Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP

Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6Rα and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFPIL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6Rα, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6·IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors. Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6Rα and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFPIL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6Rα, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6·IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors. Cytokines are important mediators in the regulation of immune responses and inflammation. Dysregulated cytokine signaling leads to chronic inflammation and cancer. Therefore, pro-inflammatory cytokines, such as tumor necrosis factor (TNF) 2The abbreviations used are: TNF, tumor necrosis factor; BSA, bovine serum albumin; CBM, cytokine binding module; CFP, cyan fluorescent protein; IL, interleukin; LIF, leukemia inhibitory factor; OSM, oncostatin M; RFP, receptor fusion protein; STAT, signal transducer and activator of transcription; YFP, yellow fluorescent protein; s, soluble; D, domain; R, receptor; ELISA, enzyme-linked immunosorbent assay; Bistris, 2-[bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)propane-1,3-diol. and interleukin-1 and -6, have been identified as promising therapeutic targets. First approaches to specifically block the action of pro-inflammatory cytokines have focused on the use of neutralizing antibodies against a specific cytokine or its receptor. Only recently, the value of soluble cytokine receptors as cytokine antagonists for the treatment of inflammatory diseases has been fully recognized. Pro-inflammatory cytokines signal through receptor proteins consisting of an extracellular part, a single transmembrane region and a cytoplasmic domain. Ligand binding to the extracellular part of the receptor results in the activation of signal transduction cascades by the cytoplasmic domain. Soluble receptors consisting only of the extracellular part are potent inhibitors of cytokine activity. They bind the cytokine with the same specificity and affinity as the membrane-bound receptors without eliciting an intracellular signal. A dimeric form of the soluble TNF receptor is currently used for the treatment of inflammatory diseases caused by elevated TNF expression (1Goldenberg M.M. Clin. Ther. 1999; 21: 75-87Abstract Full Text PDF PubMed Scopus (133) Google Scholar). Most cytokines signal through heteromeric receptor complexes consisting of two or more different receptor subunits. In such a case, inhibition of cytokine activity by soluble receptors is more challenging. Recently, we and others have shown that the appropriate fusion of different soluble receptor proteins results in highly potent antagonists (2Economides A.N. Carpenter L.R. Rudge J.S. Wong V. Koehler-Stec E.M. Hartnett C. Pyles E.A. Xu X. Daly T.J. Young M.R. Fandl J.P. Lee F. Carver S. McNay J. Bailey K. Ramakanth S. Hutabarat R. Huang T.T. Radziejewski C. Yancopoulos G.D. Stahl N. Nat. Med. 2003; 9: 47-52Crossref PubMed Scopus (340) Google Scholar, 3Ancey C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Interleukin-6 (IL-6) has been validated as a target for the treatment of several diseases, such as rheumatoid arthritis and the lymphoproliferative disorder known as Castleman disease (4Choy E. Ann. Rheum. Dis. 2003; 62: 68-69PubMed Google Scholar, 5Nishimoto N. Kanakura Y. Aozasa K. Johkoh T. Nakamura M. Nakano S. Nakano N. Ikeda Y. Sasaki T. Nishioka K. Hara M. Taguchi H. Kimura Y. Kato Y. Asaoku H. Kumagai S. Kodama F. Nakahara H. Hagihara K. Yoshizaki K. Kishimoto T. Blood. 2005; 106: 2627-2632Crossref PubMed Scopus (595) Google Scholar). IL-6 signals through two different receptor proteins. It first binds to an α-receptor subunit (IL-6Rα). The low affinity complex of IL-6 and IL-6Rα engages the signal transducing receptor subunit gp130 leading to a high affinity receptor complex and the initiation of the cytoplasmic signaling cascades. Upon ligand binding, Janus tyrosine kinases that are constitutively associated with gp130 become activated, resulting in tyrosine phosphorylation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Activated STAT3 accumulates in the nucleus where it induces IL-6 target genes (6Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Müller-Newen G. Schaper F. Biochem. J. 2003; 374: 1-20Crossref PubMed Scopus (2544) Google Scholar). Soluble gp130 (sgp130) has a moderate antagonistic effect on IL-6 activity (7Narazaki M. Yasukawa K. Saito T. Ohsugi Y. Fukui H. Koishihara Y. Yancopoulos G.D. Taga T. Kishimoto T. Blood. 1993; 82: 1120-1126Crossref PubMed Google Scholar), whereas soluble IL-6Rα (sIL-6Rα) acts even agonistically (8Taga T. Hibi M. Hirata Y. Yamasaki K. Yasukawa K. Matsuda T. Hirano T. Kishimoto T. Cell. 1989; 58: 573-581Abstract Full Text PDF PubMed Scopus (1205) Google Scholar). Together with IL-6, sIL-6Rα can be regarded as a co-ligand required for the activation of gp130. We have shown that, in the presence of sgp130, the agonistic activity of sIL-6Rα is converted to an antagonistic activity (9Müller-Newen G. Küster A. Hemmann U. Keul R. Horsten U. Martens A. Graeve L. Wijdenes J. Heinrich P.C. J. Immunol. Google Scholar). Thus, the of sIL-6Rα and results in a inhibition of IL-6 activity. The extracellular part of gp130 of a single by domains M. M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. Full Text PDF PubMed Scopus Google Scholar). and form the cytokine binding is the of the cytokine receptors Biochem. PubMed Scopus Google Scholar). The extracellular part of IL-6Rα also of an by a and K. Taga T. Hirata Y. H. Y. T. Hirano T. Kishimoto T. PubMed Scopus Google Scholar). By it was shown that IL-6 its receptors with binding A. PubMed Scopus Google Scholar). binds the of IL-6Rα H. Yasukawa K. S. M. Yamasaki K. Hibi M. Taga T. Kishimoto T. J. 1993; PubMed Scopus Google Scholar), and and with the and of I. Horsten U. S. H. Küster A. J. Heinrich P.C. Müller-Newen G. J. Immunol. 1999; Google Scholar). Recently, the structure of the soluble hexameric IL-6 receptor complex consisting of two of IL-6, sIL-6Rα and has been by 2003; PubMed Scopus Google Scholar). IL-6 with its the of and with its the of a gp130 The same is for the IL-6 leading to a highly complex. on the of the IL-6 receptor complex and the characterized antagonistic activity of the of sIL-6Rα and sgp130, we a fusion protein receptor fusion consisting of the ligand binding domains of gp130 and IL-6Rα with an appropriate C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). We have shown that this fusion protein acts as a highly potent IL-6 inhibitor. The cytokine receptor gp130 is a shared receptor subunit that is used by cytokines, such as oncostatin M and leukemia inhibitory factor J. J. PubMed Google Scholar, G. 2003; PubMed Google Scholar). Although IL-6-RFP does not C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), its activity and has not been Although the of cytokine receptors as has been Nat. Med. 2003; 9: PubMed Scopus Google Scholar), their mode of action has not been in sufficient Therefore, we the and of the complex by IL-6 and IL-6-RFP. We have shown that IL-6-RFP is a highly specific IL-6 inhibitor that does not with the of the cytokines and A for the of on blue native gel fluorescent fusion and is We have shown that a 2-fold molar excess of IL-6-RFP over IL-6 is sufficient to IL-6 in a IL-6·IL-6-RFP complex. The complex of IL-6·IL-6-RFP is more stable than the complex of IL-6 with the separate soluble receptors sIL-6Rα and As shown in a live cell bound to its cell surface is removed from the plasma membrane upon the addition of IL-6-RFP. of the by native gel and gel filtration suggests that the of the IL-6·IL-6-RFP complex is analogous to the hexameric receptor complex identified by Thus, IL-6-RFP is a promising IL-6 inhibitor for the treatment of diseases caused by IL-6 IL-6 was in and as R. F. V. A. G. G. J. Biochem. PubMed Scopus Google Scholar). The specific activity of IL-6 was by a cell J. Immunol. PubMed Scopus Google Scholar). was from and from sIL-6Rα was in cells as Hemmann U. Küster A. Müller-Newen G. J. S. J.P. S. Heinrich P.C. J. Biochem. PubMed Scopus Google Scholar). was from and of cell from the was in with with serum cells in with and cells to the and of cell by I. M. was in with with and in a in cells to the cells and with with by the and with a constitutively in for and with IL-6 or the of IL-6 and IL-6-RFP for the molar the specificity of cells in with or a of and with LIF, or a of and IL-6-RFP. to the cytokine and IL-6-RFP for to complex of and out to the of the The activity to the was determined as activity. The out in and the and of and cells on and with the cytokine or of cytokine and IL-6-RFP for cells with and The with and an against or STAT3 antibodies used in a in and and of YFP-IL-6 in cells in the cells with an expression for the was of the the and cell was removed by and filtration. YFP-IL-6 was from cell by affinity The with bovine serum to protein and against The of YFP-IL-6 in the was determined by enzyme-linked immunosorbent M. L. E. A. L. J. Immunol. PubMed Scopus Google Scholar), and YFP-IL-6 a of whereas the and was The of was used for the of the YFP-IL-6 of IL-6-RFP in IL-6-RFP was as C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The that of gp130 with of IL-6Rα of the extracellular region of IL-6Rα and is to be of low is with the C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). IL-6-RFP the expression the by the The IL-6-RFP was out from the by and and the with the same cells in with with of and from the cell by for expression of IL-6-RFP in cells by The was by cells in protein cells with the IL-6-RFP the cell IL-6-RFP was and from cells and by and filtration. of IL-6-RFP by was from by affinity with IL-6 to the cell to the and with proteins with and by affinity by and with antibodies against sIL-6Rα or The with and against The of IL-6-RFP was a of IL-6-RFP was for to without of activity. of IL-6-RFP by for the of IL-6-RFP was out as (9Müller-Newen G. Küster A. Hemmann U. Keul R. Horsten U. Martens A. Graeve L. Wijdenes J. Heinrich P.C. J. Immunol. Google Scholar). The with and was used as the The was by 2-fold of in cells and by (9Müller-Newen G. Küster A. Hemmann U. Keul R. Horsten U. Martens A. Graeve L. Wijdenes J. Heinrich P.C. J. Immunol. Google Scholar). PAGE and of of complex YFP-IL-6 and IL-6-RFP and for molar as by blue native PAGE H. G. Biochem. PubMed Scopus Google Scholar, S. A. A. H. G. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google to or to The was of Bistris, and whereas the was used as molecular In was as an YFP-IL-6 and IL-6-RFP with to protein The of was with a gel an whereas the was a the and by of cells was out a with an a an cell and a and live cell imaging, cells with for an of gp130 to C. M. S. Heinrich P.C. Müller-Newen G. J. 2005; PubMed Scopus Google Scholar). cells and the cell The and a of and a of of signals from the of the was was with and a was with and a was of with YFP-IL-6 and and and was over In a a molar excess of IL-6-RFP over YFP-IL-6 was to the cells filtration was out with a a of the was The was with of a gel filtration protein and gel filtration of YFP-IL-6 and the IL-6-RFP and STAT3 by IL-6 by the Cytokines and cells to IL-6, OSM, and with the of protein genes P.C. T. Biochem. J. PubMed Scopus Google and are to the specificity of IL-6-RFP. As shown in IL-6-RFP the of the by IL-6 in a A 2-fold molar excess of IL-6-RFP over IL-6 to a of the of A molar excess to of IL-6 by was not a molar excess of IL-6-RFP over LIF, activity was not A moderate inhibition of activity was a that IL-6-RFP acts on not only of the cytokine and the inhibitor also the inhibitor is and or with a of was cytokine whereas STAT3 tyrosine phosphorylation be STAT3 activation was IL-6 and IL-6-RFP for or In this a of the action of IL-6-RFP was not and IL-6-RFP not with STAT3 activation by or of the IL-6 and IL-6-RFP by a the of IL-6-RFP has been in C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), the complex of IL-6-RFP and IL-6 has not been In a we have shown that fusion of the yellow fluorescent protein to the of IL-6 does not its C. M. S. Heinrich P.C. Müller-Newen G. J. 2005; PubMed Scopus Google Scholar). The fluorescent cytokine YFP-IL-6 was in cells and by affinity of of was of a and was blue native PAGE the gel was by a YFP-IL-6 was as two separate and 3). the same gel was the the YFP-IL-6 is whereas the is only the YFP-IL-6 is Thus, the of the was or even the of a In was the The is the the and are also as molecular mass The molecular mass of is Therefore, the the and the to a of YFP-IL-6 whereas the the The addition of IL-6-RFP to in a of the fluorescent complex of YFP-IL-6 with IL-6-RFP We that the was with a in We this to or of the on of and in the A 2-fold molar excess of IL-6-RFP was sufficient to YFP-IL-6 a or molar of IL-6-RFP over a complex was a complex sIL-6Rα and a molar excess of sIL-6Rα is required The complex a 2-fold molar excess with The of sIL-6Rα and required a molar excess to bind whereas a 2-fold molar excess to only a of YFP-IL-6 with results that IL-6-RFP binds YFP-IL-6 more than its soluble receptors. IL-6 a with IL-6-RFP than with Soluble to the of the complexes by IL-6 and IL-6-RFP or IL-6 and its soluble receptors. In a first the complexes in the presence of urea Although the complex of IL-6·IL-6-RFP is in the presence of urea only a of the complex is in the presence of urea a of with of the of the YFP-IL-6 more that urea induces a of the YFP-IL-6 In a the complexes by YFP-IL-6 and IL-6-RFP or YFP-IL-6 and its soluble receptors with of IL-6 for to the of YFP-IL-6 by A molar excess of IL-6 over YFP-IL-6 was required to a of YFP-IL-6 in the complex of YFP-IL-6 and IL-6-RFP The of the complex a molar excess of IL-6 over YFP-IL-6 whereas the of the more In the of the receptor complex by YFP-IL-6 and its soluble of IL-6 sufficient to YFP-IL-6 The of the YFP-IL-6 IL-6 was a molar In a of sIL-6Rα and to the complex by and IL-6-RFP complex was IL-6-RFP was to a the complex of was only a molar of IL-6-RFP over sIL-6Rα and Thus, the cytokine is more trapped in complex with IL-6-RFP than in complex with the soluble receptors. IL-6-RFP YFP-IL-6 from the we as a of its superior be to IL-6 from its receptor on the plasma Therefore, we cells with an of gp130 to C. M. S. Heinrich P.C. Müller-Newen G. J. 2005; PubMed Scopus Google and cells by As shown in and the plasma membrane of cells is in the resulting from surface expression of the addition of YFP-IL-6 and sIL-6Rα cells the fluorescent cytokine the cell surface resulting in a membrane in the The of and in the of the plasma membrane are in YFP-IL-6 binding to In a a molar excess of IL-6-RFP over YFP-IL-6 was YFP-IL-6 binding was The addition of IL-6-RFP in a of cell surface of as shown in the of the The of and in the region of are in the of the addition of the YFP-IL-6 whereas The of the the plasma membrane that YFP-IL-6 is removed from the cell the receptors by Thus, IL-6-RFP has the to IL-6 from receptor complexes the cell of the of the by was from cell by affinity on In IL-6-RFP as a protein with an apparent molecular mass of We used a gel filtration to the molecular of and the complex of YFP-IL-6 and IL-6-RFP. YFP-IL-6 was by of the IL-6-RFP was by C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). YFP-IL-6 in two from the gel filtration to the to YFP-IL-6 and YFP-IL-6 is in with the two by blue native The of IL-6-RFP is shown in IL-6-RFP be The of IL-6-RFP is in a to that the IL-6-RFP molecular mass of YFP-IL-6 with a 2-fold molar excess of IL-6-RFP in a in the The of YFP-IL-6 from the to a apparent molecular that YFP-IL-6 is trapped in a complex with the results by blue native by to IL-6-RFP blue that the YFP-IL-6 also IL-6-RFP. In a of IL-6-RFP of YFP-IL-6 to the IL-6-RFP The apparent molecular mass of the complex from gel filtration is the complex is to the native hexameric receptor complex of two of IL-6, IL-6Rα, and gp130 2003; PubMed Scopus Google a complex is consisting of two of YFP-IL-6 and two of IL-6-RFP. The molecular mass for such a complex is The the and can be to the of the IL-6 receptor complex 2003; PubMed Scopus Google as to the structure of the soluble IL-6 receptor IL-6 signals through a complex consisting of two of IL-6, IL-6Rα, and gp130 2003; PubMed Scopus Google Scholar). The soluble receptors sIL-6Rα and are in in M. S. M. Yasukawa K. H. Saito T. Y. T. Kishimoto T. J. Immunol. Google Scholar, G. C. Keul R. Hemmann U. Wijdenes J. J.P. Heinrich P.C. J. Biochem. PubMed Scopus Google and (7Narazaki M. Yasukawa K. Saito T. Ohsugi Y. Fukui H. Koishihara Y. Yancopoulos G.D. Taga T. Kishimoto T. Blood. 1993; 82: 1120-1126Crossref PubMed Google Scholar, G. Küster A. Hemmann U. Keul R. Horsten U. Martens A. Graeve L. Wijdenes J. Heinrich P.C. J. Immunol. Google Scholar), We that the action of soluble receptors IL-6 responses (9Müller-Newen G. Küster A. Hemmann U. Keul R. Horsten U. Martens A. Graeve L. Wijdenes J. Heinrich P.C. J. Immunol. Google Scholar). on this we a fusion protein of the ligand binding domains of IL-6Rα and and gp130 turned out to be a highly potent inhibitor of IL-6 C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). cytokine inhibitors are promising for against chronic inflammation and diseases Nat. Med. 2003; 9: PubMed Scopus Google Scholar). Therefore, a of their mode of action is of gp130 is the shared signaling receptor subunit of the cytokines, specificity of IL-6-RFP is a signals through a of gp130 and the or a of gp130 and the M.R. J. S. PubMed Scopus Google Scholar). a excess of IL-6-RFP over does not activity and that the affinity of to the gp130 of IL-6-RFP is signals through a of gp130 and the M.R. J. S. PubMed Scopus Google Scholar). a molar excess of IL-6-RFP over LIF, activity is of IL-6-RFP can be by low affinity binding of to domains and of gp130 T. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of IL-6-RFP sufficient to IL-6 in a the fusion protein is a highly specific IL-6 inhibitor. The low activity of IL-6-RFP and is in with the of cytokines by the soluble gp130 A molar excess of over in only a inhibition of (7Narazaki M. Yasukawa K. Saito T. Ohsugi Y. Fukui H. Koishihara Y. Yancopoulos G.D. Taga T. Kishimoto T. Blood. 1993; 82: 1120-1126Crossref PubMed Google Scholar). in a that IL-6 a of IL-6-RFP is the activation of STAT3 in to IL-6, IL-6-RFP has to be results that a of STAT3 activation is not sufficient for a that the cytokine has to for a of that, for the treatment of a disease with the inhibitor can block the activity of IL-6 even the cytokine is by the results of live cell IL-6 bound to the cell surface is removed from the receptor complex upon the addition of IL-6-RFP The of fusion proteins in native can be used to S. J. C. G. A. F. PubMed Scopus (28) Google Scholar). We the of blue native gel fluorescent fusion and gel to the of IL-6-RFP with was by fusion of the to the of The of IL-6 are not in receptor binding J.P. M. J. Immunol. 1989; Google Scholar). Therefore, fusion of to the of IL-6 does not IL-6 C. M. S. Heinrich P.C. Müller-Newen G. J. 2005; PubMed Scopus Google Scholar). the of YFP-IL-6 with IL-6-RFP be by a of the fluorescent A 2-fold molar excess of IL-6-RFP over YFP-IL-6 is sufficient to YFP-IL-6 in a high molecular mass complex. In gel the separate soluble receptor proteins sIL-6Rα and are of is in with their to IL-6 C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The of in IL-6 is IL-6-RFP sIL-6Rα has affinity to IL-6 The superior activity of IL-6-RFP is also in the of the complex by the low of of YFP-IL-6 by IL-6, the of the complex an excess of the soluble receptor proteins and and the of YFP-IL-6 from receptor complexes the plasma membrane by IL-6-RFP In blue native the of the complex of YFP-IL-6 and IL-6-RFP is than the of the a of the of molecular of the the not we an apparent molecular mass of for the complex of YFP-IL-6 and IL-6-RFP. We gel filtration with a to the molecular determined by blue native filtration the by blue native We that of of a and a dimeric of YFP-IL-6 is not caused by the as a in native PAGE Thus, of the fusion protein is to the known of IL-6 to form The IL-6 has been to in the presence of urea with a of urea A. PubMed Scopus Google Scholar). The of YFP-IL-6 in gel is in with the on the IL-6 The of the IL-6·IL-6-RFP complex does not that to of the YFP-IL-6 that IL-6-RFP is with is in with the that IL-6 by of the gp130 binding of two IL-6 A. PubMed Scopus Google Scholar). are upon binding of IL-6 to IL-6-RFP IL-6-RFP from the gel filtration with a to a dimeric as as sIL-6Rα are proteins that in the presence of IL-6 G. Yasukawa K. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). in the structure of the hexameric IL-6 receptor complex 2003; PubMed Scopus Google IL-6Rα and gp130 in the dimeric are by the of the IL-6Rα and domains leading to the of an IL-6-RFP in the of IL-6 we a IL-6-RFP as in IL-6 this leading to a complex of IL-6 and IL-6-RFP in to the hexameric IL-6 receptor complex The apparent molecular determined by gel filtration and blue native PAGE are than the molecular mass of the complex. the complex in is not a highly structure a the two IL-6 the apparent molecular A and apparent molecular has also been in of the soluble IL-6 receptor complex by gel filtration and G. Yasukawa K. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). by blue native PAGE and gel filtration suggests that the IL-6·IL-6-RFP complex is in to the soluble extracellular hexameric IL-6 receptor complex. The complex is by the of and The superior of the IL-6·IL-6-RFP complex in with a complex by IL-6, and is caused by the of through a of the ligand binding domains of sIL-6Rα and Thus, a molecular is for the high specificity and superior affinity of IL-6-RFP in to IL-6 Therefore, the fusion of ligand binding domains of heteromeric cytokine receptors is a promising for anti-cytokine We Wijdenes for the of the gp130 We for with the and Küster for