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• W1987386721 abstract "Binding of interferon gamma (IFN-γ) causes oligomerization of the two interferon γ receptor (IFN-γR) subunits, receptor chain 1 (IFN-γR1, the ligand-binding chain) and the second chain of the receptor (IFN-γR2), and causes activation of two Jak kinases (Jak1 and Jak2). In contrast, the erythropoietin receptor (EpoR) requires only one receptor chain and one Jak kinase (Jak2). Chimeras between the EpoR and the IFN-γR1 and IFN-γR2 chains demonstrate that the architecture of the EpoR and the IFN-γR complexes differ significantly. Although IFN-γR1 alone cannot initiate signal transduction, the chimera EpoR/γR1 (extracellular/intracellular) generates slight responses characteristic of IFN-γ in response to Epo and the EpoR/γR1·;EpoR/γR2 heterodimer is a fully functional receptor complex. The results demonstrate that the configuration of the extracellular domains influences the architecture of the intracellular domains. Binding of interferon gamma (IFN-γ) causes oligomerization of the two interferon γ receptor (IFN-γR) subunits, receptor chain 1 (IFN-γR1, the ligand-binding chain) and the second chain of the receptor (IFN-γR2), and causes activation of two Jak kinases (Jak1 and Jak2). In contrast, the erythropoietin receptor (EpoR) requires only one receptor chain and one Jak kinase (Jak2). Chimeras between the EpoR and the IFN-γR1 and IFN-γR2 chains demonstrate that the architecture of the EpoR and the IFN-γR complexes differ significantly. Although IFN-γR1 alone cannot initiate signal transduction, the chimera EpoR/γR1 (extracellular/intracellular) generates slight responses characteristic of IFN-γ in response to Epo and the EpoR/γR1·;EpoR/γR2 heterodimer is a fully functional receptor complex. The results demonstrate that the configuration of the extracellular domains influences the architecture of the intracellular domains. The interferon γ (IFN-γ) 1The abbreviations used are: IFN-γinterferon γIFN-γRinterferon γ receptorEpoerythropoietinEpoRerythropoietin receptorEMSAelectrophoretic mobility shift assayMHCmajor histocompatibility complexPCRpolymerase chain reactionCHOChinese hamster ovaryECextracellular domainICintracellular domainGASgamma activation sequence. receptor complex consists of at least two receptor components, a ligand binding chain and a signal transducing chain, each of which is a member of the class II cytokine receptor family (1Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Google Scholar, 2Thoreau E. Petridou B. Kelly P.A. Djiane J. Mornon J.P. FEBS Lett. 1991; 282: 26-31Google Scholar). Isolation of the two chains of the interferon γ receptor (IFN-γR) has permitted an analysis of the contributions of each to the signal transduction mechanism. The first chain of the receptor (IFN-γR1) binds ligand (3Rashidbaigi A. Langer J.A. Jung V. Jones C. Morse G.H. Tischfield J.A. Trill J.J. Kung H.F. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 384-388Google Scholar, 4Aguet M. Dembic Z. Merlin G. Cell. 1988; 55: 273-280Google Scholar, 5Kumar C.S. Muthukumaran G. Frost L.J. Noe M. Ahn Y.H. Mariano T.M. Pestka S. J. Biol. Chem. 1989; 264: 17939-17946Google Scholar, 6Hemmi S. Peghiai P. Metzter M. Merlin G. Dembic Z. Aguet M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9901-9905Google Scholar, 7Gray P.W. Leong S. Fennie E.H. Farrar M.A. Pingel J.T. Fernandez-Luna J. Schreiber R.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8497-8501Google Scholar, 8Munro S. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9248-9252Google Scholar, 9Cofano F. Moore S.K. Tanaka S. Yuhki N. Landolfo S. Appella E. J. Biol. Chem. 1990; 265: 4064-4071Google Scholar). The second chain of the receptor (IFN-γR2) does not bind ligand by itself but is required for signal transduction (3Rashidbaigi A. Langer J.A. Jung V. Jones C. Morse G.H. Tischfield J.A. Trill J.J. Kung H.F. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 384-388Google Scholar, 10Jung V. Rashidbaigi A. Jones C. Tischfield J.A. Shows T.B. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4151-4155Google Scholar, 11Jung V. Jones C. Rashidbaigi A. Geyer D.D. Morse H.G. Wright R.B. Pestka S. Somat. Cell Mol. Genet. 1988; 14: 583-592Google Scholar, 12Jung V. Jones C. Kumar C.S. Stefanos S. O'Connell S. Pestka S. J. Biol. Chem. 1990; 265: 1827-1830Google Scholar, 13Soh J. Donnelly R.J. Mariano T.M. Cook J.R. Schwartz B. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8737-8741Google Scholar, 14Soh J. Donnelly R.J. Kotenko S. Mariano T.M. Cook J.R. Wang N. Emanuel S. Schwartz B. Miki T. Pestka S. Cell. 1994; 76: 793-802Google Scholar, 15Hemmi S. Böhni R. Stark G. Di Marco F. Aguet M. Cell. 1994; 76: 803-810Google Scholar, 16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar). A large body of experiments has elucidated the involvement of the Jak-Stat pathway in signaling by various cytokines (for reviews, see Refs. 17Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Google Scholar, 18Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Thierfelder W.E. Kreider B. Silvennoinen O. Trends Biochem. Sci. 1994; 19: 222-227Google Scholar, 19Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Silvennoinen O. Annu. Rev. Immunol. 1995; 13: 369-398Google Scholar, 20Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Google Scholar, 21Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Google Scholar, 22Taniguchi T. Science. 1995; 268: 251-255Google Scholar). The Janus kinases (or Jaks) are a family of receptor-associated soluble tyrosine kinases with four known members, Tyk2, Jak1, Jak2 and Jak3. Two of the kinases, Jak1 and Jak2, are required for signal transduction by IFN-γ. Further analyses of the interactions have shown that the IFN-γR1 chain binds Jak1 (16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar, 23Igarashi K. Garotta G. Ozmen L. Ziemiecki A. Wilks A.F. Harpur A.G. Larner A.C. Finbloom D.S. J. Biol. Chem. 1994; 269: 14333-14336Google Scholar, 24Sakatsume M. Igarashi K. Winestock K.D. Garotta G. Larner A.C. Finbloom D.S. J. Biol. Chem. 1995; 270: 17528-17534Google Scholar) and the intracellular domain of the IFN-γR2 chain brings Jak2 into the signal transduction complex (16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar). Upon binding of the ligand, IFN-γ, to the IFN-γR1 chain, activation of Jak1 and/or Jak2 by reciprocal transphosphorylation causes the phosphorylation of IFN-γR1 (16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar, 25Greenlund A.C. Farrar M.A. Viriano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Google Scholar). Stat1α, a latent cytoplasmic transcription factor (26Schindler C. Fu X.-Y. Improta T. Aebersold R. Darnell J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7836-7839Google Scholar), binds to the phosphorylated IFN-γR1, undergoes tyrosine phosphorylation (27Shuai K. Stark G.R. Kerr I.M. Darnell Jr., J.E. Science. 1993; 261: 1744-1746Google Scholar), and forms homodimers that translocate to the nucleus and initiate transcription of IFN-γ inducible genes (for reviews see Refs. 17Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Google Scholar and 21Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Google Scholar). interferon γ interferon γ receptor erythropoietin erythropoietin receptor electrophoretic mobility shift assay major histocompatibility complex polymerase chain reaction Chinese hamster ovary extracellular domain intracellular domain gamma activation sequence. As with other cytokine receptors, oligomerization upon ligand binding is the first step in the signaling cascade of IFN-γ. IFN-γ is a non-covalent symmetrical homodimer (28Ealick S.E. Cook W.J. Vijaykumar S. Carson M. Nagabhushan T. Trotta P.P. Bugg C.E. Science. 1991; 252: 698-700Google Scholar) that binds to IFN-γR1 with a stoichiometry of 1:2 (29Fountoulakis M. Zulauf M. Lusting A. Garotta G. Eur. J. Biochem. 1992; 208: 781-787Google Scholar, 30Greenlund A.C. Schreiber R.D. Goeddel D. Pennica D. J. Biol. Chem. 1993; 268: 18103-18110Google Scholar). It is known that a species-specific interaction between the extracellular domains of the IFN-γR1 and IFN-γR2 subunits is essential for signaling (10Jung V. Rashidbaigi A. Jones C. Tischfield J.A. Shows T.B. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4151-4155Google Scholar, 11Jung V. Jones C. Rashidbaigi A. Geyer D.D. Morse H.G. Wright R.B. Pestka S. Somat. Cell Mol. Genet. 1988; 14: 583-592Google Scholar, 12Jung V. Jones C. Kumar C.S. Stefanos S. O'Connell S. Pestka S. J. Biol. Chem. 1990; 265: 1827-1830Google Scholar, 31Gibbs V.C. Williams S.R. Gray P.W. Schreiber R.D. Pennica D. Rice G. Goeddel D.V. Mol. Cell. Biol. 1991; 11: 5860-5866Google Scholar, 32Hibino Y. Kumar C.S. Mariano T.M. Lai D. Pestka S. J. Biol. Chem. 1992; 267: 3741-3749Google Scholar, 33Hemmi S. Merlin G. Aguet M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2737-2741Google Scholar). The IFN-γR2 subunit does not by itself bind the ligand, but can be cross-linked to IFN-γ when both IFN-γR1 and IFN-γR2 chains are present (16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar). Several lines of evidence (16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar, 34Marsters S.A. Pennica D. Bach E. Schreiber R.D. Ashkenazi A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5401-5405Google Scholar) suggest that the IFN-γ signaling complex contains two IFN-γR1 chains, two IFN-γR2 chains and one IFN-γ homodimer. The erythropoietin (Epo) receptor, EpoR, is a member of the class I cytokine receptor subfamily. A single chain encodes both ligand-binding and signal-transducing functions. Epo induces homodimerization of the receptor to initiate signal transduction (for reviews, see Refs. 18Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Thierfelder W.E. Kreider B. Silvennoinen O. Trends Biochem. Sci. 1994; 19: 222-227Google Scholar, 19Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Silvennoinen O. Annu. Rev. Immunol. 1995; 13: 369-398Google Scholar, and 35Wells J.A. Curr. Opin. Cell Biol. 1994; 6: 163-173Google Scholar). Jak2 is associated with the cytoplasmic domain of the EpoR and is activated upon ligand-induced dimerization of the receptor (36Witthuhn B.A. Quelle F.W. Silvennoinen O. Yi T. Tang B. Miura O. Ihle J.N. Cell. 1993; 74: 227-236Google Scholar). Strikingly an Arg → Cys mutation in the extracellular domain of EpoR results in ligand independent dimerization/oligomerization and constitutive, ligand-independent activation of Jak2 and mitogenesis (37Watowich S.S. Yoshimura A. Longmore G.D. Hilton D.J. Yoshimura Y. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2140-2144Google Scholar, 38Yoshimura A. Longmore G.D. Lodish H.F. Nature. 1990; 348: 647-649Google Scholar). In this study we used chimeric EpoR, IFN-γR1, and IFN-γR2 constructs to investigate the differences between the architecture of Epo and IFN-γ receptor complexes and shed light on the requirement for one or two receptor-associated tyrosine kinases and the necessity for one or two distinct transmembrane chains for effective signal transduction. Recombinant human erythropoietin was a gift from Dr. Lawrence Blatt of Amgen. Restriction endonucleases were from Boehringer Mannheim and New England Biolabs; T4 DNA ligase was from U. S. Biochemical Corp.; [α-32P]dCTP was from DuPont NEN. All other reagents were of analytical grade and were purchased from Sigma. CHO-B7 cells represent the Chinese hamster ovary cell line (CHO-K1) containing a transfected human HLA-B7 gene (12Jung V. Jones C. Kumar C.S. Stefanos S. O'Connell S. Pestka S. J. Biol. Chem. 1990; 265: 1827-1830Google Scholar). The 16-9 hamster × human somatic hybrid cell line is a CHO-K1 derivative containing a translocation of the long arm of human chromosome 6 and the human HLA-B7 gene (13Soh J. Donnelly R.J. Mariano T.M. Cook J.R. Schwartz B. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8737-8741Google Scholar). These cells were maintained in Ham's F-12 medium (Life Technologies, Inc.) containing 10% heat-inactivated fetal bovine serum (Sigma). Transfections were carried out with the DOTAP transfection reagent (Boehringer Mannheim) according to the manufacturer's protocol and the transfected cells were maintained in F-12 medium containing 450 μg/ml Geneticin (antibiotic G418). Unless otherwise noted, experiments were performed with cloned cells expressing the various receptor subunits. The EpoR expression plasmid was made by cloning the EcoRI-AflIII fragment of the human EpoR cDNA p18R (39Jones S.S. D'Andrea A.D. Haines L.L. Wong G.G. Blood. 1990; 76: 31-35Google Scholar) into the EcoRI and EcoRV sites of the eukaryotic expression vector pcDNA3 (Invitrogen). The construction of plasmids expressing Hu-IFN-γR1 and Hu-IFN-γR2 chains from cDNA under the control of cytomegalovirus promoter has been previously described (5Kumar C.S. Muthukumaran G. Frost L.J. Noe M. Ahn Y.H. Mariano T.M. Pestka S. J. Biol. Chem. 1989; 264: 17939-17946Google Scholar, 12Jung V. Jones C. Kumar C.S. Stefanos S. O'Connell S. Pestka S. J. Biol. Chem. 1990; 265: 1827-1830Google Scholar, 16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar, 40Kotenko S.V. Izotova L.S. Pollack B.P. Muthukumaran G. Paukku K. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1996; 271: 17174-17182Google Scholar). For ease of construction of the various chimeric receptors, the polymerase chain reaction (PCR) was employed to incorporate a unique NheI site at the 3′ end of the extracellular domain (EC) and at the 5′ end of the transmembrane-intracellular domains (IC) of the receptors. The primers were designed to code for the three amino acids Trp, Leu, and Ala, which are commonly found in the transmembrane domain of several proteins, encompassing the NheI site. The extracellular portions of EpoR, Hu-IFN-γR1, and Hu-IFN-γR2, containing an NheI site (designated EpoREC/NheI, γR1EC/NheI, and γR2EC/NheI) were generated by PCR from the respective cDNAs as templates with the use of the T7 primer (5′-TAATACGACTCACTATA-3′) and the internal primers 5′-GCCGCTAGCCAGGGGTCCAGGTCGCTAGGCG-3′ (corresponding to nucleotides 1874-1893 of p18R EpoR cDNA; Ref. 39Jones S.S. D'Andrea A.D. Haines L.L. Wong G.G. Blood. 1990; 76: 31-35Google Scholar), 5′-GTGGCTAGCCAAGAACCTTTTATACTGCT-3′ (corresponding to nucleotides 779-785 of the Hu-IFN-γR1 cDNA; Ref. 4Aguet M. Dembic Z. Merlin G. Cell. 1988; 55: 273-280Google Scholar), and 5′-ATCGCTAGCCATTGCTGAAGCTCAGTGGAGG-3′ (corresponding to nucleotides 1370-1390 of the Hu-IFN-γR2 cDNA; Ref. 14Soh J. Donnelly R.J. Kotenko S. Mariano T.M. Cook J.R. Wang N. Emanuel S. Schwartz B. Miki T. Pestka S. Cell. 1994; 76: 793-802Google Scholar). The intracellular portions of the various receptors with the unique NheI site at the 5′ end of the transmembrane domain (designated EpoRIC/NheI, γR1IC/NheI, and γR2IC/NheI were generated by PCR on corresponding cDNA templates with the use of the SP6 primer (5′-ATTTAGGTGACACTATA-3′) and the internal primers 5′-GTGGCTAGCGACGCTCTCCCTCATCCTCG-3′ (corresponding to nucleotides 1902-1921 of plasmid p18R), 5′-GTGGCTAGCGATTCCAGTTGTTGCTGCTTTAC-3′ (corresponding to nucleotides 792-814 of the Hu-IFN-γR1 cDNA), and 5′-GTGGCTAGCGATCTCCGTGGGAACATTT-3′ (corresponding to nucleotides 1398-1416 of the Hu-IFN-γR2 cDNA). The NheI site in each primer is underlined. The PCR products encoding the extracellular domains were incubated with T4 DNA polymerase and dNTPs to generate blunt ends; then the PCR fragments, which contained the vector multiple cloning sites, were subsequently digested with the restriction endonucleases EcoRI (EpoREC/NheI and γR2EC/NheI) or BamHI (γR1EC/NheI), and cloned into the EcoRV and EcoRI/BamHI sites of the expression vector pcDNA3 (Invitrogen) to yield the plasmids pEpoREC, pγR1EC and pγR2EC. Analogously, the PCR products encoding the intracellular domains of the various receptors were treated with T4 DNA polymerase to generate blunt ends, digested with XbaI restriction endonuclease, and cloned into the EcoRV and XbaI sites of pcDNA3 to yield the plasmids pEpoRIC, pγR1IC, and pγR2IC. To introduce the Stat1α binding site of Hu-IFN-γR1 into the cytoplasmic domain of EpoR, two-step asymmetric PCR (detailed in Ref. 41Muthukumaran G. Donnelly R.J. Ebensperger C. Mariano T.M. Poast J. Baron S. Dembic Z. Pestka S. J. Interferon and Cytokine Res. 1996; 16: 1039-1045Google Scholar) was carried out sequentially on Hu-IFN-γR1 cDNA and pEpoRIC cDNA templates with vector primers and the internal primer CTTGTCCTTCTGTTTTTATTTCagagcaagccacatagetggg. The uppercase letters denote sequences corresponding to the Hu-IFN-γR1 cDNA, and the lowercase letters represent sequences corresponding to the EpoR cDNA. The Hu-IFN-γR2 chain with the Stat1α binding site of Hu-IFN-γR1 was constructed by restriction enzyme digestion of pγR2IC and IFN-γR1 cDNA with BspEI and AvaI, respectively, followed by ligation. For construction of the chimeric receptors, plasmids encoding the suitable extracellular or intracellular domains were digested with NheI and XbaI restriction endonucleases and ligated together. All constructs were sequenced for verification of the entire nucleotide sequence of the receptor. Sequencing was done in an Applied Biosystems model 373 automated DNA sequencer with dideoxy dye-terminator chemistry. EMSAs were performed with the 22-base pair sequence containing a Stat1α binding site (5′-GATCGATTTCCCCGAAATCATG-3′) corresponding to the GAS element in the promoter region of the human IRF-1 gene (42Yuan J. Wegenka U.A. Lütticken C. Buschmann J. Decker T. Schindler C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1994; 14: 1657-1668Google Scholar). Two oligonucleotides, 5′-GATCGATTTCCCCGAAAT-3′ and 5′-CATGATTTCGGGGAAATC-3′, were annealed by incubation for 10 min at 65°C, 10 min at 37°C, and 10 min at 22°C, and labeled with [α-32P]dATP and the Klenow fragment of DNA polymerase I by the filling-in reaction (43Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Whole cell extracts were prepared as follows (44Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Google Scholar). Cells were grown to confluence in six-well plates, and harvested by scraping in ice-cold phosphate-buffered saline. Cells from each well were washed with 1.0 ml of cold phosphate-buffered saline, pelleted, and resuspended in 100 μl of lysis buffer (10% glycerol, 50 mM Tris·;HCl, pH 8.0, 0.5% Nonidet P-40, 150 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 3 μg/ml aprotinin, 1 μg/ml pepstatin, and 1 μg/ml leupeptin). After 30 min on ice, the extracts were centrifuged for 5 min at full speed in a microcentrifuge and the supernatant was recovered for use in the assay and stored at −80°C. EMSA reactions contained 2.5 μl of the whole cell extracts, 1 ng 32P-labeled probe (specific activity approximately 109 cpm/μg), 24 μg/ml bovine serum albumin, 160 μg/ml poly(dI·;dC), 20 mM HEPES, pH 7.9, 1 mM MgCl2, 4.0% Ficoll (Pharmacia Biotech Inc.), 40 mM KCl, 0.1 mM EGTA, and 0.5 mM dithiothreitol in a total volume of 12.5 μl. For the supershift assay, 1 μl of a 1:10 dilution of anti-Stat1α antibody was included in the reaction. Competition experiments contained a 100-fold excess of the unlabeled oligonucleotide. Reactions were incubated at 24°C for 20 min. Then 8 μl of the reaction mixture was electrophoresed at 400 V for 3-4 h at 4°C on a 5% polyacrylamide (19:1, acrylamide:bisacrylamide) gel. The dried gel was exposed to Kodak XAR-5 film with an intensifying screen for 12 h at −80°C. Rabbit anti-Jak1 antibody was developed against a synthetic peptide (KTLIEKERFYESRCRPVTPSC) corresponding to the end of the second kinase-like domain of murine Jak1. Rabbit anti-Stat1α antibody, raised against the carboxyl-terminal region of Stat1α, was a gift from James Darnell. Rabbit anti-Jak2 antibody (catalogue no. SC-294) and rabbit anti-Stat5 antibody (catalogue no. SC-835) were from Santa Cruz Biotechnology. Monoclonal anti-phosphotyrosine antibody was purchased from Sigma (catalogue no. P3300). Cells were stimulated with Hu-IFN-γ (1,000 units/ml) or Epo (100 units/ml) for 10 min at 37°C. Immunoprecipitations and blottings were performed as described (16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar, 40Kotenko S.V. Izotova L.S. Pollack B.P. Muthukumaran G. Paukku K. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1996; 271: 17174-17182Google Scholar). Cytofluorographic analysis of cells for surface expression of class I MHC antigens was performed as described previously (13Soh J. Donnelly R.J. Mariano T.M. Cook J.R. Schwartz B. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8737-8741Google Scholar, 41Muthukumaran G. Donnelly R.J. Ebensperger C. Mariano T.M. Poast J. Baron S. Dembic Z. Pestka S. J. Interferon and Cytokine Res. 1996; 16: 1039-1045Google Scholar, 45Cook J.R. Jung V. Schwartz B. Wang P. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11317-11321Google Scholar) with mouse anti-human-HLA B-7 monoclonal antibody (W6/32) and fluorescein isothiocyanate-conjugated goat anti-mouse IgG. The schematic illustration of the various chimeric receptor molecules that were produced is shown in Fig. 1. In one set of chimeric constructs, the extracellular domain of the EpoR was spliced to the transmembrane domain and the cytoplasmic domain of each of the two IFN-γR subunits. In the other set of chimeras, the transmembrane and intracellular domain of EpoR was fused to the extracellular domain of IFN-γR1 and IFN-γR2. To investigate the role of the intracellular domain of IFN-γR2 in the signal transduction complex of IFN-γ, we constructed a chimeric receptor chain consisting of the extracellular domain of IFN-γR2 and the intracellular domain of EpoR. This chimeric construct, γR2/EpoR, and the native IFN-γR2 subunit were separately transfected into CHO-B7 as well as CHO-16-9 cells. The ability of the transfected chimeric cDNA to transduce a signal upon induction with Hu-IFN-γ was assayed by measurement of enhanced MHC class I antigen expression in the transfected cells and by activation of Stat1α. CHO-B7 cells transfected with IFN-γR2 or γR2/EpoR cDNA showed no response to Hu-IFN-γ as they lack the ligand-binding receptor subunit, Hu-IFN-γR1 (data not shown). Parental CHO-16-9 cells, which contain human chromosome 6q and express the Hu-IFN-γR1 subunit, showed no induction of MHC class I antigens in response to Hu-IFN-γ (Fig. 2, panel A) but when stably transfected with expression vectors encoding Hu-IFN-γR2 cDNA or γR2/EpoR chimera, exhibited enhanced cell surface expression of class I MHC antigens in response to Hu-IFN-γ (Fig. 2, panels B and C). To assess how effectively the intracellular domain of EpoR could substitute for the intracellular domain of the IFN-γR2 subunit, we measured the induction of MHC class I antigens as a function of IFN-γ concentration. As depicted in Fig. 3, there was a slightly lower induction of MHC class I antigens in the cells containing the chimeric γR2/EpoR than in the cells containing the native Hu-IFN-γR2 chain at each concentration of Hu-IFN-γ used. Nevertheless, the fact that the EpoR intracellular domain can be substituted for the Hu-IFN-γR2 intracellular domain shows that another sequence that can recruit Jak2 into the signal transduction complex can substitute for the intracellular domain of Hu-IFN-γR2.Fig. 3Induction of expression of class I MHC surface antigens as a function of interferon concentration. Cells were treated with Hu-IFN-γ at concentrations of 0, 1, 10, 100, and 1,000 units/ml for 72 h. Class I MHC antigens were detected as described in the legend to Fig. 2. Relative fluorescence values are based on the mean fluorescence of cell populations (n = 10,000). The data were normalized so that the mean fluorescence intensity was adjusted to 1.0 for cells in the absence of Hu-IFN-γ.View Large Image Figure ViewerDownload (PPT) Various chimeric receptors between the EpoR and Hu-IFN-γR1 and Hu-IFN-γR2 subunits were constructed in order to gain an understanding of the events leading to signal transduction. CHO-16-9 cells were stably transfected with expression vectors coding for EpoR, EpoR/γR1, EpoR/γR2, the combination of EpoR/γR1 and EpoR/γR2, and γR1/EpoR(p91). In response to Epo, the EpoR transfectants showed no response (Fig. 2, panel D). The EpoR/γR1 transfectants showed a slight enhancement of expression of MHC class I antigens (Fig. 2, panel E), which shows that the intracellular domain of the Hu-IFN-γR1 chain, by itself, can recruit all the requisite components for signal transduction. At lower concentrations of Epo (less than 100 units/ml), there was little or no increased MHC class I antigen expression in these cells (Fig. 4). The transfectants containing both EpoR/γR1 and EpoR/γR2 chains exhibited substantial expression of MHC class I antigens (Fig. 2, panel F; Fig. 4). Cells transfected with the expression vector coding for EpoR(p91) chimeric cDNA (EpoR with the p91 recruitment site from IFN-γR1) respond to Epo with enhanced expression of class I MHC antigens, while the γR1/EpoR(p91) transfectants were unresponsive (Fig. 2, panels H and G, respectively). Furthermore, the γR1/γR2(p91) receptor chain is unable to transduce a signal upon binding ligand, 2S. Kotenko, unpublished observation. whereas the cells expressing the EpoR/γR2(p91) chimeric receptor exhibited enhanced class I MHC antigen expression in response to activation by Epo (Fig. 2, panel I). We analyzed Stat activation in cells expressing wild-type and chimeric receptors in response to IFN-γ and Epo. As shown in Fig. 5, IFN-γ stimulation resulted in Stat1α activation in transfected 16-9 cells expressing native IFN-γR2 or γR2/EpoR chains. Similarly, Epo caused activation of Stat1α in transfected cell lines expressing EpoR/γR1 and both EpoR/γR1 and EpoR/γR2 receptor chains (Fig. 6). Consistent with the small enhancement in surface expression of class I MHC antigens in cells expressing EpoR/γR1 in response to Epo, Stat1α activation was also lower in these cells compared to cells expressing both EpoR/γR1 and EpoR/γR2 chains. Activation of p91 was also observed in cells expressing the EpoR(p91) and EpoR/γR2(p91) chains (Fig. 6). Furthermore, cells expressing those chimeric receptors containing the EpoR intracellular domain, except γR1/EpoR(p91) and γR1/EpoR, exhibited activation of Stat5 in addition to Stat1α (Fig. 5; data with γR1/EpoR were negative similar to results with γR1/EpoR(p91)). 3G. Muthukumaran, S. Kotenko, R. Donnelly, J. N. Ihle, and S. Pestka, unpublished results. Stat5 is phosphorylated on tyrosine in response to Epo (19Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Silvennoinen O. Annu. Rev. Immunol. 1995; 13: 369-398Google Scholar, 20Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Google Scholar). Both Stat1α and Stat5 are supershifted by the addition of anti-Stat1α and anti-Stat5 antibodies, respectively. Addition of 100-fold molar excess of unlabeled GAS oligonucleotide eliminates both Stat1α and Stat5 activated complexes.Fig. 6Electrophoretic mobility shift assays of cells expressing chimeric receptors. Clones of transfected 16-9 cells stably expressing EpoR/γR1, EpoR(p91), or EpoR/γR2(p91) chimeric receptor subunits, or both EpoR/γR1 and EpoR/γR2 chimeric receptors were treated with erythropoietin at 100 units/ml for 15 min at 37°C. Whole cell extracts were made and the electrophoretic mobility shift assay performed. As shown in the figure, induction with Epo causes activation of Stat1α in cells expressing EpoR/γR1, EpoR(p91), and EpoR/γR2(p91), as well as in those cells expressing both EpoR/γR1 and EpoR/γR2. Addition of anti-Stat1α antibody to the reaction mixture caused the Stat1α complex to be shifted.View Large Image Figure ViewerDownload (PPT) IFN-γ activates Jak1 and Jak2 kinases (46Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Google Scholar), whereas Epo activates Jak2 (36Witthuhn B.A. Quelle F.W. Silvennoinen O. Yi T. Tang B. Miura O. Ihle J.N. Cell. 1993; 74: 227-236Google Scholar) during signal transduction. Thus, we tested the ability of the various chimeric receptors to activate Jak1 and Jak2 kinases in response to binding of ligand. Phosphorylation of Jak1 and Jak2 (Fig. 7) was examined by immunoprecipitation of cellular lysates with anti-phosphotyrosine antibodies, followed by a Western blot visualized with specific anti-Jak1 and anti-Jak2 antibodies. Both Jak1 and Jak2 were phosphorylated in response to Hu-IFN-γ treatment in 16-9 cells expressing parental IFN-γR2 or chimeric γR2/EpoR receptors. Induction with Epo phosphorylated both Jak1 and Jak2 kinase in the cell line expressing both EpoR/γR1 and EpoR/γR2 chains. In the cell line expressing only the chimeric EpoR/γR1 receptor, only Jak1 kinase was phosphorylated in response to Epo. The cell line transfected with the γR1/EpoR chimeric receptor did not exhibit phosphorylation of either Jak1 or Jak2 kinase upon IFN-γ treatment. For hormones, growth factors and cytokines, the conversion of the extracellular ligand-binding event to the intracellular signal involves a change in the oligomeric structure of the receptor. Depending on the ligand, this can take the form of receptor homodimers (Epo, growth hormone), heterodimers (ciliary neurotrophic factor, leukemia inhibitory factor), homotrimers (tumor necrosis factor), and more complex assemblies (reviewed in Ref. 47Stahl N. Yancopoulous G.D. Cell. 1993; 74: 587-590Google Scholar). In the case of IFN-γ, the oligomerization involving IFN-γR1 and IFN-γR2 initiates the signal transduction events: activation of Jak1 and Jak2, phosphorylation of IFN-γR1 on Tyr-457 (16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar, 25Greenlund A.C. Farrar M.A. Viriano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Google Scholar), followed by phosphorylation and activation of Stat1α (27Shuai K. Stark G.R. Kerr I.M. Darnell Jr., J.E. Science. 1993; 261: 1744-1746Google Scholar). A major function of receptor dimerization is to bring two receptor-associated kinases together for transactivation and phosphorylation of the receptor chains. The cytoplasmic domain of the IFN-γR2 subunit serves to bring Jak2 kinase into the signal transduction complex (16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar). This is a crucial event since deletion of the membrane-proximal region of the intracellular domain of the IFN-γR2 chain, which encompasses the Jak2 association site, completely abrogates its ability to transduce signals in response to IFN-γ (16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar), and cells lacking Jak2 do not respond to IFN-γ (46Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Google Scholar). This is further supported by the observation that the IFN-γR2/EpoR chimeric receptor, which recruits Jak2, is almost as effective as the native IFN-γR2 chain in supporting signal transduction in response to IFN-γ (Figs. 2, 5, and 7). The IFN-γR2 subunit is a helper receptor subunit with a Jak2 association site, but no Stat recruitment site; its intracellular domain can be substituted with the cytoplasmic domain of any receptor subunit that can bring a Jak kinase to the IFN-γ receptor complex to support signal transduction (40Kotenko S.V. Izotova L.S. Pollack B.P. Muthukumaran G. Paukku K. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1996; 271: 17174-17182Google Scholar). The requirement for two distinct Jak kinases in the IFN-γ signaling pathway was demonstrated with the use of kinase-deficient cell lines (46Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Google Scholar, 48Watling D. Guschin D. Müller M. Silvennoinen O. Witthuhn B.A. Quelle F.W. Rogers N.C. Schindler C. Stark G.R. Ihle J.N. Kerr I.M. Nature. 1993; 366: 166-170Google Scholar). Based on our results with the chimeric erythropoietin-interferon γ receptors, we propose that this reflects two features characteristic of the IFN-γ receptor complex: the unique properties of the receptor relative to the positioning of the Jaks, and the idea that Jak1 is relatively ineffective in one or more of the following phosphorylation steps (trans-phosphorylation of itself, phosphorylation of IFN-γR1, and activation of Stat1α). The presence of Jak2 facilitates effective phosphorylation of the above steps. In contrast to the growth hormone receptor (49De Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Google Scholar) and the EpoR (37Watowich S.S. Yoshimura A. Longmore G.D. Hilton D.J. Yoshimura Y. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2140-2144Google Scholar) complexes, when one IFN-γ homodimer binds two IFN-γR1 molecules, the two receptor subunits do not interact with one another and are separated by 27 Å (50Walter M.R. Windsor W.T. Nagabhushan T.L. Lundell D.J. Lunn C.A. Zanodry P.J. Narula S.K. Nature. 1995; 376: 230-235Google Scholar) at their closest point. Therefore, although the IFN-γR1 chain possesses both a Jak1 association site and a Stat1α recruitment site, alone it is unable to transduce a signal on homodimerization as the two Jak1 kinases are not in physical proximity to permit transphosphorylation (Fig. 8A). Crystallographic analysis of the IFN-γ·;IFN-γR1 complex suggests that each monomer of the IFN-γ homodimer binds one IFN-γR1 and one IFN-γR2 subunit (50Walter M.R. Windsor W.T. Nagabhushan T.L. Lundell D.J. Lunn C.A. Zanodry P.J. Narula S.K. Nature. 1995; 376: 230-235Google Scholar). Thus the signal-transducing complex of IFN-γ consists of the IFN-γ homodimer bound to two IFN-γR1 and two IFN-γR2 chains, which recruit Jak1 and Jak2, respectively (16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar, 40Kotenko S.V. Izotova L.S. Pollack B.P. Muthukumaran G. Paukku K. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1996; 271: 17174-17182Google Scholar); and Jak2 phosphorylates Jak1, following which either kinase phosphorylates Tyr-457 of the IFN-γR1 chain (Fig. 8B; see also Refs. 25Greenlund A.C. Farrar M.A. Viriano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Google Scholar, 45Cook J.R. Jung V. Schwartz B. Wang P. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11317-11321Google Scholar, and 51Farrar M.A. Campbell J.D. Schreiber R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11706-11710Google Scholar). The phosphorylated segment of each IFN-γR1 chain recruits Stat1α, which is then phosphorylated by Jak1 or Jak2, then released to dimerize and form the active Stat1α. In contrast, with the EpoR/γR1 dimer, two Jak1 kinases are brought sufficiently close together to activate one another (Fig. 8C), albeit inefficiently. In the case of the EpoR/γR1·;EpoR/γR2 dimer, one Jak1 and one Jak2 are in close apposition for Jak2 to phosphorylate Jak1 and initiate efficient downstream signaling events (Fig. 8D). Cells expressing the EpoR/γR2(p91) chimeric receptor (Fig. 2I) exhibit a stronger biological response than cells expressing both EpoR/γR1 and EpoR/γR2 (Fig. 2F) or even the native IFN-γ receptor (γR2, Fig. 2B), which supports a modulating role for Jak1 in the IFN-γR complex. In cells expressing both EpoR/γR1 and EpoR/γR2 chains, binding of Epo can induce the formation of three types of receptor dimers: EpoR/γR1 homodimers, EpoR/γR2 homodimers, and EpoR/γR1·;EpoR/γR2 heterodimers. The EpoR/γR1 homodimer is barely active (Figs. 2E and 4), and the EpoR/γR2 homodimer is inactive. The major functional receptor complex therefore must be the EpoR/γR1·;EpoR/γR2 heterodimer (Fig. 8D). That Jak1 is relatively ineffective in transphosphorylation is supported by the observation that cells expressing the EpoR/γR1 chimera show a smaller response than the cells expressing both EpoR/γR1 and EpoR/γR2 or the EpoR/γR2(p91) chimeric receptor chains. Thus, even though homodimerization of the EpoR/γR1 receptor by Epo brings the cytoplasmic domains of the two γR1 subunits into close proximity (Fig. 8C), the data of Figs. 2, 4, and 7 indicate that Jak2 is more effective at phosphorylating Jak1 than the latter is at cross-phosphorylating itself. This is consistent with the results of Briscoe et al. (52Briscoe J. Rogers N. Witthuhn B. Watling D. Harpur A. Wilks A. Horn F. Heinrich P. Stark G.R. Ihle J. Kerr I.M. J. Interferon and Cytokine Res. 1995; 15,: S56Google Scholar), who reported that a Jak1 molecule with an inactive kinase domain can replace the normal Jak1 in signal transduction by IFN-γ and suggested a structural role for Jak1 in the receptor complex. As noted above, the Jak kinases do not mediate Stat selectivity and are promiscuous in their activity; each of the Jak kinases can substitute for Jak2 in signal transduction by IFN-γ (40Kotenko S.V. Izotova L.S. Pollack B.P. Muthukumaran G. Paukku K. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1996; 271: 17174-17182Google Scholar). Selectivity is likely maintained from the extracellular receptor-ligand interaction to the final signal transduction mechanism by other regions of the intracellular domains. For example, Heim et al. (53Heim M.H. Kerr I.M. Stark G.R. Darnell J.E.J. Science. 1995; 267: 1347-1349Google Scholar) suggested that the SH2 recognition domain of Stat1α maintains some of the specificity. It remains to be established, however, how Stat1α can be activated by many different cytokines and maintain specificity through transcription. Other molecules that interact with Jaks and Stats may contribute to the specificity of the interaction (54Pollack B.P. Kotenko S.V. Izotova L. Krause C.D. Mehnert J.M. Pestka S. Eur. Cytokine Network. 1996; 7: 478Google Scholar). 4C. Schindler, personal communication. We propose that the multichain cytokine class II receptors have two major chains exemplified by the IFN-γ receptor complex (Fig. 8B). The ligand binding chain (IFN-γR1) and the accessory chain (IFN-γR2; helper receptor) serve as a foundation for the functional IFN-γR complex (16Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Google Scholar, 40Kotenko S.V. Izotova L.S. Pollack B.P. Muthukumaran G. Paukku K. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1996; 271: 17174-17182Google Scholar). The geometry of the IFN-γR1 chain is such that its homodimerization yields a non-functional intracellular receptor complex. The accessory chain completes this function (Fig. 8A). The question arises: why should two separate chains have evolved when one in the correct configuration would suffice? We postulate that the presence of two distinct chains provides for more effective control and fine tuning of responses to ligand. For example, the differences in response of TH1 and TH2 cells to IFN-γ result from the lack of expression of the IFN-γR2 chain in the TH1 subset (55Bach E.A. Szabo S.J. Dighe A.S. Ashkenazi A. Aguet M. Murphy K.M. Schreiber R.D. Science. 1995; 269: 1215-1217Google Scholar–57Skrenta H. Peritt D. Cook J.R. Garotta G. Trinchieri G. Pestka S. Eur. Cytokine Network. 1996; 7: 622Google Scholar) and allows exquisite fine tuning of sensitivity to IFN-γ. It is also possible that receptors with multiple chains could recruit additional factors into the complex to generate a wider variety of intracellular signals. This could explain how receptors with multiple subunits could activate a greater number of specific pathways and signals than those with fewer elements in the receptor complex. Our experiments begin to provide an insight into these possibilities. We thank Brian Pollack for providing the pγR2EC cDNA, Dr. Lawrence Blatt of Amgen for the gift of recombinant human erythropoietin, and Dr. Simon Jones for the EpoR cDNA. We are grateful to Dr. James Darnell, Jr. for the anti-Stat1α antibodies. We thank Dr. Jerry Langer for critical review of the manuscript and Eleanor Kells for its preparation." @default.
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• W1987386721 title "Chimeric Erythropoietin-Interferon γ Receptors Reveal Differences in Functional Architecture of Intracellular Domains for Signal Transduction" @default.
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