNaR2 construct carrying these mutations was either (i) overexpressed in 293T cells or (ii) used to complement IFNaR2-deficient U5A cells. Unexpectedly, the activity of an IFNα-dependent reporter gene was not reduced but, instead, was enhanced up to 2-fold. This suggests that this particular IFNaR2-Stat2 interaction is not required for IFNα signaling, but might act to negatively inhibit signaling. Finally, a doubly truncated recombinant fragment of Stat2, spanning residues 136–702, associated with IFNaR2 in vitro, indicating that the interaction with IFNaR2 is direct and occurs in a central region of Stat2 marked by a hydrophobic core. interferon Janus tyrosine kinase signal transducers and activators of transcription interferon-stimulated responsive element glutathione S-transferase wild type mutant 1 Src homology 2 domain hemaggluttinin Type I interferons (IFNs),1 including multiple interferon-α (IFNα) isoforms and interferon-β (IFNβ), bind a common receptor complex consisting of two subunits, interferon-α receptor 1 (IFNaR1) and interferon-α receptor 2 (IFNaR2). IFNaR1 displays low affinity for most human IFNα isoforms and appears to be primarily a signal transducing subunit (1Constantinescu S. Croze E. Wang C. Murti A. Basu L. Mullersman J.E. Pfeffer L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9602-9606Crossref PubMed Scopus (97) Google Scholar, 2Hwang S.Y. Hertzog P.J. Holland K.A. Sumarsono S.H. Tymms M.J. Hamilton J.A. Whitty G. Bertoncello I. Kola I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11284-11288Crossref PubMed Scopus (321) Google Scholar). The other subunit, IFNaR2, mediates both ligand binding and signaling (3Domanski P. Witte M. Kellum M. Rubinstein M. Hackett R. Pitha P. Colamonici O.R. J. Biol. Chem. 1995; 270: 21606-21611Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 4Lutfalla G. Holland S.J. Cinato E. Monneron D. Reboul J. Rogers N.C. Smith J.M. Stark G.R. Gardiner K. Mogensen K.E. Kerr I.M. Uzé G. EMBO J. 1995; 14: 5100-5108Crossref PubMed Scopus (222) Google Scholar, 5Novick D. Cohen B. Rubinstein M. Cell. 1994; 77: 391-400Abstract Full Text PDF PubMed Scopus (573) Google Scholar). It is expressed as three variants: a soluble receptor (5Novick D. Cohen B. Rubinstein M. Cell. 1994; 77: 391-400Abstract Full Text PDF PubMed Scopus (573) Google Scholar), a short transmembrane form (5Novick D. Cohen B. Rubinstein M. Cell. 1994; 77: 391-400Abstract Full Text PDF PubMed Scopus (573) Google Scholar), and a long transmembrane form believed to be the physiologically relevant receptor (3Domanski P. Witte M. Kellum M. Rubinstein M. Hackett R. Pitha P. Colamonici O.R. J. Biol. Chem. 1995; 270: 21606-21611Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 4Lutfalla G. Holland S.J. Cinato E. Monneron D. Reboul J. Rogers N.C. Smith J.M. Stark G.R. Gardiner K. Mogensen K.E. Kerr I.M. Uzé G. EMBO J. 1995; 14: 5100-5108Crossref PubMed Scopus (222) Google Scholar). The short and long transmembrane forms are referred to as IFNaR2-1 and IFNaR2-2, respectively. IFNaR2-1 is usually expressed at lower levels than IFNaR2-2 and may exert a dominant negative effect on IFNα signaling, although its precise role is unclear (6Pfeffer L.M. Basu L. Pfeffer S.R. Yang C.H. Murti A. Russell-Harde D. Croze E. J. Biol. Chem. 1997; 272: 11002-11005Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). IFNα receptor subunits lack intrinsic enzymatic activity, instead relying on members of the Janus tyrosine kinase (JAK) family to transduce signals. Genetic complementation experiments have linked two JAKs, Tyk2 and Jak1, to IFNα signaling (7Velazquez L. Fellous M. Stark G.R. Pelligrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (698) Google Scholar, 8Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C.W. Pelligrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Crossref PubMed Scopus (635) Google Scholar), and biochemical studies subsequently demonstrated constitutive and direct association of these JAKs with the IFNaR1 and IFNaR2 subunits, respectively (9Yan H. Krishnan K. Lim J.T.E. Contillo L.G. Krolewski J.J. Mol. Cell. Biol. 1996; 16: 2074-2082Crossref PubMed Scopus (85) Google Scholar, 10Yan H. Krishnan K. Greenlund A.C. Gupta S. Lim J.T.E. Schreiber R.D. Schindler C.W. Krolewski J.J. EMBO J. 1996; 15: 1064-1074Crossref PubMed Scopus (157) Google Scholar, 11Colamonici O.R. Uyttendaele H. Domanski P. Yan H. Krolewski J.J. J. Biol. Chem. 1994; 269: 3518-3522Abstract Full Text PDF PubMed Google Scholar, 12Colamonici O.R. Yan H. Domanski P. Handa R. Smalley D. Mullersman J. Witte M. Krishnan K. Krolewski J.J. Mol. Cell. Biol. 1994; 14: 8133-8142Crossref PubMed Google Scholar). Two members of the signal transducer and activator of transcription (STAT) family, Stat1 and Stat2, have also been implicated (13Fu X.-Y. Cell. 1992; 70: 323-335Abstract Full Text PDF PubMed Scopus (303) Google Scholar, 14Schindler C.W. Shuai K. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (713) Google Scholar). Signaling begins with IFNα binding, triggering receptor oligomerization and juxtaposing associated JAKs. A series of auto- and/or transphosphorylations result in Jak1 and Tyk2 activation (8Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C.W. Pelligrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Crossref PubMed Scopus (635) Google Scholar, 11Colamonici O.R. Uyttendaele H. Domanski P. Yan H. Krolewski J.J. J. Biol. Chem. 1994; 269: 3518-3522Abstract Full Text PDF PubMed Google Scholar,15Gauzzi M.C. Velazquez L. McKendry R. Mogensen K.E. Fellous M. Pelligrini S. J. Biol. Chem. 1996; 271: 20494-20500Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and phosphorylation of tyrosine residue 466 on IFNaR1 (10Yan H. Krishnan K. Greenlund A.C. Gupta S. Lim J.T.E. Schreiber R.D. Schindler C.W. Krolewski J.J. EMBO J. 1996; 15: 1064-1074Crossref PubMed Scopus (157) Google Scholar, 12Colamonici O.R. Yan H. Domanski P. Handa R. Smalley D. Mullersman J. Witte M. Krishnan K. Krolewski J.J. Mol. Cell. Biol. 1994; 14: 8133-8142Crossref PubMed Google Scholar, 16Krishnan K. Yan H. Lim J.T.E. Krolewski J.J. Oncogene. 1996; 13: 125-133PubMed Google Scholar). Once phosphorylated, this tyrosine recruits Stat2 in an SH2-dependent manner (10Yan H. Krishnan K. Greenlund A.C. Gupta S. Lim J.T.E. Schreiber R.D. Schindler C.W. Krolewski J.J. EMBO J. 1996; 15: 1064-1074Crossref PubMed Scopus (157) Google Scholar, 17Krishnan K. Singh B. Krolewski J.J. J. Biol. Chem. 1998; 273: 19495-19501Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Activated Tyk2 or Jak1 phosphorylate Stat2 on tyrosine that, in turn, recruits Stat1 to the receptor (10Yan H. Krishnan K. Greenlund A.C. Gupta S. Lim J.T.E. Schreiber R.D. Schindler C.W. Krolewski J.J. EMBO J. 1996; 15: 1064-1074Crossref PubMed Scopus (157) Google Scholar, 18Leung S. Qureshi S.A. Kerr I.M. Darnell Jr., J.E. Stark G.R. Mol. Cell. Biol. 1995; 15: 1312-1317Crossref PubMed Google Scholar). Following Stat1 phosphorylation, the two STATs subsequently heterodimerize via SH2-phosphotyrosine interactions (19Shuai K. Horvath C.M. Huang L.H.T. Qureshi S.A. Cowburn D. Darnell Jr., J.E. Cell. 1994; 76: 821-826Abstract Full Text PDF PubMed Scopus (677) Google Scholar,20Li X. Leung S. Qureshi S.A. Darnell Jr., J.E. Stark G.R. J. Biol. Chem. 1996; 271: 5790-5794Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). With the p48/IRF9 protein, Stat1-Stat2 heterodimers form the interferon-stimulated gene factor 3 complex (14Schindler C.W. Shuai K. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (713) Google Scholar, 21Qureshi S.A. Salditt-Georgieff M. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3829-3833Crossref PubMed Scopus (195) Google Scholar, 22Horvath C.M. Stark G.R. Kerr I.M. Darnell Jr., J.E. Mol. Cell. Biol. 1996; 16: 6957-6964Crossref PubMed Scopus (160) Google Scholar), which binds to the interferon-stimulated responsive element (ISRE) to direct transcription. Latent STATs were initially believed to exist as monomers in the cytoplasm, but recent studies indicate that they exist primarily in high molecular weight complexes (23Ndubuisi M.I. Guo G.G. Fried V.A. Etlinger J.D. Sehgal P.B. J. Biol. Chem. 1999; 274: 25499-25509Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 24Lackmann M. Harpur A.G. Oates A.C. Mann R.J. Gabriel A. Meutermans W. Alewood P.F. Kerr I.M. Stark G.R. Wilks A.F. Growth Factors. 1998; 16: 39-51Crossref PubMed Scopus (47) Google Scholar). In this regard, constitutive association of Stat2 with IFNaR2 has been reported, and termed “pre-docking” (25Li X. Leung S. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1997; 17: 2048-2056Crossref PubMed Scopus (162) Google Scholar). Although it has been suggested that this interaction facilitates the subsequent SH2 domain-dependent Stat2 recruitment, the biological significance of this association remains unclear. Therefore, we sought to delineate the Stat2 interaction domain on IFNaR2 and investigate its role in IFNα signaling. We have found that the minimal Stat2 binding region is between residues 418 and 444 of IFNaR2, although a larger domain (residues 340–462) is required for maximal binding. Mutation of conserved acidic amino acids corresponding to residues 435–438 disrupts IFNaR2 binding to Stat2. Importantly, expression of these mutated IFNaR2 constructs in two separate cell systems demonstrates that ISRE-driven reporter gene activity is increased relative to cells expressing the wild-type receptor. Thus, this interaction is dispensable for effective IFNα signaling and instead might function in the negative regulation of such signaling. The following antibodies were used: 4G10, against phosphotyrosine (Upstate Biotechnology); SC-138, against glutathione S-transferase (GST) (Santa Cruz Biotechnology); SC-805, against the influenza virus hemagglutinin (HA) epitope (Santa Cruz Biotechnology) (26Kolodziej P.A. Young R.A. Methods Enzymol. 1991; 194: 508-519Crossref PubMed Scopus (423) Google Scholar); H15, against the polyhistidine tag epitope (Santa Cruz Biotechnology); T20220, against Tyk2 (BD Transduction Laboratories); J24320, against Jak1 (BD Transduction Laboratories); polyclonal rabbit antisera against the carboxyl terminus of Tyk2 (11Colamonici O.R. Uyttendaele H. Domanski P. Yan H. Krolewski J.J. J. Biol. Chem. 1994; 269: 3518-3522Abstract Full Text PDF PubMed Google Scholar); polyclonal rabbit antiserum against Stat2 (C. Schindler, Columbia University, College of Physicians and Surgeons, New York, NY) (14Schindler C.W. Shuai K. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (713) Google Scholar); and AC-15 against β-actin (Sigma). Recombinant IFNα2 was from M. Brunda (Hoffmann-La Roche, Nutley, NJ). Baculoviruses encoding Stat1, Stat2, and Jak2 (12Colamonici O.R. Yan H. Domanski P. Handa R. Smalley D. Mullersman J. Witte M. Krishnan K. Krolewski J.J. Mol. Cell. Biol. 1994; 14: 8133-8142Crossref PubMed Google Scholar) were used to infect Sf9 cells (Invitrogen) (27O'Reilly D.R. Miller L.K. Luckow V.A. Baculovirus Expression Vectors: a Laboratory Manual. W. H. Freeman, New York1992Google Scholar). Four human cell lines, embryonic kidney 293T cells (H. Young, Columbia University, College of Physicians and Surgeons, New York, NY), osteogenic sarcoma U20S cells (ATCC, Manassas, VA), IFNaR2-deficient U5A cells (4Lutfalla G. Holland S.J. Cinato E. Monneron D. Reboul J. Rogers N.C. Smith J.M. Stark G.R. Gardiner K. Mogensen K.E. Kerr I.M. Uzé G. EMBO J. 1995; 14: 5100-5108Crossref PubMed Scopus (222) Google Scholar), and 2fTGH cells, the parental line for U5A cells, containing wild-type IFNaR2 (the latter two lines are from G. Stark, Lerner Research Institute, Cleveland Clinic Foundation), were all maintained as adherent cultures in Dulbecco's modified Eagle's medium plus 10% heat-inactivated fetal calf serum. Fragments of the IFNaR2 cytoplasmic domain were generated by PCR and were cloned, sequenced, and transferred into an appropriate pGEX vector (Amersham Biosciences) to encode GST-IFNaR2 fusion proteins. To create site-specific mutants the overlapping PCR technique (28Horton R.M. Cai Z.L. Ho S.N. Pease L.R. Biotechniques. 1990; 8: 528-535PubMed Google Scholar) was used to generate DNA fragments (spanning residues 376–462) with alanine substitutions at positions 435–438 (mutant 1, DDED to AAAA), and 440–443 (mutant 2, DDLE to AAAA) of IFNaR2, respectively. The full-length IFNaR2 cDNA and the corresponding 435–438 mutant were cloned into pMT2T for expression in human cells (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). A DNA fragment flanked by XhoI sites, encoding amino acids 136–702 of Stat2, was PCR-amplified usingPfu polymerase (Stratagene), cloned, sequenced, and then transferred into pET15b (Novagen) for expression in bacteria. Truncated Stat2 constructs (corresponding to residues 1–293, 1–323, and 123–517, respectively) were generated using convenient restriction sites and cloned into pMT2T. The eukaryotic expression construct encoding a constitutively expressed β-galactosidase gene has been previously described (30Herbomel P. Bourachot B. Yaniv M. Cell. 1984; 39: 653-662Abstract Full Text PDF PubMed Scopus (556) Google Scholar). The luciferase gene construct is under the control of an ISRE from the ISG-15 gene (31Improta T. Pine R. Cytokine. 1997; 9: 383-393Crossref PubMed Scopus (15) Google Scholar). Escherichia coli DH5α, containing the appropriate pGEX-IFNaR2 construct, was grown at 37 °C to log phase and induced at 30 °C with 0.1 mmisothiogalactopyranoside (IPTG) for 3–4 h. Pelleted bacteria, resuspended in STE (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 1 mm EDTA) containing 150 μg/ml of lysozyme and 1 mm phenylmethylsulfonyl fluoride, were sonicated. Inclusion bodies were pelleted and solubilized in Sarkosyl (32Grieco F. Hay J.M. Hull R. Biotechniques. 1992; 13: 856-857PubMed Google Scholar) and stored at −80 °C in STE containing 1% Triton X-100, 0.1% sarcosyl, 10% glycerol. Purification of His-tagged Stat2-(136–702) will be described in detail. 2A. Z. Saleh and J. J. Krolewski, manuscript in preparation. Briefly, E. coli BL21 (λDE3) expressing His-tagged Stat2 protein were grown at 37 °C and induced at 30 °C with IPTG. Following cell lysis and centrifugation, the protein was purified by chromatography on hydroxylapatite (Bio-Rad) and nickel resin (Invitrogen). Stat2 was prepared from baculovirus-infected Sf9 cells 48 h post-infection or from calcium phosphate transfected 293T cells 48 h post-transfection, by lysing cells in TBES/1% Nonidet P-40 buffer (20 mm Tris-HCl, pH 7.5, 137 mm NaCl, 5 mm EDTA, 1% Nonidet P-40) followed by centrifugation to remove nuclei and debris. GST-IFNaR2 fusions, immobilized on glutathione-agarose beads (Sigma), were washed with STE, 1% Triton X-100, 0.1% Sarkosyl, and incubated with recombinant Stat2. Following washes in TBES/1% Nonidet P-40 buffer, complexes were eluted in sample buffer and immunoblotted as described below. Transient transfection of subconfluent 293T cells (∼8 × 106 cells on 15-cm dishes), U2OS cells (∼1 × 106 cells on 10-cm dishes), stable U5A derivatives created as described below (∼8 × 105 cells on 10-cm dishes), or 2fTGH cells (∼8 × 105 cells on 10-cm dishes) was performed using calcium-phosphate precipitates and the indicated amount(s) of plasmid DNA. In some experiments, 10-cm dishes of 293T cells (∼3 × 106 cells) were transiently transfected with 20 μg of the appropriate plasmid DNA employing LipofectAMINE Plus (Invitrogen), according to the manufacturer's instructions. Forty-eight hours post-transfection, transiently transfected cultures were subjected to immunoprecipitation and/or immunoblotting or reporter gene analysis, as described below. Stable transfectants of U5A cells were created by co-transfecting 10-cm dishes (∼8 × 105 cells) with 10 μg of HA-tagged pMT2T-IFNaR2 expression constructs and 1 μg of pcDNA3.1(+), which carries a neomycin resistance gene. Forty-eight hours post-transfection cells were diluted into medium containing 400 μg/ml G418 (Invitrogen). Individual clones were propagated and screened by immunoblotting cytoplasmic lysates with an anti-HA antibody to identify lines expressing IFNaR2 proteins. Stable derivative cell lines were periodically cultured in the presence of G418 and monitored by immunoblotting with anti-HA antibodies to ensure the continued expression of the exogenous IFNaR2 constructs. Nearly confluent cultures were lysed in TBES/1% Nonidet P-40 plus 0.2 mmphenylmethylsulfonyl fluoride. Nuclei, debris, and cell membranes were removed by centrifugation to yield a mainly cytoplasmic protein extract. In some cases portions of these crude extracts were mixed with sample buffer, resolved by SDS-PAGE, transferred to nitrocellulose (Osmonics), blocked in either 5% nonfat dry milk or 3% bovine serum albumin, and sequentially probed with an appropriate primary antibody, followed by a secondary antibody linked to horseradish peroxidase. Bands were visualized by chemiluminescence (Pierce Super Signal Substrate). In other cases proteins were first immunoprecipitated by incubating the cytoplasmic lysates with appropriate antibodies and protein A-Sepharose beads (Sigma). The antibody-protein complexes were collected by centrifugation, washed, eluted by boiling in sample buffer, and immunoblotted as described above. The primary antibody dilutions used for immunoblotting were: 1:3000 for 4G10, 1:10,000 for SC805, H15, SC-138, and AC-15, and 1:20,000 for the anti-Stat2 polyclonal antibody. To monitor protein loading on some immunoblots, membranes were stripped, washed, and reprobed with appropriate antibodies. In the case of U2OS cells, duplicate 10-cm dishes were transfected with 10 μg of a β-galactosidase construct, 10 μg of an ISRE-luciferase construct, and 30 μg of the appropriate expression construct. Three to four sets of duplicate dishes were used for each expression construct in a single experiment, which were performed on four separate occasions (n = 13). Thirty-four hours post-transfection, one dish was treated with 300 units of IFNa2/ml for 14 h, whereas the other dish was left untreated. Cells were washed, lysed in Reporter Lysis Buffer (Promega), and frozen on dry ice. After thawing, debris were removed by centrifugation, and a portion of each lysate was assayed for luciferase activity using firefly luciferase assay substrate (Promega) in an auto-injecting luminometer (Turner Design). The remainder of each lysate was assayed for β-galactosidase activity using a previously described protocol (17Krishnan K. Singh B. Krolewski J.J. J. Biol. Chem. 1998; 273: 19495-19501Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) adapted for microtiter plates. Fold-induction is calculated by dividing luciferase activity in IFNα-treated cultures by the activity in companion untreated cultures after normalizing each luciferase value with the appropriate β-galactosidase value. Reporter gene assays on stable transfectants of U5A cells or on 2fTGH cells were performed similarly on 10-cm dishes using 5 μg of the β-galactosidase construct and 10 μg of the ISRE-luciferase construct. IFNα treatment (1000 units/ml) was for 6 h. Data from the luciferase reporter gene assays were analyzed by an unpaired Student's t test employing Statview software (Abacus Concepts). Non-saturating exposures of immunoblot autoradiograms were scanned in Photoshop version 6.0 (Adobe Systems) and pixel density quantitated using NIH Image version 1.62 software. Background pixel density was sampled in multiple spots, and the average background density was subtracted to obtain a corrected pixel density for each scanned band. Relative Stat2 protein levels were determined by dividing the corrected Stat2 pixel density by the corresponding corrected β-actin pixel density. IFNaR2-1 and IFNaR2-2 (3Domanski P. Witte M. Kellum M. Rubinstein M. Hackett R. Pitha P. Colamonici O.R. J. Biol. Chem. 1995; 270: 21606-21611Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 4Lutfalla G. Holland S.J. Cinato E. Monneron D. Reboul J. Rogers N.C. Smith J.M. Stark G.R. Gardiner K. Mogensen K.E. Kerr I.M. Uzé G. EMBO J. 1995; 14: 5100-5108Crossref PubMed Scopus (222) Google Scholar) are identical across their extracellular and transmembrane domains, as well as within the first 15 amino acids of the cytoplasmic domain, but then they diverge. To determine whether Stat2 interacts specifically with IFNaR2-2, immobilized GST fusion proteins containing the cytoplasmic domain of IFNaR2-2, IFNaR2-1, or IFNaR1 were incubated with a human Stat2-containing Sf9 cell extract, and complexes were immunoblotted with anti-Stat2 antiserum. A strong interaction was detected with GST-IFNaR2-2 (Fig.1 A, lane 5), but not with GST, GST-IFNaR1, or GST-IFNaR2-1 (Fig. 1 A,lanes 2–4), essentially as previously observed (25Li X. Leung S. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1997; 17: 2048-2056Crossref PubMed Scopus (162) Google Scholar, 33Nadeau O.W. Domanski P. Usacheva A. Uddin S. Platanias L.C. Pitha P. Raz R. Levy D. Majchrzak B. Fish E. Colamonici O.R. J. Biol. Chem. 1999; 274: 4045-4052Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). We did not observe consistent binding of baculovirus-produced Stat1 and GST-IFNaR2-2 (data not shown). Next, to investigate whether phosphorylated Stat2 can bind IFNaR2-2, we coinfected Sf9 cells with recombinant baculoviruses encoding Jak1 and Stat2. Under these conditions Stat2 is tyrosine-phosphorylated in a manner similar to that seen following IFNα treatment (10Yan H. Krishnan K. Greenlund A.C. Gupta S. Lim J.T.E. Schreiber R.D. Schindler C.W. Krolewski J.J. EMBO J. 1996; 15: 1064-1074Crossref PubMed Scopus (157) Google Scholar, 34Gupta S. Yan H. Wong L.H. Ralph S. Krolewski J.J. Schindler C.W. EMBO J. 1996; 15: 1075-1084Crossref PubMed Scopus (134) Google Scholar). Employing the same in vitro binding assay, we observed a strong association between phosphorylated Stat2 and IFNaR2-2 (Fig. 1 B, lane 3). Docking of Stat2 to IFNaR1 requires phosphorylation of tyrosine residue 466 on IFNaR1 as well as an intact Stat2 SH2 domain (10Yan H. Krishnan K. Greenlund A.C. Gupta S. Lim J.T.E. Schreiber R.D. Schindler C.W. Krolewski J.J. EMBO J. 1996; 15: 1064-1074Crossref PubMed Scopus (157) Google Scholar). Because it is unlikely that the bacterially expressed GST fusions employed in Fig. 1are tyrosine-phosphorylated, it is similarly unlikely that IFNaR2 phosphorylation is required for the interaction with Stat2. Thus, we anticipated that the Stat2 SH2 domain would also be superfluous for the Stat2-IFNaR2-2 interaction. As expected, a strong signal was observed (Fig. 1 C, lane 3) when the in vitrobinding assay was performed with lysate from cells infected with a Stat2 baculovirus bearing an inactivating SH2 domain mutation (R601K) (10Yan H. Krishnan K. Greenlund A.C. Gupta S. Lim J.T.E. Schreiber R.D. Schindler C.W. Krolewski J.J. EMBO J. 1996; 15: 1064-1074Crossref PubMed Scopus (157) Google Scholar). Fig. 1indicates that Stat2 specifically interacts with IFNaR2-2. Therefore, we employed the IFNaR2-2 subunit, referred to hereafter as IFNaR2 for the remainder of our studies. To delineate the Stat2 binding domain on IFNaR2, a panel of GST fusion proteins encoding portions of the cytoplasmic domain was used in similar in vitro binding assays (Fig. 2 A). Truncation of IFNaR2 from the carboxyl terminus to residue 444 (Fig.2 B, lane 4), or from the amino terminus to residue 340 (Fig. 2 B, lane 7) did not affect binding. However, a doubly truncated construct (340–444) displayed some reduction in binding (Fig. 3, compare lanes 4 and 3). Further truncation from the amino terminus revealed that a 25-amino acid fragment (418–444) of IFNaR2 bound Stat2, albeit at reduced efficiency (Fig. 2 B,lane 11). These data suggest that residues in this small region are critical for the interaction. Alignment of human and murine IFNaR2 revealed substantial homology, including a block (435–438; DDED) containing four acidic residues (Fig. 3 A). To test the role of these acidic residues two mutants were created by substituting alanine for either these four amino acids (designated mutant 1), or for another, non-conserved block of mainly acidic amino acids (mutant 2). Stat2 binding to IFNaR2 was diminished when alanine substitution was made at positions 435–438 (Fig. 3 B, compare lanes 7 and 6) but not when similar changes were made at the non-conserved residues (Fig. 3 B, lane 8).Figure 3Conserved acidic IFNaR2 residues are required for Stat2 binding. A, comparison of human and murine IFNaR2 between residues 417 and 449. Dashes represent gaps introduced to optimize alignment. Vertical bars indicate identical residues. Residues that were converted to alanine in mutants 1 and 2 are underlined. B, mutation of acidic IFNaR2 residues disrupt Stat2 binding in vitro. In vitrobinding was performed as described in Figs. 1 and 2. The lower half of each immunoblot was probed with anti-GST antibody to verify equal loading (data not shown). The molecular masses (in kDa) of co-migrated standards are indicated on the left. The diffuse bands inlanes 5 and 6 are apparently artifacts, as they were not observed in replicate experiments (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The in vitro binding data prompted us to examine if mutation of residues 435–438 also decreased binding in vivo. Constructs encoding Stat2 and HA-tagged wild-type (wt) or mutant 1 (m1) IFNaR2 proteins were transfected alone or in combination into 293T cells. Co-immunoprecipitation of Stat2 and IFNaR2 was significantly reduced in cells expressing m1 IFNaR2 (Fig.4 A, compare lanes 5and 6), indicating that acidic residues at positions 435–438 of IFNaR2 are critical for association both in vitro and in vivo. As seen in Figs. Figure 1, Figure 2, Figure 3, Figure 4, the Stat2-IFNaR2 association appears to be constitutive and therefore ligand-independent. However, to investigate the effect of IFNα treatment on the interaction, transfected cells overexpressing Stat2 and HA-tagged wt or m1 IFNaR2 were treated with IFNα. Co-immunoprecipitation analysis indicates that Stat2 association with wt IFNaR2 increases following ligand binding (Fig.5 A, compare lanes 4and 3). However, in other experiments we observed little or no increase (Fig. 5 E, compare lanes 3 and4), suggesting that IFNα treatment has a slight effect on the amount of Stat2 bound to IFNaR2. Stat2 binding to the m1 IFNaR2 construct was minimally detectable following IFNα treatment (Fig.5 A, lane 6) but, as seen in Fig. 4, was greatly reduced compared with wt IFNaR2 (Fig. 5 A, comparelanes 4 and 6). Both phosphorylated and non-phosphorylated Stat2 bound GST-IFNaR2in vitro (Fig. 1, A and B). To determine whether this occurs in vivo cells transien" @default.
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• W1972508857 title "Stat2 Binding to the Interferon-α Receptor 2 Subunit Is Not Required for Interferon-α Signaling" @default.
• W1972508857 cites W131140191 @default.
• W1972508857 cites W1479767677 @default.
• W1972508857 cites W1521367633 @default.
• W1972508857 cites W1544033113 @default.
• W1972508857 cites W1594864284 @default.
• W1972508857 cites W1747682552 @default.
• W1972508857 cites W1931571693 @default.
• W1972508857 cites W1965398762 @default.
• W1972508857 cites W1968134447 @default.
• W1972508857 cites W1968177644 @default.
• W1972508857 cites W1968333833 @default.
• W1972508857 cites W1969979508 @default.
• W1972508857 cites W1974163527 @default.
• W1972508857 cites W1987274954 @default.
• W1972508857 cites W1987485969 @default.
• W1972508857 cites W1994810882 @default.
• W1972508857 cites W2002708146 @default.
• W1972508857 cites W2006061341 @default.
• W1972508857 cites W2008123904 @default.
• W1972508857 cites W2013279950 @default.
• W1972508857 cites W2015440478 @default.
• W1972508857 cites W2030227166 @default.
• W1972508857 cites W2043803403 @default.
• W1972508857 cites W2052982889 @default.
• W1972508857 cites W2059891790 @default.
• W1972508857 cites W2078971599 @default.
• W1972508857 cites W2082783596 @default.
• W1972508857 cites W2088622701 @default.
• W1972508857 cites W2088988430 @default.
• W1972508857 cites W2095536071 @default.
• W1972508857 cites W2111377245 @default.
• W1972508857 cites W2122702335 @default.
• W1972508857 cites W2140783929 @default.
• W1972508857 cites W2142512966 @default.
• W1972508857 cites W2149560334 @default.
• W1972508857 cites W2151712105 @default.
• W1972508857 cites W218554609 @default.
• W1972508857 cites W2799941631 @default.
• W1972508857 cites W5404037 @default.
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