Chimeric Erythropoietin-Interferon gamma  Receptors Reveal Differences in Functional Architecture of Intracellular Domains for Signal Transduction*

(Received for publication, September 24, 1996, and in revised form, December 9, 1996)

Geetha Muthukumaran , Serguei Kotenko , Robert Donnelly , James N. Ihle Dagger and Sidney Pestka

From the Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635 and the Dagger  Department of Biochemistry, St. Jude Children's Hospital, Memphis, Tennessee 38105

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Binding of interferon gamma (IFN-gamma ) causes oligomerization of the two interferon gamma  receptor (IFN-gamma R) subunits, receptor chain 1 (IFN-gamma R1, the ligand-binding chain) and the second chain of the receptor (IFN-gamma R2), 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-gamma R1 and IFN-gamma R2 chains demonstrate that the architecture of the EpoR and the IFN-gamma R complexes differ significantly. Although IFN-gamma R1 alone cannot initiate signal transduction, the chimera EpoR/gamma R1 (extracellular/intracellular) generates slight responses characteristic of IFN-gamma in response to Epo and the EpoR/gamma R1·EpoR/gamma R2 heterodimer is a fully functional receptor complex. The results demonstrate that the configuration of the extracellular domains influences the architecture of the intracellular domains.


INTRODUCTION

The interferon gamma  (IFN-gamma )1 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 (1, 2). Isolation of the two chains of the interferon gamma  receptor (IFN-gamma R) has permitted an analysis of the contributions of each to the signal transduction mechanism. The first chain of the receptor (IFN-gamma R1) binds ligand (3-9). The second chain of the receptor (IFN-gamma R2) does not bind ligand by itself but is required for signal transduction (3, 10-16). A large body of experiments has elucidated the involvement of the Jak-Stat pathway in signaling by various cytokines (for reviews, see Refs. 17-22). 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-gamma . Further analyses of the interactions have shown that the IFN-gamma R1 chain binds Jak1 (16, 23, 24) and the intracellular domain of the IFN-gamma R2 chain brings Jak2 into the signal transduction complex (16). Upon binding of the ligand, IFN-gamma , to the IFN-gamma R1 chain, activation of Jak1 and/or Jak2 by reciprocal transphosphorylation causes the phosphorylation of IFN-gamma R1 (16, 25). Stat1alpha , a latent cytoplasmic transcription factor (26), binds to the phosphorylated IFN-gamma R1, undergoes tyrosine phosphorylation (27), and forms homodimers that translocate to the nucleus and initiate transcription of IFN-gamma inducible genes (for reviews see Refs. 17 and 21).

As with other cytokine receptors, oligomerization upon ligand binding is the first step in the signaling cascade of IFN-gamma . IFN-gamma is a non-covalent symmetrical homodimer (28) that binds to IFN-gamma R1 with a stoichiometry of 1:2 (29, 30). It is known that a species-specific interaction between the extracellular domains of the IFN-gamma R1 and IFN-gamma R2 subunits is essential for signaling (10-12, 31-33). The IFN-gamma R2 subunit does not by itself bind the ligand, but can be cross-linked to IFN-gamma when both IFN-gamma R1 and IFN-gamma R2 chains are present (16). Several lines of evidence (16, 34) suggest that the IFN-gamma signaling complex contains two IFN-gamma R1 chains, two IFN-gamma R2 chains and one IFN-gamma 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. 18, 19, and 35). Jak2 is associated with the cytoplasmic domain of the EpoR and is activated upon ligand-induced dimerization of the receptor (36). Strikingly an Arg right-arrow Cys mutation in the extracellular domain of EpoR results in ligand independent dimerization/oligomerization and constitutive, ligand-independent activation of Jak2 and mitogenesis (37, 38).

In this study we used chimeric EpoR, IFN-gamma R1, and IFN-gamma R2 constructs to investigate the differences between the architecture of Epo and IFN-gamma 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.


MATERIALS AND METHODS

Reagents, Restriction Endonucleases, and Other Enzymes

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.; [alpha -32P]dCTP was from DuPont NEN. All other reagents were of analytical grade and were purchased from Sigma.

Cells and Media

CHO-B7 cells represent the Chinese hamster ovary cell line (CHO-K1) containing a transfected human HLA-B7 gene (12). 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 (13). 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.

Construction of Chimeric Receptors

The EpoR expression plasmid was made by cloning the EcoRI-AflIII fragment of the human EpoR cDNA p18R (39) into the EcoRI and EcoRV sites of the eukaryotic expression vector pcDNA3 (Invitrogen). The construction of plasmids expressing Hu-IFN-gamma R1 and Hu-IFN-gamma R2 chains from cDNA under the control of cytomegalovirus promoter has been previously described (5, 12, 16, 40). 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-gamma R1, and Hu-IFN-gamma R2, containing an NheI site (designated EpoREC/NheI, gamma R1EC/NheI, and gamma R2EC/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'-GCC<UNL>GCTAGC</UNL>CAGGGGTCCAGGTCGCTAGGCG-3' (corresponding to nucleotides 1874-1893 of p18R EpoR cDNA; Ref. 39), 5'-GTG<UNL>GCTAGC</UNL>CAAGAACCTTTTATACTGCT-3' (corresponding to nucleotides 779-785 of the Hu-IFN-gamma R1 cDNA; Ref. 4), and 5'-ATC<UNL>GCTAGC</UNL>CATTGCTGAAGCTCAGTGGAGG-3' (corresponding to nucleotides 1370-1390 of the Hu-IFN-gamma R2 cDNA; Ref. 14). The intracellular portions of the various receptors with the unique NheI site at the 5' end of the transmembrane domain (designated EpoRIC/NheI, gamma R1IC/NheI, and gamma R2IC/NheI were generated by PCR on corresponding cDNA templates with the use of the SP6 primer (5'-ATTTAGGTGACACTATA-3') and the internal primers 5'-GTG<UNL>GCTAGC</UNL>GACGCTCTCCCTCATCCTCG-3' (corresponding to nucleotides 1902-1921 of plasmid p18R), 5'-GTG<UNL>GCTAGC</UNL>GATTCCAGTTGTTGCTGCTTTAC-3' (corresponding to nucleotides 792-814 of the Hu-IFN-gamma R1 cDNA), and 5'-GTG<UNL>GCTAGC</UNL>GATCTCCGTGGGAACATTT-3' (corresponding to nucleotides 1398-1416 of the Hu-IFN-gamma R2 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 gamma R2EC/NheI) or BamHI (gamma R1EC/NheI), and cloned into the EcoRV and EcoRI/BamHI sites of the expression vector pcDNA3 (Invitrogen) to yield the plasmids pEpoREC, pgamma R1EC and pgamma R2EC. 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, pgamma R1IC, and pgamma R2IC. To introduce the Stat1alpha binding site of Hu-IFN-gamma R1 into the cytoplasmic domain of EpoR, two-step asymmetric PCR (detailed in Ref. 41) was carried out sequentially on Hu-IFN-gamma R1 cDNA and pEpoRIC cDNA templates with vector primers and the internal primer CTTGTCCTTCTGTTTTTATTTCagagcaagccacatagetggg. The uppercase letters denote sequences corresponding to the Hu-IFN-gamma R1 cDNA, and the lowercase letters represent sequences corresponding to the EpoR cDNA. The Hu-IFN-gamma R2 chain with the Stat1alpha binding site of Hu-IFN-gamma R1 was constructed by restriction enzyme digestion of pgamma R2IC and IFN-gamma R1 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.

Electrophoretic Mobility Shift Assays (EMSA)

EMSAs were performed with the 22-base pair sequence containing a Stat1alpha binding site (5'-GATCGATTTCCCCGAAATCATG-3') corresponding to the GAS element in the promoter region of the human IRF-1 gene (42). 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 [alpha -32P]dATP and the Klenow fragment of DNA polymerase I by the filling-in reaction (43). Whole cell extracts were prepared as follows (44). 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-Stat1alpha 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.

Antibodies

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-Stat1alpha antibody, raised against the carboxyl-terminal region of Stat1alpha , 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).

Immunoprecipitations and Blottings

Cells were stimulated with Hu-IFN-gamma (1,000 units/ml) or Epo (100 units/ml) for 10 min at 37 °C. Immunoprecipitations and blottings were performed as described (16, 40).

Cytofluorographic Analysis

Cytofluorographic analysis of cells for surface expression of class I MHC antigens was performed as described previously (13, 41, 45) with mouse anti-human-HLA B-7 monoclonal antibody (W6/32) and fluorescein isothiocyanate-conjugated goat anti-mouse IgG.


RESULTS

Construction of Chimeric Receptors

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-gamma R subunits. In the other set of chimeras, the transmembrane and intracellular domain of EpoR was fused to the extracellular domain of IFN-gamma R1 and IFN-gamma R2.


Fig. 1. Schematic representation of native and chimeric receptors. EpoR, gamma R1, and gamma R2 represent the wild-type human erythropoietin receptor, Hu-IFN-gamma R1, and Hu-IFN-gamma R2, respectively. The various chimeric constructs were made by joining the extracellular domain (top) with the transmembrane and cytoplasmic domains (bottom) of the receptors indicated. The EpoR(p91) and EpoR/gamma R2(p91) chimeras contain the 5-amino acid Stat1alpha recruitment site of Hu-IFN-gamma R1 (25, 45, 50) at the 3' end of the intracellular domain of EpoR and IFN-gamma R2, respectively.
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Class I MHC Antigen Induction

To investigate the role of the intracellular domain of IFN-gamma R2 in the signal transduction complex of IFN-gamma , we constructed a chimeric receptor chain consisting of the extracellular domain of IFN-gamma R2 and the intracellular domain of EpoR. This chimeric construct, gamma R2/EpoR, and the native IFN-gamma R2 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-gamma was assayed by measurement of enhanced MHC class I antigen expression in the transfected cells and by activation of Stat1alpha . CHO-B7 cells transfected with IFN-gamma R2 or gamma R2/EpoR cDNA showed no response to Hu-IFN-gamma as they lack the ligand-binding receptor subunit, Hu-IFN-gamma R1 (data not shown). Parental CHO-16-9 cells, which contain human chromosome 6q and express the Hu-IFN-gamma R1 subunit, showed no induction of MHC class I antigens in response to Hu-IFN-gamma (Fig. 2, panel A) but when stably transfected with expression vectors encoding Hu-IFN-gamma R2 cDNA or gamma R2/EpoR chimera, exhibited enhanced cell surface expression of class I MHC antigens in response to Hu-IFN-gamma (Fig. 2, panels B and C). To assess how effectively the intracellular domain of EpoR could substitute for the intracellular domain of the IFN-gamma R2 subunit, we measured the induction of MHC class I antigens as a function of IFN-gamma concentration. As depicted in Fig. 3, there was a slightly lower induction of MHC class I antigens in the cells containing the chimeric gamma R2/EpoR than in the cells containing the native Hu-IFN-gamma R2 chain at each concentration of Hu-IFN-gamma used. Nevertheless, the fact that the EpoR intracellular domain can be substituted for the Hu-IFN-gamma R2 intracellular domain shows that another sequence that can recruit Jak2 into the signal transduction complex can substitute for the intracellular domain of Hu-IFN-gamma R2.


Fig. 2. Induction of class I MHC surface antigens. Induction of class I MHC surface antigens by Hu-IFN-gamma or Epo, as indicated, of the parental 16-9 cells expressing only Hu-IFN-gamma R1 (panel A) and of 16-9 cells expressing Hu-IFN-gamma R1 along with various transfected receptor chains: IFN-gamma R2 (panel B), gamma R2/EpoR (panel C), EpoR (panel D), EpoR/gamma R1 (panel E), both EpoR/gamma R1 and EpoR/gamma R2 (panel F), gamma R1/EpoR(p91) (panel G), EpoR(p91) (panel H), and EpoR/gamma R2(p91) (panel I). Cells were treated with Hu-IFN-gamma at 1,000 units/ml, or Epo at 100 units/ml for 72 h as described (32). Class I MHC antigens were detected by flow cytometry after treatment of cells with mouse anti-human-HLA-B7 monoclonal antibody (W6/32), followed by treatment with fluorescein isothiocyanate-conjugated goat anti-mouse IgG. The unshaded regions represent untreated cells, and shaded regions represent cells treated with Hu-IFN-gamma (panels A-C and G) or Epo (panels D-F, H, and I).
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Fig. 3. Induction of expression of class I MHC surface antigens as a function of interferon concentration. Cells were treated with Hu-IFN-gamma 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-gamma .
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Various chimeric receptors between the EpoR and Hu-IFN-gamma R1 and Hu-IFN-gamma R2 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/gamma R1, EpoR/gamma R2, the combination of EpoR/gamma R1 and EpoR/gamma R2, and gamma R1/EpoR(p91). In response to Epo, the EpoR transfectants showed no response (Fig. 2, panel D). The EpoR/gamma R1 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-gamma R1 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/gamma R1 and EpoR/gamma R2 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-gamma R1) respond to Epo with enhanced expression of class I MHC antigens, while the gamma R1/EpoR(p91) transfectants were unresponsive (Fig. 2, panels H and G, respectively). Furthermore, the gamma R1/gamma R2(p91) receptor chain is unable to transduce a signal upon binding ligand,2 whereas the cells expressing the EpoR/gamma R2(p91) chimeric receptor exhibited enhanced class I MHC antigen expression in response to activation by Epo (Fig. 2, panel I).


Fig. 4. Induction of class I MHC antigens as a function of Epo concentration. The induction of expression of class I MHC antigens as a function of Epo concentration was assessed by treating cells with varying concentrations of Epo at 0, 0.1, 1, 10, and 100 units/ml for 72 h. 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 Epo.
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Activation of Stat Proteins

We analyzed Stat activation in cells expressing wild-type and chimeric receptors in response to IFN-gamma and Epo. As shown in Fig. 5, IFN-gamma stimulation resulted in Stat1alpha activation in transfected 16-9 cells expressing native IFN-gamma R2 or gamma R2/EpoR chains. Similarly, Epo caused activation of Stat1alpha in transfected cell lines expressing EpoR/gamma R1 and both EpoR/gamma R1 and EpoR/gamma R2 receptor chains (Fig. 6). Consistent with the small enhancement in surface expression of class I MHC antigens in cells expressing EpoR/gamma R1 in response to Epo, Stat1alpha activation was also lower in these cells compared to cells expressing both EpoR/gamma R1 and EpoR/gamma R2 chains. Activation of p91 was also observed in cells expressing the EpoR(p91) and EpoR/gamma R2(p91) chains (Fig. 6). Furthermore, cells expressing those chimeric receptors containing the EpoR intracellular domain, except gamma R1/EpoR(p91) and gamma R1/EpoR, exhibited activation of Stat5 in addition to Stat1alpha (Fig. 5; data with gamma R1/EpoR were negative similar to results with gamma R1/EpoR(p91)).3 Stat5 is phosphorylated on tyrosine in response to Epo (19, 20). Both Stat1alpha and Stat5 are supershifted by the addition of anti-Stat1alpha and anti-Stat5 antibodies, respectively. Addition of 100-fold molar excess of unlabeled GAS oligonucleotide eliminates both Stat1alpha and Stat5 activated complexes.


Fig. 5. Electrophoretic mobility shift assays of cells expressing chimeric receptors. Clones of transfected 16-9 cells stably expressing native IFN-gamma R2, chimeric IFN-gamma R2/EpoR, or chimeric IFN-gamma R1/EpoR(p91) receptor subunits were induced with 1,000 units/ml IFN-gamma . Whole cell extracts were prepared, incubated with 32P-labeled GAS probe, and complexes resolved by separation on 5% polyacrylamide gels (16, 41) and detected by autoradiography. Competition experiments contained a 100-fold molar excess of unlabeled GAS oligonucleotide. Supershift assays were performed by the addition of 0.1 µg of antibody specific to Stat1alpha (anti-Stat 1alpha ) or Stat5 (anti-Stat 5). Arrow A marks the position of the Stat1alpha complex, and arrow B marks the position of the Stat5 complex activated only in those cells expressing the intracellular domain of EpoR. Arrows C and D designate the positions of the Stat1alpha and Stat5 complexes supershifted by their respective antibodies.
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Fig. 6. Electrophoretic mobility shift assays of cells expressing chimeric receptors. Clones of transfected 16-9 cells stably expressing EpoR/gamma R1, EpoR(p91), or EpoR/gamma R2(p91) chimeric receptor subunits, or both EpoR/gamma R1 and EpoR/gamma R2 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 Stat1alpha in cells expressing EpoR/gamma R1, EpoR(p91), and EpoR/gamma R2(p91), as well as in those cells expressing both EpoR/gamma R1 and EpoR/gamma R2. Addition of anti-Stat1alpha antibody to the reaction mixture caused the Stat1alpha complex to be shifted.
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Activation of Jak Kinases

IFN-gamma activates Jak1 and Jak2 kinases (46), whereas Epo activates Jak2 (36) 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-gamma treatment in 16-9 cells expressing parental IFN-gamma R2 or chimeric gamma R2/EpoR receptors. Induction with Epo phosphorylated both Jak1 and Jak2 kinase in the cell line expressing both EpoR/gamma R1 and EpoR/gamma R2 chains. In the cell line expressing only the chimeric EpoR/gamma R1 receptor, only Jak1 kinase was phosphorylated in response to Epo. The cell line transfected with the gamma R1/EpoR chimeric receptor did not exhibit phosphorylation of either Jak1 or Jak2 kinase upon IFN-gamma treatment.


Fig. 7. Phosphorylation of Jak1 and Jak2 kinases. Untreated cells or cells treated with Hu-IFN-gamma or Epo were lysed and immunoprecipitated (I.P.) with monoclonal anti-phosphotyrosine antibodies (anti-P-Tyr panels), polyclonal anti-Jak1 antibodies (anti-Jak1 panels) or anti-Jak2 antibodies (anti-Jak2 panels) as described under "Materials and Methods." The cell lines are indicated on the figure and are defined in the legend to Fig. 1. Immunoprecipitates were resolved on SDS-PAGE, transferred to PVDF membranes and the blots probed with anti-Jak1 or anti-Jak2 antibodies as noted under the respective horizontal panels.
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DISCUSSION

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. 47). In the case of IFN-gamma , the oligomerization involving IFN-gamma R1 and IFN-gamma R2 initiates the signal transduction events: activation of Jak1 and Jak2, phosphorylation of IFN-gamma R1 on Tyr-457 (16, 25), followed by phosphorylation and activation of Stat1alpha (27). 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-gamma R2 subunit serves to bring Jak2 kinase into the signal transduction complex (16). This is a crucial event since deletion of the membrane-proximal region of the intracellular domain of the IFN-gamma R2 chain, which encompasses the Jak2 association site, completely abrogates its ability to transduce signals in response to IFN-gamma (16), and cells lacking Jak2 do not respond to IFN-gamma (46). This is further supported by the observation that the IFN-gamma R2/EpoR chimeric receptor, which recruits Jak2, is almost as effective as the native IFN-gamma R2 chain in supporting signal transduction in response to IFN-gamma (Figs. 2, 5, and 7). The IFN-gamma R2 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-gamma receptor complex to support signal transduction (40).

The requirement for two distinct Jak kinases in the IFN-gamma signaling pathway was demonstrated with the use of kinase-deficient cell lines (46, 48). Based on our results with the chimeric erythropoietin-interferon gamma  receptors, we propose that this reflects two features characteristic of the IFN-gamma 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-gamma R1, and activation of Stat1alpha ). The presence of Jak2 facilitates effective phosphorylation of the above steps. In contrast to the growth hormone receptor (49) and the EpoR (37) complexes, when one IFN-gamma homodimer binds two IFN-gamma R1 molecules, the two receptor subunits do not interact with one another and are separated by 27 Å (50) at their closest point. Therefore, although the IFN-gamma R1 chain possesses both a Jak1 association site and a Stat1alpha 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-gamma ·IFN-gamma R1 complex suggests that each monomer of the IFN-gamma homodimer binds one IFN-gamma R1 and one IFN-gamma R2 subunit (50). Thus the signal-transducing complex of IFN-gamma consists of the IFN-gamma homodimer bound to two IFN-gamma R1 and two IFN-gamma R2 chains, which recruit Jak1 and Jak2, respectively (16, 40); and Jak2 phosphorylates Jak1, following which either kinase phosphorylates Tyr-457 of the IFN-gamma R1 chain (Fig. 8B; see also Refs. 25, 45, and 51). The phosphorylated segment of each IFN-gamma R1 chain recruits Stat1alpha , which is then phosphorylated by Jak1 or Jak2, then released to dimerize and form the active Stat1alpha . In contrast, with the EpoR/gamma R1 dimer, two Jak1 kinases are brought sufficiently close together to activate one another (Fig. 8C), albeit inefficiently. In the case of the EpoR/gamma R1·EpoR/gamma R2 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/gamma R2(p91) chimeric receptor (Fig. 2I) exhibit a stronger biological response than cells expressing both EpoR/gamma R1 and EpoR/gamma R2 (Fig. 2F) or even the native IFN-gamma receptor (gamma R2, Fig. 2B), which supports a modulating role for Jak1 in the IFN-gamma R complex. In cells expressing both EpoR/gamma R1 and EpoR/gamma R2 chains, binding of Epo can induce the formation of three types of receptor dimers: EpoR/gamma R1 homodimers, EpoR/gamma R2 homodimers, and EpoR/gamma R1·EpoR/gamma R2 heterodimers. The EpoR/gamma R1 homodimer is barely active (Figs. 2E and 4), and the EpoR/gamma R2 homodimer is inactive. The major functional receptor complex therefore must be the EpoR/gamma R1·EpoR/gamma R2 heterodimer (Fig. 8D).


Fig. 8. Schematic representation of receptor complexes. A represents the IFN-gamma R1 homodimer bound to IFN-gamma . The cytoplasmic domains of the two chains are too far apart to permit transactivation of the two Jak1 kinases. B represents the active heteromeric IFN-gamma receptor complex with two IFN-gamma R1 and two IFN-gamma R2 subunits per complex. The IFN-gamma homodimer binds to two IFN-gamma R1 chains, followed by its interaction with two IFN-gamma R2 chains. The associated Jak2 and Jak1 kinases activate one another by transphosphorylation, with subsequent phosphorylation and dimerization of Stat1alpha . C depicts the EpoR/gamma R1 homodimer, which, unlike the IFN-gamma R1 homodimer, permits transactivation of the two Jak1 molecules. D illustrates the structure of the heterodimer of EpoR/gamma R1 and EpoR/gamma R2, which is the putative active receptor complex.
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That Jak1 is relatively ineffective in transphosphorylation is supported by the observation that cells expressing the EpoR/gamma R1 chimera show a smaller response than the cells expressing both EpoR/gamma R1 and EpoR/gamma R2 or the EpoR/gamma R2(p91) chimeric receptor chains. Thus, even though homodimerization of the EpoR/gamma R1 receptor by Epo brings the cytoplasmic domains of the two gamma R1 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. (52), who reported that a Jak1 molecule with an inactive kinase domain can replace the normal Jak1 in signal transduction by IFN-gamma 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-gamma (40). 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. (53) suggested that the SH2 recognition domain of Stat1alpha maintains some of the specificity. It remains to be established, however, how Stat1alpha 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 (54).4

We propose that the multichain cytokine class II receptors have two major chains exemplified by the IFN-gamma receptor complex (Fig. 8B). The ligand binding chain (IFN-gamma R1) and the accessory chain (IFN-gamma R2; helper receptor) serve as a foundation for the functional IFN-gamma R complex (16, 40). The geometry of the IFN-gamma R1 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-gamma result from the lack of expression of the IFN-gamma R2 chain in the TH1 subset (55-57) and allows exquisite fine tuning of sensitivity to IFN-gamma . 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.


FOOTNOTES

*   This study was supported in part by United States Public Health Service Grants RO1 CA46465, RO1 CA52363, and R01 AI36450 from the National Institutes of Health and Grant VM-76731 from the American Cancer Society (all to S. P.), and by a postdoctoral fellowship from The Governor's Council on the Prevention of Mental Retardation and Developmental Disabilities, New Jersey (to G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1    The abbreviations used are: IFN-gamma , interferon gamma ; IFN-gamma R, interferon gamma  receptor; Epo, erythropoietin; EpoR, erythropoietin receptor; EMSA, electrophoretic mobility shift assay; MHC, major histocompatibility complex; PCR, polymerase chain reaction; CHO, Chinese hamster ovary; EC, extracellular domain; IC, intracellular domain; GAS, gamma activation sequence.
2    S. Kotenko, unpublished observation.
3    G. Muthukumaran, S. Kotenko, R. Donnelly, J. N. Ihle, and S. Pestka, unpublished results.
4    C. Schindler, personal communication.

Acknowledgments

We thank Brian Pollack for providing the pgamma R2EC 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-Stat1alpha antibodies. We thank Dr. Jerry Langer for critical review of the manuscript and Eleanor Kells for its preparation.


REFERENCES

  1. Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934-6938 [Abstract]
  2. Thoreau, E., Petridou, B., Kelly, P. A., Djiane, J., and Mornon, J. P. (1991) FEBS Lett. 282, 26-31 [CrossRef][Medline] [Order article via Infotrieve]
  3. Rashidbaigi, A., Langer, J. A., Jung, V., Jones, C., Morse, G. H., Tischfield, J. A., Trill, J. J., Kung, H. F., and Pestka, S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 384-388 [Abstract]
  4. Aguet, M., Dembic, Z., and Merlin, G. (1988) Cell 55, 273-280 [Medline] [Order article via Infotrieve]
  5. Kumar, C. S., Muthukumaran, G., Frost, L. J., Noe, M., Ahn, Y. H., Mariano, T. M., and Pestka, S. (1989) J. Biol. Chem. 264, 17939-17946 [Abstract/Free Full Text]
  6. Hemmi, S., Peghiai, P., Metzter, M., Merlin, G., Dembic, Z., and Aguet, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9901-9905 [Abstract]
  7. Gray, P. W., Leong, S., Fennie, E. H., Farrar, M. A., Pingel, J. T., Fernandez-Luna, J., and Schreiber, R. D. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8497-8501 [Abstract]
  8. Munro, S., and Maniatis, T. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9248-9252 [Abstract]
  9. Cofano, F., Moore, S. K., Tanaka, S., Yuhki, N., Landolfo, S., and Appella, E. (1990) J. Biol. Chem. 265, 4064-4071 [Abstract/Free Full Text]
  10. Jung, V., Rashidbaigi, A., Jones, C., Tischfield, J. A., Shows, T. B., and Pestka, S. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4151-4155 [Abstract]
  11. Jung, V., Jones, C., Rashidbaigi, A., Geyer, D. D., Morse, H. G., Wright, R. B., and Pestka, S. (1988) Somat. Cell Mol. Genet. 14, 583-592 [Medline] [Order article via Infotrieve]
  12. Jung, V., Jones, C., Kumar, C. S., Stefanos, S., O'Connell, S., and Pestka, S. (1990) J. Biol. Chem. 265, 1827-1830 [Abstract/Free Full Text]
  13. Soh, J., Donnelly, R. J., Mariano, T. M., Cook, J. R., Schwartz, B., and Pestka, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8737-8741 [Abstract/Free Full Text]
  14. Soh, J., Donnelly, R. J., Kotenko, S., Mariano, T. M., Cook, J. R., Wang, N., Emanuel, S., Schwartz, B., Miki, T., and Pestka, S. (1994) Cell 76, 793-802 [Medline] [Order article via Infotrieve]
  15. Hemmi, S., Böhni, R., Stark, G., Di Marco, F., and Aguet, M. (1994) Cell 76, 803-810 [Medline] [Order article via Infotrieve]
  16. Kotenko, 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., and Pestka, S. (1995) J. Biol. Chem. 270, 20915-20921 [Abstract/Free Full Text]
  17. Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Science 264, 1415-1421 [Medline] [Order article via Infotrieve]
  18. Ihle, J. N., Witthuhn, B. A., Quelle, F. W., Yamamoto, K., Thierfelder, W. E., Kreider, B., and Silvennoinen, O. (1994) Trends Biochem. Sci. 19, 222-227 [CrossRef][Medline] [Order article via Infotrieve]
  19. Ihle, J. N., Witthuhn, B. A., Quelle, F. W., Yamamoto, K., and Silvennoinen, O. (1995) Annu. Rev. Immunol. 13, 369-398 [CrossRef][Medline] [Order article via Infotrieve]
  20. Ihle, J. N., and Kerr, I. M. (1995) Trends Genet. 11, 69-74 [CrossRef][Medline] [Order article via Infotrieve]
  21. Schindler, C., and Darnell, J. E., Jr. (1995) Annu. Rev. Biochem. 64, 621-651 [CrossRef][Medline] [Order article via Infotrieve]
  22. Taniguchi, T. (1995) Science 268, 251-255 [Medline] [Order article via Infotrieve]
  23. Igarashi, K., Garotta, G., Ozmen, L., Ziemiecki, A., Wilks, A. F., Harpur, A. G., Larner, A. C., and Finbloom, D. S. (1994) J. Biol. Chem. 269, 14333-14336 [Abstract/Free Full Text]
  24. Sakatsume, M., Igarashi, K., Winestock, K. D., Garotta, G., Larner, A. C., and Finbloom, D. S. (1995) J. Biol. Chem. 270, 17528-17534 [Abstract/Free Full Text]
  25. Greenlund, A. C., Farrar, M. A., Viriano, B. L., and Schreiber, R. D. (1994) EMBO J. 13, 1591-1600 [Abstract]
  26. Schindler, C., Fu, X.-Y., Improta, T., Aebersold, R., and Darnell, J. E. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7836-7839 [Abstract]
  27. Shuai, K., Stark, G. R., Kerr, I. M., and Darnell, J. E., Jr. (1993) Science 261, 1744-1746 [Medline] [Order article via Infotrieve]
  28. Ealick, S. E., Cook, W. J., Vijaykumar, S., Carson, M., Nagabhushan, T., Trotta, P. P., and Bugg, C. E. (1991) Science 252, 698-700 [Medline] [Order article via Infotrieve]
  29. Fountoulakis, M., Zulauf, M., Lusting, A., and Garotta, G. (1992) Eur. J. Biochem. 208, 781-787 [Abstract]
  30. Greenlund, A. C., Schreiber, R. D., Goeddel, D., and Pennica, D. (1993) J. Biol. Chem. 268, 18103-18110 [Abstract/Free Full Text]
  31. Gibbs, V. C., Williams, S. R., Gray, P. W., Schreiber, R. D., Pennica, D., Rice, G., and Goeddel, D. V. (1991) Mol. Cell. Biol. 11, 5860-5866 [Medline] [Order article via Infotrieve]
  32. Hibino, Y., Kumar, C. S., Mariano, T. M., Lai, D., and Pestka, S. (1992) J. Biol. Chem. 267, 3741-3749 [Abstract/Free Full Text]
  33. Hemmi, S., Merlin, G., and Aguet, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2737-2741 [Abstract]
  34. Marsters, S. A., Pennica, D., Bach, E., Schreiber, R. D., and Ashkenazi, A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5401-5405 [Abstract]
  35. Wells, J. A. (1994) Curr. Opin. Cell Biol. 6, 163-173 [Medline] [Order article via Infotrieve]
  36. Witthuhn, B. A., Quelle, F. W., Silvennoinen, O., Yi, T., Tang, B., Miura, O., and Ihle, J. N. (1993) Cell 74, 227-236 [Medline] [Order article via Infotrieve]
  37. Watowich, S. S., Yoshimura, A., Longmore, G. D., Hilton, D. J., Yoshimura, Y., and Lodish, H. F. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2140-2144 [Abstract]
  38. Yoshimura, A., Longmore, G. D., and Lodish, H. F. (1990) Nature 348, 647-649 [CrossRef][Medline] [Order article via Infotrieve]
  39. Jones, S. S., D'Andrea, A. D., Haines, L. L., and Wong, G. G. (1990) Blood 76, 31-35 [Abstract]
  40. Kotenko, S. V., Izotova, L. S., Pollack, B. P., Muthukumaran, G., Paukku, K., Silvennoinen, O., Ihle, J. N., and Pestka, S. (1996) J. Biol. Chem. 271, 17174-17182 [Abstract/Free Full Text]
  41. Muthukumaran, G., Donnelly, R. J., Ebensperger, C., Mariano, T. M., Poast, J., Baron, S., Dembic, Z., and Pestka, S. (1996) J. Interferon and Cytokine Res. 16, 1039-1045 [Medline] [Order article via Infotrieve]
  42. Yuan, J., Wegenka, U. A., Lütticken, C., Buschmann, J., Decker, T., Schindler, C., Heinrich, P. C., and Horn, F. (1994) Mol. Cell. Biol. 14, 1657-1668 [Abstract]
  43. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  44. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489 [Abstract]
  45. Cook, J. R., Jung, V., Schwartz, B., Wang, P., and Pestka, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11317-11321 [Abstract]
  46. Müller, 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., and Kerr, I. M. (1993) Nature 366, 129-135 [CrossRef][Medline] [Order article via Infotrieve]
  47. Stahl, N., and Yancopoulous, G. D. (1993) Cell 74, 587-590 [Medline] [Order article via Infotrieve]
  48. Watling, D., Guschin, D., Müller, M., Silvennoinen, O., Witthuhn, B. A., Quelle, F. W., Rogers, N. C., Schindler, C., Stark, G. R., Ihle, J. N., and Kerr, I. M. (1993) Nature 366, 166-170 [CrossRef][Medline] [Order article via Infotrieve]
  49. De Vos, A. M., Ultsch, M., and Kossiakoff, A. A. (1992) Science 255, 306-312 [Medline] [Order article via Infotrieve]
  50. Walter, M. R., Windsor, W. T., Nagabhushan, T. L., Lundell, D. J., Lunn, C. A., Zanodry, P. J., and Narula, S. K. (1995) Nature 376, 230-235 [CrossRef][Medline] [Order article via Infotrieve]
  51. Farrar, M. A., Campbell, J. D., and Schreiber, R. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11706-11710 [Abstract]
  52. Briscoe, J., Rogers, N., Witthuhn, B., Watling, D., Harpur, A., Wilks, A., Horn, F., Heinrich, P., Stark, G. R., Ihle, J., and Kerr, I. M. (1995) J. Interferon and Cytokine Res. 15, Suppl. 1, S56
  53. Heim, M. H., Kerr, I. M., Stark, G. R., and Darnell, J. E. J. (1995) Science 267, 1347-1349 [Medline] [Order article via Infotrieve]
  54. Pollack, B. P., Kotenko, S. V., Izotova, L., Krause, C. D., Mehnert, J. M., and Pestka, S. (1996) Eur. Cytokine Network 7, 478
  55. Bach, E. A., Szabo, S. J., Dighe, A. S., Ashkenazi, A., Aguet, M., Murphy, K. M., and Schreiber, R. D. (1995) Science 269, 1215-1217
  56. Pernis, A., Gupta, S., Gollob, K. J., Garfein, E., Coffman, R. L., Schindler, C., and Rothman, P. (1995) Science 269, 245-247 [Medline] [Order article via Infotrieve]
  57. Skrenta, H., Peritt, D., Cook, J. R., Garotta, G., Trinchieri, G., and Pestka, S. (1996) Eur. Cytokine Network 7, 622

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