Species-specific Agonist/Antagonist Activities of Human Interleukin-4 Variants Suggest Distinct Ligand Binding Properties of Human and Murine Common Receptor γ Chain

Interleukin-4 (IL-4) is a pleiotropic cytokine eliciting various responses in target cells upon binding to its receptor. Activation of the IL-4 receptor probably involves interaction of the ligand with both the IL-4 receptor α subunit (IL-4Rα) and the common γ chain (cγ). Although human and murine IL-4 receptor α chains are specific for IL-4 from the same species, murine cγ can form a signal-competent complex with human IL-4Rα (hIL-4Rα) and human IL-4 (hIL-4). We have generated a hIL-4 responsive murine myeloid cell line (FDC-4G) expressing a chimera comprising the extracellular domain of human IL-4Rα and the intracellular domain of human granulocyte colony-stimulating factor receptor (hG-CSFR). This hybrid receptor was shown to form a complex with hIL-4 and the murine cγ-chain. Biological activities of human IL-4 variants on murine FDC-4G cells and on the human erythroleukemic cell line TF-1 displayed a strikingly different pattern. Single amino acid replacements at two different positions in the C-terminal helix of hIL-4, the region of the previously defined “signaling site,” lead to an inverse agonist/antagonist behavior of the resulting cytokines in the two cellular systems. From these findings we conclude that upon formation of the activated IL-4 receptor complex murine and human cγ interact with hIL-4 in a geometrically different fashion. Interleukin-4 (IL-4) is a pleiotropic cytokine eliciting various responses in target cells upon binding to its receptor. Activation of the IL-4 receptor probably involves interaction of the ligand with both the IL-4 receptor α subunit (IL-4Rα) and the common γ chain (cγ). Although human and murine IL-4 receptor α chains are specific for IL-4 from the same species, murine cγ can form a signal-competent complex with human IL-4Rα (hIL-4Rα) and human IL-4 (hIL-4). We have generated a hIL-4 responsive murine myeloid cell line (FDC-4G) expressing a chimera comprising the extracellular domain of human IL-4Rα and the intracellular domain of human granulocyte colony-stimulating factor receptor (hG-CSFR). This hybrid receptor was shown to form a complex with hIL-4 and the murine cγ-chain. Biological activities of human IL-4 variants on murine FDC-4G cells and on the human erythroleukemic cell line TF-1 displayed a strikingly different pattern. Single amino acid replacements at two different positions in the C-terminal helix of hIL-4, the region of the previously defined “signaling site,” lead to an inverse agonist/antagonist behavior of the resulting cytokines in the two cellular systems. From these findings we conclude that upon formation of the activated IL-4 receptor complex murine and human cγ interact with hIL-4 in a geometrically different fashion. Interleukin-4 exerts its activity on target cells by interaction with at least two membrane-bound receptor chains, i.e. the IL-4Rα 1The abbreviations used are:IL-4interleukin-4IL-4Rαthe IL-4 receptor α subunithIL-4human interleukin-4hIL-4Rhuman interleukin-4 receptormIL-4murine interleukin-4mIL-3murine interleukin-3hIL-4Rαhuman interleukin-4 receptor α subunitcγcommon receptor γ chainhcγhuman common receptor γ chainmcγmurine common receptor γ chainhG-CSFRhuman granulocyte-colony-stimulating factor receptorGM-CSFhuman granulocyte/macrophage colony-stimulating factorIL-2interleukin-2IL-2Rβγinterleukin-2 receptor β and γ chainDMEMDulbecco,'s modified essential mediumFCSfetal calf serumG418GeniticinMTT3-{4,5-dimethythiazol-2-yl}-2,5-diphenyl-tetrazolium bromide-tetrazolium4GhIL-4Rα/hG-CSFR hybrid receptorIgGimmunoglobulin GPBSphosphate-buffered saline. subunit (formerly termed IL-4R) (1Idzerda R.L. March C.J. Mosley B. Lyman S.D. Vanden Bos T. Gimpel S.D. Din W.S. Grabstein K.H. Widmer M.B. Park L.S. Cosman D. Beckman M.P. J. Exp. Med. 1990; 171: 861-873Crossref PubMed Scopus (362) Google Scholar), and the common receptor γ chain (cγ) (2Kondo M. Takeshita T. Ishii N. Nakamura M. Watanabe S. Arai K. Sugamura K. Science. 1993; 262: 1874-1879Crossref PubMed Scopus (735) Google Scholar, 3Russell S.M. Keegan A.D. Harada N. Nakamura Y. Noguchi M. Leland P. Friedman M.C. Miyajima A. Puri R.K. Paul W.E. Leonard W.J. Science. 1993; 262: 1880-1883Crossref PubMed Scopus (742) Google Scholar), both of which are members of the hematopoietin receptor superfamily (4Bazan J.F. Immunol. Today. 1990; 11: 350-354Abstract Full Text PDF PubMed Scopus (511) Google Scholar). The induction of productive IL-4 receptor complex formation by binding of the ligand is not yet understood; however, mutational analysis of hIL-4 indicated that two distinct structural determinants of the cytokine are important for this process (5Kruse N. Tony H.-P. Sebald W. EMBO J. 1992; 11: 3237-3244Crossref PubMed Scopus (155) Google Scholar, 6Kruse N. Shen B.-J. Arnold S. Tony H.-P. Müller T. Sebald W. EMBO J. 1993; 12: 5121-5129Crossref PubMed Scopus (115) Google Scholar). Amino acids located within helixes A and C of the hIL-4 molecule have been shown to be essential for the interaction with hIL-4Rα, whereas three positions in the C-terminal helix have proven crucial for signaling. Replacement of residues Arg121, Tyr124, or Ser125yielded high affinity partial agonists or antagonists of hIL-4 in cellular assays employing human B or T cells (6Kruse N. Shen B.-J. Arnold S. Tony H.-P. Müller T. Sebald W. EMBO J. 1993; 12: 5121-5129Crossref PubMed Scopus (115) Google Scholar), most probably by interfering with a productive interaction of hIL-4 and cγ. hIL-4 does not detectably bind to murine IL-4Rα (1Idzerda R.L. March C.J. Mosley B. Lyman S.D. Vanden Bos T. Gimpel S.D. Din W.S. Grabstein K.H. Widmer M.B. Park L.S. Cosman D. Beckman M.P. J. Exp. Med. 1990; 171: 861-873Crossref PubMed Scopus (362) Google Scholar, 7Morrison B.W. Leder P. J. Biol. Chem. 1992; 267: 11957-11963Abstract Full Text PDF PubMed Google Scholar). Several groups have shown, however, that human IL-4Rα can confer hIL-4 responsiveness to murine lymphoid cells when expressed by gene transfer, thus indicating that murine cγ is able to form a signaling competent receptor complex with hIL-4 and hIL-4Rα (1Idzerda R.L. March C.J. Mosley B. Lyman S.D. Vanden Bos T. Gimpel S.D. Din W.S. Grabstein K.H. Widmer M.B. Park L.S. Cosman D. Beckman M.P. J. Exp. Med. 1990; 171: 861-873Crossref PubMed Scopus (362) Google Scholar, 8Harada N. Yang G. Miyajima A. Howard M. J. Biol. Chem. 1992; 267: 22752-22758Abstract Full Text PDF PubMed Google Scholar, 9Skoda R.C. Seldin D.C. Chiang M.K. Peichel C.L. Vogt T.F. Leder P. EMBO J. 1993; 12: 2645-2653Crossref PubMed Scopus (168) Google Scholar, 10Sakamaki K. Wang H.-M. Miyajima I. Kitamura T. Todokoro K. Harada N. Miyajima A. J. Biol. Chem. 1993; 268: 15833-15839Abstract Full Text PDF PubMed Google Scholar, 11Koettnitz K. Kalthoff F.S. Eur. J. Immunol. 1993; 23: 988-991Crossref PubMed Scopus (25) Google Scholar). interleukin-4 the IL-4 receptor α subunit human interleukin-4 human interleukin-4 receptor murine interleukin-4 murine interleukin-3 human interleukin-4 receptor α subunit common receptor γ chain human common receptor γ chain murine common receptor γ chain human granulocyte-colony-stimulating factor receptor human granulocyte/macrophage colony-stimulating factor interleukin-2 interleukin-2 receptor β and γ chain Dulbecco,'s modified essential medium fetal calf serum Geniticin 3-{4,5-dimethythiazol-2-yl}-2,5-diphenyl-tetrazolium bromide-tetrazolium hIL-4Rα/hG-CSFR hybrid receptor immunoglobulin G phosphate-buffered saline. In order to investigate the interactions between hIL-4 and the two hIL-4 receptor components, we have established a hIL-4-responsive murine myeloid precursor cell line. A chimera of the extracellular domain of hIL-4Rα fused to the cytoplasmic portion of hG-CSFR was expressed in factor-dependent FDC-P1 cells and shown to become associated with murine cγ upon binding of hIL-4. Subsequently the formation of productive receptor complexes resulting in cell proliferation was studied using mutant variants of hIL-4. Comparison of the results with those obtained in a parallel set of experiments employing the hIL-4-responsive human cell line TF-1 revealed a different activity pattern of hIL-4 variants. These findings are discussed with respect to the putative interaction of hIL-4 with murine and human cγ. Total RNA was isolated from 1 g of fresh human placenta (obtained from Dr. J. Martius, Department of Gynecology, University of Würzburg) according to the guanidinium thiocyanate-phenol-chloroform extraction method (12Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63189) Google Scholar), and used as a template for cDNA synthesis which was performed using Superscript Reverse Transcriptase (Life Technologies, Inc.) following the manufacturer's instructions. The cDNA clone providing the extracellular domain of hIL-4Rα has been described previously (5Kruse N. Tony H.-P. Sebald W. EMBO J. 1992; 11: 3237-3244Crossref PubMed Scopus (155) Google Scholar). Oligonucleotide synthesis, polymerase chain reactions, and other enzymatic manipulations of DNA were done following standard procedures (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For transfection experiments SV40-based expression vector pKCR (14O'Hare K. Benoist C. Breathnach R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1527-1531Crossref PubMed Scopus (114) Google Scholar) and neomycin resistance plasmid pSV2neo (15Southern P.J. Berg P. J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google Scholar) were employed. Other reagents and enzymes were from Boehringer (Mannheim, Germany), Fermentas (Vilnius, Lithuania), Amersham-Buchler (Braunschweig, Germany), and Merck (Darmstadt, Germany). Both the murine myeloid precursor cell line FDC-P1 (16Dexter T.M. Garland J. Scott D. Scolnick E. Metcalf D. J. Exp. Med. 1980; 152: 1036-1047Crossref PubMed Scopus (534) Google Scholar) and the human erythroleukemic cell line TF-1 (17Kitamura T. Tange T. Terasawa T. Chiba S. Kuwaki T. Miyagawa K. Piao Y.-F. Miyazono K. Urabe A. Takaku F. J. Cell. Physiol. 1989; 140: 323-334Crossref PubMed Scopus (715) Google Scholar) were described previously. Cells were routinely grown in DMEM, 8% FCS (FDC-P1) or RPMI 1640, 8% FCS (TF-1). Media were supplemented with 5% culture supernatant of mIL-3 producing X63Ag8-653 BPV mIL-3 cells (18Karasuyama H. Melchers F. Eur. J. Immunol. 1988; 18: 97-104Crossref PubMed Scopus (1080) Google Scholar) (FDC-P1) or recombinant GM-CSF basically produced as described for hIL-4 (19Kruse N. Lehrnbecher T. Sebald W. FEBS Lett. 1991; 286: 58-60Crossref PubMed Scopus (48) Google Scholar) at a final concentration of 5 n M (TF-1). Transfection of FDC-P1 cells was performed by electroporation using a Easyject+electroporator (Eurogentec). 3.5 × 106cells were washed and resuspended in 400 μl of DMEM, 5% FCS containing 50 μg of pKCR derivative and 2 μg of pSV2neo and subsequently subjected to an electric pulse of 250 V at 1500 microfarads. Cells were then kept in DMEM, 8% plus mIL-3 for 48 h before adding G418 to a final concentration of 1 mg/ml. After cultivation in 24-well plates for 2 weeks (medium was renewed twice), G418-resistant cell clones were propagated in 75-ml flasks and further screened for receptor expression by flow cytometry. For cytometric identification of hIL-4Rα expression on transfectants, 106cells were stained with monoclonal concentration 400 n the extracellular domain of hIL-4Rα H.-P. Shen B.-J. P. Sebald W. Eur. J. Biochem. PubMed Scopus (115) Google Scholar) in μl of for at with amino and using a flow with a of recombinant hIL-4 was done as described J. Arai N. T. Arai K. J. Biochem. 1987; PubMed Scopus Google Scholar), specific activity of the cytokine 5 was by binding employing a binding on the recombinant extracellular domain of hIL-4Rα (6Kruse N. Shen B.-J. Arnold S. Tony H.-P. Müller T. Sebald W. EMBO J. 1993; 12: 5121-5129Crossref PubMed Scopus (115) Google Scholar). binding experiments were performed a standard L.S. D. Grabstein K. Proc. Natl. Acad. Sci. U. S. A. 1987; PubMed Scopus Google Scholar). of 2 × 106cells were with of in a of μl at for 2 Cells were then from by a and was using a γ binding of as in a parallel at least of ligand was before the of × were with RPMI medium and in μl of serum containing 1 n hIL-4 at for 1 Cells were then washed with with 1 M in at for washed with and subsequently in μl of M M 1 M 5% M 2 M After an at for the cell were at for h and on was by at × g for were for 1 h at of 1 of or μl of containing (2Kondo M. Takeshita T. Ishii N. Nakamura M. Watanabe S. Arai K. Sugamura K. Science. 1993; 262: 1874-1879Crossref PubMed Scopus (735) Google Scholar) from Dr. K. University of of was performed by at × g for 2 an for 1 h at with μl of The were washed in in M M and for 5 in 2 × were then by and subjected to After in for were and subsequently for using a hIL-4 and and have been described previously (5Kruse N. Tony H.-P. Sebald W. EMBO J. 1992; 11: 3237-3244Crossref PubMed Scopus (155) Google Scholar, 6Kruse N. Shen B.-J. Arnold S. Tony H.-P. Müller T. Sebald W. EMBO J. 1993; 12: 5121-5129Crossref PubMed Scopus (115) Google Scholar). and and which have been and in the same and are described in H.-P. Shen B.-J. P. Sebald W. Eur. J. Biochem. PubMed Scopus (115) Google Scholar). a of murine IL-4 the culture supernatant of an cell line by Dr. W. of University of was proliferation of FDC-4G and TF-1 cells was by of formation from bromide-tetrazolium or by DNA as described previously A. U. C. Sebald W. Eur. 1992; Google Scholar). In both cells were washed in medium to mIL-3 or and with or μl of medium containing various of IL-4 or IL-4 variants for h or h by with or for of proliferation and binding were done using the A hybrid gene comprising the of the human hIL-4Rα gene the and the extracellular (1Idzerda R.L. March C.J. Mosley B. Lyman S.D. Vanden Bos T. Gimpel S.D. Din W.S. Grabstein K.H. Widmer M.B. Park L.S. Cosman D. Beckman M.P. J. Exp. Med. 1990; 171: 861-873Crossref PubMed Scopus (362) Google Scholar) fused to of the hG-CSFR gene R. Y. S. S. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar) the and intracellular was in two a was from hIL-4Rα cDNA by polymerase chain with the and the and the of the expression vector pKCR (14O'Hare K. Benoist C. Breathnach R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1527-1531Crossref PubMed Scopus (114) Google Scholar). for both and the were subsequently used to clone the hG-CSFR gene which was from human placenta RNA with the and The resulting a hybrid receptor of amino acids two to the at the with the plasmid pSV2neo (15Southern P.J. Berg P. J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google Scholar) was FDC-P1 cells by After in the of a clone was stained with an hIL-4Rα specific monoclonal and was for expression of hIL-4Rα by flow cytometry. The cells from this termed displayed an in when with cells thus indicating a We the G418-resistant clone FDC-4G become responsive to hIL-4. FDC-P1 cells have been shown to to murine IL-4 by proliferation C. S. A.D. 1991; PubMed Scopus Google we used as a in the proliferation that FDC-P1 cells were responsive to clone FDC-4G in a This was with the and that this cell line the hybrid receptor. the can a factor-dependent FDC-P1 cells when activated by G. R. S. Science. 1992; PubMed Scopus Google Scholar, E. R. S. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar), we hIL-4 mIL-3 to for culture of FDC-4G a of hIL-4 to that hIL-4, can cell for h not We to the affinity for hIL-4 of the hybrid receptor as as the of ligand binding the binding shown in was The to be which is in the same as was for human T cells (5Kruse N. Tony H.-P. Sebald W. EMBO J. 1992; 11: 3237-3244Crossref PubMed Scopus (155) Google Scholar). A of hIL-4 binding to FDC-4G cells was at a concentration of M This is to hIL-4 binding of hIL-4 to FDC-4G cells by using was performed in order to the of the receptor shown in monoclonal the extracellular domain of hIL-4Rα a complex with a of This from hIL-4 to the hIL-4Rα/hG-CSFR hybrid to the of the chimera and does not in from FDC-P1 cells not and TF-1 The a which can be to a of hIL-4 and of the extracellular domain of hIL-4Rα in both complexes be proven by of with not a murine common γ chain be in the hIL-4 binding receptor complex on FDC-4G In to the a at was by to its to the receptor was on This the same behavior as the from TF-1 for with the hybrid when of FDC-4G cells were with both and were of a of in which most the complex In order to the of the hybrid receptor ligand we subjected FDC-4G cells to proliferation experiments using a set of hIL-4 mutant has been shown previously that hIL-4 variants with amino acid positions Arg121, Tyr124, and for acid were in signaling on hIL-4-responsive human cells (5Kruse N. Tony H.-P. Sebald W. EMBO J. 1992; 11: 3237-3244Crossref PubMed Scopus (155) Google Scholar, 6Kruse N. Shen B.-J. Arnold S. Tony H.-P. Müller T. Sebald W. EMBO J. 1993; 12: 5121-5129Crossref PubMed Scopus (115) Google Scholar, S.M. F. B. G. EMBO J. 1993; 12: PubMed Scopus Google Scholar). we variants and (6Kruse N. Shen B.-J. Arnold S. Tony H.-P. Müller T. Sebald W. EMBO J. 1993; 12: 5121-5129Crossref PubMed Scopus (115) Google Scholar) as as the and and H.-P. Shen B.-J. P. Sebald W. Eur. J. Biochem. PubMed Scopus (115) Google Scholar) to these The TF-1 cell line expressing the human was used as a The was that activities of hIL-4 were different in the two cellular systems. in and hIL-4 on FDC-4G whereas as as and hIL-4 containing the for at displayed mutant an partial has been that hIL-4 as a high affinity when in proliferation with both human T cells (5Kruse N. Tony H.-P. Sebald W. EMBO J. 1992; 11: 3237-3244Crossref PubMed Scopus (155) Google Scholar) and TF-1 cells S.M. F. B. G. EMBO J. 1993; 12: PubMed Scopus Google Scholar). results with TF-1 were with these In to FDC-4G TF-1 cells to to a activity of which was to FDC-4G partial activity in the TF-1 and were of experiments were performed in order to hIL-4 variants with the were on FDC-4G cells of binding to the derivative expressed on these cells or to of the mutant that as as the and and proliferation of FDC-4G The results obtained with that this a hIL-4 for TF-1 cells S.M. F. B. G. EMBO J. 1993; 12: PubMed Scopus Google Scholar), has activity on FDC-4G cells with that of hIL-4. IL-4 receptor α chain has been shown to murine cells responsive to hIL-4 when expressed gene N. Yang G. Miyajima A. Howard M. J. Biol. Chem. 1992; 267: 22752-22758Abstract Full Text PDF PubMed Google Scholar, 9Skoda R.C. Seldin D.C. Chiang M.K. Peichel C.L. Vogt T.F. Leder P. EMBO J. 1993; 12: 2645-2653Crossref PubMed Scopus (168) Google Scholar, 10Sakamaki K. Wang H.-M. Miyajima I. Kitamura T. Todokoro K. Harada N. Miyajima A. J. Biol. Chem. 1993; 268: 15833-15839Abstract Full Text PDF PubMed Google Scholar, 11Koettnitz K. Kalthoff F.S. Eur. J. Immunol. 1993; 23: 988-991Crossref PubMed Scopus (25) Google Scholar). and IL-4Rα bind to IL-4 from the same in B.W. Leder P. J. Biol. Chem. 1992; 267: 11957-11963Abstract Full Text PDF PubMed Google Scholar), IL-4Rα of the activated IL-4 receptor. however, at least receptor that was to be with the interleukin-2 receptor γ chain or cγ (2Kondo M. Takeshita T. Ishii N. Nakamura M. Watanabe S. Arai K. Sugamura K. Science. 1993; 262: 1874-1879Crossref PubMed Scopus (735) Google Scholar, 3Russell S.M. Keegan A.D. Harada N. Nakamura Y. Noguchi M. Leland P. Friedman M.C. Miyajima A. Puri R.K. Paul W.E. Leonard W.J. Science. 1993; 262: 1880-1883Crossref PubMed Scopus (742) Google Scholar). hIL-4 responsiveness of murine cells to expression of hIL-4Rα thus that murine cγ can both and hIL-4. In order to a hIL-4-responsive cell we have expressed a chimera of hIL-4Rα and in FDC-P1 The for this was in the of is to become the cytoplasmic of IL-4Rα and cγ to an cellular the of cytoplasmic of the in the of signaling is as yet of hIL-4Rα have been with in hIL-4 proliferation N. Yang G. Miyajima A. Howard M. J. Biol. Chem. 1992; 267: 22752-22758Abstract Full Text PDF PubMed Google Scholar, 11Koettnitz K. Kalthoff F.S. Eur. J. Immunol. 1993; 23: 988-991Crossref PubMed Scopus (25) Google Scholar, A.D. K. M. Wang Paul W.E. Cell. Full Text PDF PubMed Scopus Google Scholar). has been shown that a in the intracellular domain of IL-4Rα is for interaction with receptor 1 A.D. K. M. Wang Paul W.E. Cell. Full Text PDF PubMed Scopus Google Scholar), a which probably other to an of and the to a signaling with to a set of by the activated receptor. from the intracellular domain of hIL-4Rα were to interact with the factor IL-4 J. U. W.J. M. Science. PubMed Scopus Google Scholar). In of members of the with the IL-4 receptor complex was M. K. PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar). results obtained on the and the an interaction of with cγ and a binding of to IL-4Rα T. A.D. J. Science. PubMed Scopus Google Scholar, S.M. Noguchi M. M. Friedman M. Berg M. Leonard W.J. Science. PubMed Scopus Google Scholar, T. A. H. Y. Y. I. T. Science. PubMed Scopus Google Scholar). Both become as a of receptor whereas is The of the IL-4 receptor complex has not yet been expressing a hIL-4Rα/hG-CSFR chimera we a hIL-4 receptor in a of hIL-4Rα has been shown to upon ligand and the intracellular domain of was used to a of a fused extracellular human receptor domain FDC-P1 resulting in human cell proliferation (12Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63189) Google Scholar, H. Melchers F. Eur. J. Immunol. 1988; 18: 97-104Crossref PubMed Scopus (1080) Google Scholar). The of hIL-4 to of FDC-4G cells a of hIL-4Rα in formation of the activated with the that hIL-4 binding to an of hIL-4Rα and cγ murine a receptor an which is for signaling. FDC-4G cells a behavior in to and hIL-4 both and hIL-4 by of a hybrid receptor the intracellular domain of hIL-4Rα, this the common intracellular are in both The cytoplasmic portion of receptor a of with both IL-4Rα and receptor β subunit M. M. M. H. K. M. T. T. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). For a of amino acids comprising this was to be essential for interaction with C. PubMed Scopus Google Scholar). for and other of members and other has been upon of its probably to activity T. T. PubMed Google Scholar). that of FDC-4G cells by hIL-4 results in of the hybrid receptor. and K. We that by with extracellular hIL-4 the intracellular of receptor and to The of the hG-CSFR is associated with a which in a of this a is generated by the receptor subunit which that produced by the murine We used the FDC-4G line as a cellular for the analysis of interactions between hIL-4, hIL-4Rα, and cγ in the of receptor mutational analysis of hIL-4 (5Kruse N. Tony H.-P. Sebald W. EMBO J. 1992; 11: 3237-3244Crossref PubMed Scopus (155) Google Scholar, 6Kruse N. Shen B.-J. Arnold S. Tony H.-P. Müller T. Sebald W. EMBO J. 1993; 12: 5121-5129Crossref PubMed Scopus (115) Google Scholar). The most results of this were obtained when the of FDC-4G cells and TF-1 cells to hIL-4 variants were These that the of human or murine cγ in the formation of a productive receptor complex is not of and for the of amino acid chains in helix of hIL-4 in a fashion. hIL-4 displayed activity on murine FDC-4G cells expressing a chimera and as an in experiments with hIL-4. In when with the human cell For hIL-4 an activity in the two cell was Although proliferation of FDC-4G cells was by to an from that of hIL-4, the cytokine a activity on is to that the two most amino acids of hIL-4 for signaling in FDC-4G and TF-1 are by of helix which results in an of chains T. T. Sebald W. H. J. Mol. Biol. PubMed Scopus Google Scholar) a C by amino acids of with hIL-4 7Morrison B.W. Leder P. J. Biol. Chem. 1992; 267: 11957-11963Abstract Full Text PDF PubMed Google Scholar, is to that the interaction of is by has been that of the three C-terminal amino acids of in a cytokine with B.W. Leder P. J. Biol. Chem. 1992; 267: 11957-11963Abstract Full Text PDF PubMed Google Scholar). In the of the findings in this be to and the murine of hIL-4 and hIL-4 with to activity on murine We the that murine and human cγ have different in IL-4 signaling. In the receptor human is of binding whereas murine is not S. M. Takeshita T. H. Nakamura M. Sugamura K. Biochem. 1993; PubMed Scopus Google Scholar). on the interaction of IL-4 and cγ are important to the The of C. Müller is We Dr. W. Sebald for W. and W. Müller for providing with cell Dr. C. for on cell culture the of the C. and H. for recombinant hIL-4 mutant and Dr. P. for Dr. K. Sugamura for Dr. A. and B. for on Dr. J. for human and W. for synthesis and DNA