Other kinases can substitute for Jak2 in signal transduction by interferon-gamma.

Each cytokine which utilizes the Jak-Stat signal transduction pathway activates a distinct combination of members of the Jak and Stat families. Thus, either the Jaks, the Stats, or both could contribute to the specificity of ligand action. With the use of chimeric receptors involving the interferon γ receptor (IFN-γR) complex as a model system, we demonstrate that Jak2 activation is not an absolute requirement for IFN-γ signaling. Other members of the Jak family can functionally substitute for Jak2. IFN-γ can signal through the activation of Jak family members other than Jak2 as measured by Statlα homodimerization and major histocompatibility complex class I antigen expression. This indicates that Jaks are interchangeable and indiscriminative in the Jak-Stat signal transduction pathway. The necessity for the activation of one particular kinase during signaling can be overcome by recruiting another kinase to the receptor complex. The results may suggest that the Jaks do not contribute to the specificity of signal transduction in the Jak-Stat pathway to the same degree as Stats.

The Jak-Stat signal transduction pathway was first discovered for interferon ␣ (IFN-␣) 1 and interferon ␥ (IFN-␥) by the complementation of mutant cell lines defective in response to IFN-␥ and/or IFN-␣ (Velazquez et al., 1992;Watling et al., 1993;Mü ller et al., 1993aMü ller et al., , 1993bDarnell et al., 1994;Leung et al., 1995). It has subsequently been shown that the same general pathway is activated by most cytokines and some growth factors (for review, see Ihle and Kerr (1995) and Taniguchi (1995)). This pathway is activated predominantly through receptors which do not possess intrinsic intracellular kinase domains and belong to the class I or class II cytokine receptor superfamily. The lack of inherent catalytic activity in these receptors is overcome through the use of receptor-associated kinases of the Janus kinase (Jak) family. Upon ligand binding, the receptor chains oligomerize allowing the associated kinases to interact and likely cross-activate each other by tyrosine phosphorylation. Subsequently, the activated Jaks directly phosphorylate the intracellular domains of the receptors on specific tyrosine residues. This phosphorylation allows the selective recruitment of SH2-domain containing proteins, particularly Stats (signal transducers and activators of transcription), through a specific interaction between the Stat SH2 domains and the phosphotyrosines within the Stat recruitment sites of the intracellular domains of the receptor chains. These receptor-associated Stats are then rapidly phosphorylated, likely by the activated Jaks . The phosphorylation of the Stats is followed by Stat dimerization, translocation to the nucleus, and activation of cytokine inducible genes.
The Jak kinases are characterized by seven conserved domains: two PTK-related domains and five domains with unknown functions . The main difference between Jaks and other protein tyrosine kinases (PTK) is that along with a kinase domain, shown to be active (Wilks et al., 1991), they also contain a PTK-like domain with substitutions of several residues essential for kinase activity. Thus, the second domain is expected to be inactive as a PTK and probably has some other function. Another feature of this family is the lack of any detectable SH2 or SH3 domains. The functions of the other five regions of homology are also unknown.
Stats represent proteins containing SH2, SH3, and DNAbinding domains (for reviews, see Darnell et al. (1994) and Fu (1995)). The highly selective and specific interaction between Stat SH2 domains and the phosphotyrosine containing Stat recruitment sites on the intracellular domains of the cytokine receptors determines which Stats are to be recruited to a particular receptor complex (Heim et al., 1995;Stahl et al., 1995).
The intracellular domain of each cytokine receptor specifically associates with one or more distinct Jaks. While some Jaks and Stats participate in several cytokine signaling path-* This study was supported by United States Public Health Services Grants RO1-CA46465 and RO1-CA52363 from the National Cancer Institute, National Institute of Allergy and Infectious Diseases Grant RO1 AI36450, American Cancer Society Grant VM-135 (to S. P.), Predoctoral Fellowship 94-2006-CCR00 from the New Jersey Commission on Cancer Research (to B. P. P.), a postdoctoral fellowship from The Governor's Council on the Prevention of Mental Retardation and Developmental Disabilities, New Jersey (to L. S. I.), and a grant by the Academy of Finland and Cancer Society of Finland (to O. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This 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: INF, interferon; Stats, signal transducers and activators of transcription; PTK, protein tyrosine kinase; IL, interleukin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; MHC, major histocompatibility complex. ways, others are more restricted. In the case of Jaks, Jak2, and, especially Jak1, associate with receptors participating in different apparently unrelated cytokine-receptor systems (for reviews, see Ziemiecki et al. (1994), Ihle et al. (1994, Taniguchi (1995), and Ihle and Kerr (1995)). Jak3 appears to be restricted to the ligand receptor systems through its association with the IL-2R␥ C chain Russell et al., 1994;Miyazaki et al., 1994;Tanaka et al., 1994). Tyk2 was shown to be activated during IFN-␣ signaling (Velazquez, et al., 1992;Barbieri et al., 1994) and also during ciliary neurotrophic factor-related cytokine signaling, albeit only in certain cell types (Stahl et al., 1994;. Recently, the activation of Tyk2 by IL-10 and IL-12 was shown Bacon et al., 1995;Ho et al., 1995). Thus, the Jaks may contribute to the specificity of signal transduction at some level.
In the case of IFN-␥ and IFN-␣ signal transduction, the activation of Jak1 and Stat1 occurs in both pathways, however, Tyk2 and Stat2 are activated only during IFN-␣ signaling; and Jak2 only in the IFN-␥ signal transduction pathway. Thus, it was proposed that there may be substrate specificity of Jaks for Stats .
To assess whether the specificity of signal transduction resides in the Jaks, we prepared a variety of chimeric receptors based on the human IFN-␥ receptor complex. The extracellular domain of the second human IFN-␥ receptor chain, designated Hu-IFN-␥R2 or AF-1 (Soh et al., 1994a), was fused to the transmembrane and intracellular domains of various receptors. We then determined which chimeric receptors could support signal transduction. The result of these studies are reported here.

EXPERIMENTAL PROCEDURES
Reagents, Restriction Endonucleases, and Other Enzymes-Taq polymerase and all restriction endonucleases were from Boehringer Mannheim Biochemicals or New England Biolabs; Sequenase 2.0 and T4 DNA ligase were from U. S. Biochemical Corp. The [␣-32 P]dATP and [␥-32 P]ATP were from DuPont NEN. The cross-linker bis(sulfosuccinimidyl)suberate was from Pierce Chemical Co. All other chemical reagents were analytical grade and purchased from U. S. Biochemical Corp.
Plasmid Construction-The vector p␥R2 expressing the Hu-IFN-␥R2 chain under control of cytomegalovirus promoter was constructed as described previously (Soh et al., 1994a;Kotenko et al., 1995). The vector p␥R1 expressing the Hu-IFN-␥R1 chain was constructed as follows. The pHu-IFN-␥R8 plasmid (Kumar et al., 1989) was digested with HindIII restriction endonuclease, incubated with the large fragment of DNA polymerase I and dNTPs to fill in the ends, and then digested with BamHI restriction endonuclease. The fragment containing the Hu-IFN-␥R1 cDNA was ligated into EcoRV and BamHI sites of the pcDNA3 vector (Invitrogen).
The Hu-IFN-␣R1 cDNA was recloned from HuIFNAR1/pcDNAI (Lim, 1995) into pcDNA3 with EcoRI and XbaI restriction endonucleases. The expression vector was designated p␣R1. The PCR products for Hu-IFN-␣R2 cDNA were obtained by a nested PCR procedure with a pCEV15 phage DNA mixture isolated from about 5 ϫ 10 6 phage clones from the human M426 cell library (Miki et al., 1989) as template. The primers for the first round of PCR were: 5Ј-GCCGCAAGGC-GAGAGCTGC-3Ј specific for the 5Ј end of Hu-IFN-␣R2 cDNA (Novick et al., 1994); and pCEV15 vector primer 5Ј-AGATCTAAGCTTGGC-CGAGG-3Ј. For the second round we used the same 5Ј primer and primer 5Ј-GCGGAATTCTTAATCACTGGGGCACAG-3Ј specific for the 3Ј end (bases 1204 -1222) of Hu-IFN-␣R2 cDNA (boldface) containing an EcoRI site within the primer. The PCR product was digested with EcoRI restriction endonuclease and ligated into EcoRI and blunt ended BamHI sites of the pcDNA3 vector. The expression vector was designated p␣R2.
To isolate the Hu-CRFB4 cDNA an oligonucleotide 5Ј-GTCCATG-GCGTGGAGCCTTGGGAG-3Ј homologous to the 5Ј end of Hu-CRFB4 cDNA (Lutfalla et al., 1993) was labeled with [␣-32 P]dATP with terminal deoxynucleotide transferase to a specific activity of 5 ϫ 10 6 cpm/g. It was used to screen about 10 6 phages from the human M426 cell library (Miki et al., 1989). Two positive clones were purified and plas-mids were rescued from these two phages. The CRFB4 cDNA was released from one of the rescued plasmids designated pCEV15-CRFB4 -1 by digestion with ThaI and SalI restriction endonucleases and cloned into EcoRV and XhoI sites of the pcDNAIneo vector (Invitrogen). The expression vector for the Hu-CRFB4 chain was designated pCRF.
The vector expressing the chimeric receptor Hu-IFN-␥R2/Hu-IFN-␥R1 (␥R2/␥R1) was constructed as followed. The asymmetric PCR reaction was performed with a specific primer 5Ј-GATGCCTCCAC-TGAGCTTCAGCAACTTTGGATTCCAGTTGTTGC-3Ј and 100-fold excess of T7 primer with plasmids pHu-IFN-␥R8 (the Hu-IFN-␥R1 cDNA) and p␥R2 as templates in the same reaction mixture. The second round of the PCR was performed with the set of primers 5Ј-GACCCTCTTTC-CCAGCTGC-3Ј and 5Ј-GCCACACATCCTCTTTACGC-3Ј with 1 l of PCR reaction mixture from the first round of PCR as template. The final ␥R2/␥R1 PCR product was digested with BstEII restriction endonuclease and cloned into BstEII and blunt-ended XbaI sites of the p␥R2 plasmid. The expression vector was designated p␥R2/␥R1.
To introduce an NheI site in the beginning of the transmembrane domain of the Hu-IFN-␥R2 cDNA clone, the PCR reaction was performed with two primers 5Ј-GCCTTTTTTAGTTATTATGTC-3Ј and 5Ј-ATCGCTAGCCATTGCTGAAGCTCAGTGGAGG-3Ј and plasmid p␥R2 as a template according to a standard protocol (Sambrook et al., 1989). The PCR product was digested with BstXI restriction endonuclease and ligated into BstXI and EcoRV sites of the p␥R2. The plasmid was designated p␥R2NheI.
To create chimera Hu-IFN-␥R2/IL-2R␥ C , the ␥ C chain of IL-2R (Takeshita et al., 1992) was obtained by reverse transcriptase-PCR as follows. The first strand cDNA synthesis in reverse transcriptase-PCR was performed with poly(dT) 18 primer with Moloney murine leukemia virus reverse transcriptase with total RNA isolated from peripheral blood leukocytes as template. Two PCR rounds were performed. The primers 5Ј-CGGTTCAGGAACAATCGG-3Ј and 5Ј-CAAGCGCCATGTT-GAAGCC-3Ј were used for the first round. For the second round the PCR product from the first round was diluted 100-fold and used as a template for the second round with primers 5Ј-GTTAGTACCACT-TAGGGC-3Ј and 5Ј-GTGGCTAGCATGGGAAGCCGTGGTTATC-3Ј. The IL-2R␥ C PCR product was digested with NheI restriction endonuclease and ligated into the NheI and blunt ended XbaI sites of plasmid p␥R2NheI. The resultant expression vector was designated p␥R2/␥ C . The nucleotide sequences of the modified regions of all the constructs were verified in their entirety.
The 16-9 cells were stably transfected with the expression vectors (1-3 g of super-coiled plasmid DNA per 10 5 -10 6 cells) with Lipo-fectAMINE TM Reagent (Life Technologies) according to the manufacturer's instructions for stable transfection of adherent cells. For cotransfection we used 1-3 g of plasmid DNA with the neo R gene and a 10-fold excess of plasmid DNA without neo R gene per 10 5 -10 6 cells. All cell lines transfected with plasmids carrying the neo R gene were selected and maintained in complete F-12 medium containing 450 g/ml antibiotic G418. COS-1 cells were transiently transfected with the expression vectors by the DEAE-dextran procedure with dimethyl sulfoxide shock (Seed and Aruffo, 1987;Sussman and Milman, 1984).
Cytofluorographic Analysis-Cytofluorographic analysis of cells for expression of the HLA-B7 surface antigen was performed as described previously (Jung et al., 1988;Cook et al., 1992;Hibino et al., 1992). Hu-IFN-␣A/D, a chimeric human interferon active on hamster cells (Rehberg et al., 1982), was used as a control to demonstrate the integrity of the HLA-B7 gene in various cell lines.
Antibodies-Rabbit anti-Jak1, anti-Jak2, and anti-Jak3 antibodies were developed against synthetic peptides KTLIEKERFYES-RCRPVTPSC, DSQRKLQFYEDKHQLPAPKC, and AKLLPLDKDYYV-VREPG corresponding to the end of the kinase-like domains of murine Jak1 and Jak2, and to a sequence within the kinase domain of murine Jak3, respectively. Rabbit anti-Tyk2 antibody was from Santa Cruz Biotechnology (catalog number SC-169). Rabbit anti-Stat1␣ antibody, raised against the C terminus of Stat1␣, was a gift from James Darnell. Monoclonal anti-phosphotyrosine antibody was from Sigma (catalog number P3300). Rabbit anti-Hu-IFN-␥R2 antibody, prepared with the extracellular domain of Hu-IFN-␥R2 as antigen, was a gift of Gianni Garotta.
Immunoprecipitations, Blottings, and Kinase Assay-Cells were starved overnight in serum-free media and subsequently stimulated with Hu-IFN-␥ (1000 units/ml) for 10 min at 37°C. Preparation of cell lysates, immunoprecipitations, blottings, and in vitro kinase activation assay were performed as described .
Electrophoretic Mobility Shift Assays-Electrophoretic mobility shift assays were performed with a 22-base pair sequence containing a Stat1␣ binding site corresponding to the GAS element in the promoter region of the human IRF-1 gene (5Ј-GATCGATTTCCCCGAAATCATG-3Ј) as described .

Construction of Chimeric Receptors, Expression in COS Cells,
and Cross-linking-To investigate the specific requirements for the intracellular domain of Hu-IFN-␥R2 we created chimeric receptors with the extracellular domain of Hu-IFN-␥R2 attached to the transmembrane and intracellular domains of different human receptors as shown in Fig. 1. In our studies we used the following receptors: two chains of the Hu-IFN-␥ receptor complex, Hu-IFN-␥R1(␥R1) (Aguet et al., 1988), or human IFN-␥R1t 456 (␥R1t 456 ) the truncated ␥R1 with the intracellular domain terminated by premature stop codon after amino acid 456 (Cook et al., 1992), and Hu-IFN-␥R2 (␥R2) (Soh et al., 1994a); two chains of human IFN-␣ receptor complex, Hu-IFN-␣R1 (␣R1) (Uzé et al., 1990) and Hu-IFN-␣R2 (␣R2) (Novick et al., 1994); Hu-CRFB4, a class II cytokine receptor with unknown function (Lutfalla et al., 1993); and Hu-IL-2 receptor ␥ C chain (Takeshita et al., 1992). To confirm that all chimeric receptors can be expressed properly we first determined the ability of expression vectors encoding chimeric receptors to express the proteins. All plasmids were transiently transfected into COS-1 cells and the expression of the receptors were evaluated. The cellular lysates from COS-1 cells transiently transfected with the expression vectors were resolved on SDS-PAGE, transferred to membrane and probed with antibodies to the Hu-IFN-␥R2 extracellular domain as all these chimeric receptors contain the Hu-IFN-␥R2 extracellular domain. In all cases specific bands were detected (Fig. 2). Thus, all vectors encoding the chimeric receptors express these proteins.
It was previously shown that the Hu-IFN-␥R2 chain is a part of the human IFN-␥ receptor ligand binding complex, although by itself does not bind Hu-IFN-␥; nevertheless, the Hu-IFN-␥R2 chain can be detected by cross-linking to Hu-IFN-␥ . Therefore, we used cross-linking to ascertain that the chimeric receptors were expressed on the cell surface and were able to participate in the human IFN-␥ receptor ligand binding complex (Fig. 3). All chimeric receptors were stably expressed in 16-9 cells, hamster cells expressing the Hu-IFN-␥R1 chain (Soh et al., 1993), and antibiotic G418resistant cell populations were used in cross-linking experiments. The cell lines were designated according to the extracellular/intracellular domains (e.g. ␥R2/␥R1) of the chimeric receptors expressed (Fig. 1). It was previously shown that the cross-linking of labeled IFN-␥ to the parental 16-9 cells results in formation of a single cross-linked band on the SDS-PAGE migrating in the region of 120 kDa and corresponding to the Hu-IFN-␥:Hu-IFN-␥R1 (IFN-␥:␥R1) complex . In the ␥R2 cells, in addition to the IFN-␥:␥R1 band the appearance of another cross-linked band migrating in the region of 60 kDa was observed and it was shown that this additional band corresponds to the Hu-IFN-␥:Hu-IFN-␥R2 (IFN-␥: ␥R2) complex Fig. 3). In cross-linking experiments with cells expressing chimeric receptors the IFN-␥:␥R1 cross-linked complex was observed in all cell lines (Fig.  3). Depending on the chimeric receptor expressed in the 16-9 cells, the mobility of the second band corresponding to the Hu-IFN-␥:Hu-IFN-␥R2/X (IFN-␥:␥R2/X) complex (where X represents the transmembrane and intracellular domains of the chimeric receptor expressed) was different for each chimeric receptor (Fig. 3, left panels). However, the IFN-␥:␥R2/X complex was observed for all chimeric receptors, indicating that  (Uzé et al., 1990); ␥R2/␣R2, Hu-IFN-␣R2 (Novick et al., 1994); ␥R2/CRF, CRFB4 (Lutfalla et al., 1993); ␥R2/␥ C , IL-2 receptor ␥ C chain (Takeshita et al., 1992). Although it was reported that the short form of Hu-IFN-␣R2 we used binds Jak1 (Novick et al., 1995), we have placed a "?" under the ␥R1/␣R2 chimera because only the long form of Hu-IFN-␣R2 is functional (Lutfalla et al., 1995;Domanski et al., 1995; also see text).  . Cells were harvested after 3 days and lysed as described under "Experimental Procedures." Lysates were resolved on SDS-PAGE, transferred to polyvinylidine difluoride membranes, and Western blots probed with anti-Hu-IFN-␥R2 antibodies. they all were expressed on the cell surface and formed ternary ligand-receptor complexes (␥R1:IFN-␥:␥R2/X) in all cell lines. Because the IFN-␥:␥R2/␥R1 complex has almost the same mobility as the IFN-␥:␥R1 complex formed from the endogenous Hu-IFN-␥R1, in ␥R2/␥R1 cells we observed a single crosslinked band in the region of 120 kDa (Fig. 3, right panels). However, in the 16-9 cells transfected with the ␥R2/␥R1t 456 (␥R2/␥R1 chimera, containing a truncated ␥R1 intracellular domain, ␥R1t 456 ) we observed the appearance of an additional band migrating faster than the IFN-␥:␥R2/␥R1 complex and representing the IFN-␥:␥R2/␥R1t 456 complex (Fig. 3, right panels). The additional complexes observed in all cell lines migrating slower than the IFN-␥:␥R1 complex were formed by crosslinking the IFN-␥ homodimer to one or two molecules of ␥R1 and one or two molecules of ␥R2 as shown previously (Marsters et al., 1995).
The Recruitment of Different Jaks into the IFN-␥ Receptor Complex-It was reported that different receptors are associated with different Jak family members. Particularly, Hu-IFN-␣R1 associates with Tyk2 Colamonici et al., 1994aColamonici et al., , 1994b, Hu-IFN-␥R2 associates with Jak2 , and IL-2R␥ C associates with Jak3 (Russell et al., 1994;Miyazaki et al., 1994;Tanaka et al., 1994). Jak1 was shown to associate Hu-IFN-␥R1 Sakatsume et al., 1995) and reported to associate with Hu-IFN-␣R2 (Novick et al., 1994). In addition, Hu-IFN-␥R1 probably weakly associates with Jak2 after oligomerization upon ligand binding . We tested the ability of the intracellular domains of different receptors fused to the Hu-IFN-␥R2 extracellular domain to recruit different members of the Jak family into the Hu-IFN-␥ receptor complex and to determine if the recruited Jaks can be activated upon Hu-IFN-␥ treatment. First, we determined the phosphorylation of Jak2 upon Hu-IFN-␥ treatment in the cell lines expressing various chimeric receptors (Fig. 5A). The phosphorylation of Jak2 in response to Hu-IFN-␥ in cells was examined by immunoprecipitation with specific anti-Jak2 antibodies, followed by a Western blot visualized with anti-phosphotyrosine antibodies. We were able to detect the phosphorylation of Jak2 only in ␥R2 cells (Fig. 5A).
Inability to detect Jak2 activation in 16-9 cell lines responsive to Hu-IFN-␥ containing the chimeric receptors ␥R2/␣R1 and ␥R2/CRF suggested involvement of other Jaks in these cells. Since the Hu-IFN-␣R1 associates with Tyk2 Colamonici et al., 1994aColamonici et al., , 1994b, we evaluated Tyk2 activation in cells expressing the chimeric ␥R2/␣R1 chain. With anti-Tyk2 antibodies which were developed against human Tyk2, we were able to detect weak phosphorylation of a protein of ϳ130 kDa which was precipitated with anti-Tyk2 antibodies in the cells expressing ␥R2/␣R1 and ␥R2/CRF (Fig. 5B). When the same blot was reprobed with anti-Tyk2 antibodies, these phosphorylated proteins corresponded to a band recognizable by anti-Tyk2 antibodies and likely represented hamster Tyk2 (Fig. 5B, lower panels), but these proteins migrated slightly slower than human Tyk2 from lysates of COS-1 cells transiently transfected with a plasmid encoding human Tyk2 (data not shown). We observed the phosphorylated proteins of the same size in ␥R2/␣R1 and ␥R2/CRF cells when immunoprecipitation was performed with anti-phosphotyrosine antibodies and the blot was probed with anti-Tyk2 antibodies (Fig. 5C). The same differences in mobility of hamster Tyk2 from human Tyk2 from control lysates of COS-1 cells transiently transfected with a plasmid encoding human Tyk2 were observed (Fig. 5C).
To confirm that the phosphorylated protein in ␥R2/␣R1 and ␥R2/CRF cells was activated Tyk2, we stably cotransfected a plasmid encoding ␥R2/␣R1 or ␥R2/CRF with a plasmid encoding human Tyk2 into the 16-9 cells. The new cell lines were designated ␥R2/␣R1ϩTyk2 and ␥R2/CRFϩTyk2, respectively. As controls we also cotransfected a plasmid encoding ␥R2, ␥R2/␥R1, or ␥R2/␣R2 with a plasmid encoding human Tyk2 into the 16-9 cells. The resultant cell lines were designated ␥R2ϩTyk2, ␥R2/␥R1ϩTyk2, and ␥R2/␣R2ϩTyk2, respectively. First, we performed immunoprecipitation with anti-Tyk2 antibodies from cellular lysates prepared from ␥R2/␣R1ϩTyk2 and ␥R2/␣R1 cells to evaluate the expression of exogenous human Tyk2 and endogenous hamster Tyk2. After blotting with anti-Tyk2 antibodies we observed that human Tyk2 was expressed at a much higher level or was able to be precipitated with the antibodies to a greater extent than hamster Tyk2. We also observed the same differences in mobility of human Tyk2 expressed in the 16-9 cells and hamster Tyk2 as we observed above with Tyk2 expressed in COS-1 cells (Fig. 5D). With these cell lines, we observed weak phosphorylation of Tyk2 in untreated ␥R2/␣R1ϩTyk2 and ␥R2/CRFϩTyk2 cells as well as in untreated and treated ␥R2ϩTyk2, ␥R2/␥R1ϩTyk2, and ␥R2/ ␣R2ϩTyk2 cells. This is in agreement with the observation that overexpression of members of the Jak family causes a low spontaneous level of phosphorylation of kinases in the absence of ligand (Watling et al., 1993;Mü ller et al., 1993a;Silvennoinen et al., 1993a). However, only in ␥R2/␣R1ϩTyk2 and ␥R2/CRFϩTyk2 cells did we observe an enhancement of Tyk2 phosphorylation after Hu-IFN-␥ treatment (Fig. 5E). Thus, we showed that the intracellular domains of Hu-IFN-␣R1 and Hu-CRFB4 linked to the extracellular domain of ␥R2 causes activation of Tyk2 instead of Jak2 which is regularly observed during IFN-␥ signaling.
The chimeric receptor ␥R2/␥ C was able to render 16-9 cells responsive to human IFN-␥ to a small extent as measured by class I MHC antigen induction (Fig. 4H). It was shown that the IL-2R␥ C chain associates with Jak3 (Russell et al., 1994;Miyazaki et al., 1994;Tanaka et al., 1994). Thus, we examined whether Jak3 participates in the IFN-␥ receptor complex in ␥R2/␥ C cells. We hypothesized that failure of Hu-IFN-␥ to induce strong MHC class I antigen induction is due to low level of endogenous Jak3 expression in the 16-9 hamster ovary cells since Jak3 is normally only expressed in hemopoietic cells. To test the hypothesis that the low level of Jak3 limited the IFN-␥ signaling in ␥R2/␥ C cells, we stably cotransfected a plasmid encoding ␥R2/␥ C with a plasmid encoding Jak1, Jak2, Jak3, or Tyk2 into the 16-9 cells. Cell lines were designated ␥R2/ ␥ C ϩJak1, ␥R2/␥ C ϩJak2, ␥R2/␥ C ϩJak3, and ␥R2/␥ C ϩTyk2, respectively. Upon Hu-IFN-␥ treatment, only ␥R2/␥ C ϩJak3 cells showed strong enhancement in MHC class I antigen induction upon Hu-IFN-␥ treatment (Fig. 4I). All other cell lines showed the same level of responsiveness as ␥R2/␥ C cells (Fig. 4H). We confirmed the participation of Jak3 in IFN-␥ signaling in the ␥R2/␥ C ϩJak3 cells by immunoprecipitation experiments. Only in ␥R2/␥ C ϩJak3 cells did we observe enhancement in Jak3 phosphorylation after Hu-IFN-␥ treatment. After longer exposure, a low spontaneous level of Jak3 phosphorylation was observed in untreated ␥R2/␥ C ϩJak3 cells and in treated and untreated ␥R2ϩJak3 cells used as a control similar to Tyk2 phosphorylation observed in cells overexpressing Tyk2 (see above). Thus, we showed that the ␥R2/␥ C chimeric receptor recruits Jak3 into the IFN-␥ receptor complex due to association of the IL-2R␥ C intracellular domain with Jak3, and that IFN-␥ induces phosphorylation of Jak3 in ␥R2/␥ C ϩJak3 cells instead of phosphorylation of Jak2. Therefore, the substitution of the intracellular domain of ␥ C for ␥R2 accordingly substitutes Jak3 for Jak2 in the functional IFN-␥ receptor complex.
Jak1 was shown to be activated upon IFN-␥ treatment by the in vitro kinase activation assay only in cell lines positive in MHC class I antigen induction. 2 IFN-␥ Activates Stat1␣ through Jak Family Members Other Than Jak2-It was proposed that Jaks can contribute to the specificity of signal transduction by different IFNs, particularly, Tyk2 (which is active only during Type I IFN signaling) and Jak2 (which is active only during IFN-␥ signaling) . To evaluate this hypothesis, we performed electrophoretic mobility shift assays with cell lysates prepared from the various transformants before and after treatment with Hu-IFN-␥ (Fig. 6). Since Stat1␣ homodimer formation occurs during IFN-␥ signaling and Stat1␣ homodimers bind the GAS element with high specificity, we used oligonucleotides corresponding to the GAS element in the promoter region of the human IRF-1 gene as the phosphorylated probe . We observed the formation of Stat1␣ DNA binding complexes (Fig. 6) in all cell lines responsive to Hu-IFN-␥ as determined by induction of MHC class I antigen (Fig. 4). The Hu-IFN-␥ induced activation of Stat1␣ in ␥R2/␥ C ϩJak3 cells was increased to the level comparable to the level of Stat1␣ activation in all other Hu-IFN-␥ responsive cell lines (data not shown). Because it was shown that other Stats can bind the same GAS element with different affinity (Seidel et al., 1995), we performed supershift assays with specific anti-Stat1␣ antibodies to determine whether the GAS binding complexes observed in the tested cells are formed by Stat1␣ homodimers. In addition, the ␥R2/XϩTyk2 cell lines represent 16-9 cells cotransfected with the designated expression vectors encoding ␥R2/X receptors and human Tyk2; and the ␥R2/ XϩJak3 cell line, 16-9 cells cotransfected with designated expression vectors encoding ␥R2/X receptors and Jak3. Immunoprecipitates were resolved on SDS-PAGE, transferred to polyvinylidine difluoride membranes, and probed with various antibodies: anti-phosphotyrosine antibodies, first panels in A, B, E, and F; with anti-Jak2 antibodies, second panel in A; with anti-Tyk2 antibodies, C, D; and second panels in B and E; and with anti-Jak3 antibodies, second panel in F.
The GAS binding complexes in all tested cells were shifted, indicating that Stat1␣ was activated in all Hu-IFN-␥ responsive cells (Fig. 6).

DISCUSSION
Type I interferons (IFN-␣ and IFN-␤) activate Jak1, Tyk2, Stat1, and Stat2 during signal transduction; Type II interferon (IFN-␥) uses Jak1, Jak2, and Stat1 for signaling (Velazquez et al., 1992;Watling et al., 1993;Mü ller et al., 1993aMü ller et al., , 1993bLeung et al., 1995). It was proposed that the utilization of different kinases by a particular receptor complex could contribute to the specificity of signaling by different IFNs . To evaluate this hypothesis, we undertook a series of experiments with chimeric receptors.
As a model we used the IFN-␥ receptor complex whose components and signal partners are now well defined. It has been shown that the active receptor complex consists of two chains of IFN-␥R1 (␥R1) and likely two chains of IFN-␥R2 (␥R2) oligomerized upon primary binding of the IFN-␥ homodimer to the IFN-␥R1 (Greenlund et al., 1993;Langer et al., 1994;Marsters et al., 1995;Walter et al., 1995;Kotenko et al., 1995). IFN-␥R1 primarily associates with Jak1 Sakatsume et al., 1995) and, perhaps, weakly with Jak2 after IFN-␥R1 homodimerization . The intracellular domain of IFN-␥R2 associates with Jak2 and brings Jak2 into the complex upon ligand binding Sakatsume et al., 1995). The expression of a kinase negative Jak1 mutant in a Jak1 negative (U4A) cell line can sustain an IFN-␥ response, indicating that Jak1 predominantly plays a structural role in the normal functional IFN-␥ receptor complex rather than catalytical role (Briscoe et al., 1995). Thus PTK activity of Jak1 is dispensable for IFN-␥ signaling. In contrast, the expression of a kinase negative Jak2 mutant in a Jak2 negative (␥2A) cell line cannot sustain an IFN-␥ response (Briscoe et al., 1995), indicating that the PTK activity of Jak2 is absolutely necessary for IFN-␥ signaling. Thus, we tested whether substitution of Jak2 by other kinases would change the specificity of signal transduction by IFN-␥. For this purpose we substituted the intracellular domains of other receptor chains for the intracellular domain of Hu-IFN-␥R2 to recruit kinases other than Jak2 into the IFN-␥ receptor complex. The extracellular domain of Hu-IFN-␥R2 was fused to the transmembrane and intracellular domains of either the Hu-IFN-␥R1 (␥R2/␥R1), Hu-IFN-␣R1 (␥R2/␣R1), Hu-IFN-␣R2 (␥R2/␣R2), Hu-CRFB4 (␥R2/CRF), or Hu-IL-2R␥ C chain (␥R2/␥ C ) (Fig. 1). The chimeric chains were expressed in 16-9 cells, Chinese hamster ovary cells expressing Hu-IFN-␥R1. By cross-linking  we showed that all chimeric receptors were expressed on the cell surface and were able to participate in formation of the extracellular IFN-␥ receptor complex (Fig.  3). We then determined biological responsiveness of the cells to Hu-IFN-␥ as measured by MHC class I antigen induction (Fig.  4). The chimeric receptors ␥R2/␥R1, ␥R2/␣R1, ␥R2/CRF, and ␥R2/␥ C rendered the 16-9 cells responsive to Hu-IFN-␥ (Fig. 4,  C, E, G, and H). In contrast, the chimeric receptor ␥R2/␣R2 did not support signal transduction by Hu-IFN-␥ (Fig. 4F). Finally, we investigated the activation of Jaks and Stats to determine whether the specificity of signal transduction is altered in cells expressing these chimeric receptors.
In ␥R2/␣R1 cells we detected phosphorylation of Tyk2, but not Jak2 (Fig. 5, A , B, C, and E). This is in agreement with observation that the IFN-␣R1 (␣R1) intracellular domain associates with Tyk2 Colamonici et al., 1994aColamonici et al., , 1994b. Similarly, we showed that Hu-IFN-␥ induces phosphorylation of Tyk2, but not Jak2 in ␥R2/CRF cells (Fig. 5, A , B, C, and E). Thus, the intracellular domain of CRFB4 or Hu-IFN-␣R1 fused to the extracellular domain of Hu-IFN-␥R2 recruits Tyk2 into the IFN-␥ receptor complex and allows Hu-IFN-␥ to signal through activation of Tyk2 instead of Jak2, as usually occurs during IFN-␥ signaling. Thus, the activation of Jak2 per se is not necessary for signal transduction by IFN-␥ and Tyk2 can substitute for Jak2.
The ␥R2/␥R1 chimeric chain brings an additional molecule of Jak1 into the IFN-␥ receptor complex. Although we could not detect the phosphorylation of Jak2 in ␥R2/␥R1 cells (Fig. 5A), there may be some involvement of Jak2 in signal transduction by IFN-␥ in ␥R2/␥R1 cells, arising through a weak interaction between dimerized IFN-␥R1 and Jak2 . There are two regions of the IFN-␥R1 intracellular domain important for signaling, the membrane proximal region, with a possible Jak1 association site (Farrar et al., 1991), and the membrane-distal region around Tyr-457 (Cook et al., 1992;Farrar et al., 1992), the Stat1␣ recruitment site (Greenlund et al., 1994). We showed that the presence of the Stat1␣ recruitment site on this chimeric receptor chain is not required for activity because ␥R2/␥R1t 456 cells that also express the normal Hu-IFN-␥R1 chain are still responsive to Hu-IFN-␥ (Fig. 4D).
To investigate further the ability of Jaks to substitute for each other we examined ␥R2/␥ C cells. The IL-2R␥ C chain is a common chain for a broad range of receptor complexes for such cytokines as IL-2, IL-4, IL-7, IL-9 (for review, see ), and IL-15 (Giri et al., 1994). The intracellular domain of the IL-2R␥ C chain associates with Jak3 (Russell et al., 1994;Miyazaki et al., 1994;Tanaka et al., 1994). The expression of both the IL-2R␥ C chain and Jak3 are restricted to certain cell types (Minami et al., 1993;Johnston et al., 1994;Witthuhn et al., 1994). We observed MHC class I antigen induction in ␥R2/␥ C cells in response to Hu-IFN-␥ (Fig. 4H), but the induction was weaker than in all other cell lines expressing chimeric receptors (Fig. 4, B, C, E, and G). However, by coexpression of ␥R2/␥ C together with Jak1, Jak2, Jak3, or Tyk2 we showed that only Jak3 was able to restore the biological responsiveness of ␥R2/␥ C cells to Hu-IFN-␥ (Fig. 4, H and I). We further showed that Jak3 is phosphorylated upon Hu-IFN-␥ treatment of these cells (Fig. 5F). Thus, Jak3 can functionally substitute for Jak2 in the active IFN-␥ receptor complex.
Since the formation of the Stat1␣ homodimeric DNA-binding complex as the only binding complex is specific for IFN-␥ signaling, we investigated whether substitution of other kinases for Jak2 would change the specificity of the Jak-Stat signal transduction pathway. The formation of Stat1␣ DNA binding complexes was observed in all cell lines positive in MHC class FIG. 6. Electrophoretic mobility shift assay. Electrophoretic mobiliby shift assays were performed as described under "Experimental Procedures" with the 22-base pair labeled sequence containing the Stat1␣ binding site corresponding to the GAS element in the promoter region of the human IRF-1 gene  with nuclear extracts from the cells indicated on the figure and defined in the legend to Fig. 4. In addition, HEp-2 cells, a human epidermoid larynx carcinoma cell line, were used as a positive control. Supershift assays were performed with specific anti-Stat1␣ antibodies. The position of the Stat1␣ DNA-binding complexes are indicated by the arrow. I antigen induction (Fig. 6). By the supershift assay with specific anti-Stat1␣ antibodies we further showed that the GAS binding complexes consisted of Stat1␣ homodimers (Fig. 6). Thus, we concluded that other members of the Jak family recruited to the IFN-␥ receptor complex can substitute for Jak2 without changing the specificity of IFN-␥ signaling in the Jak-Stat pathway. That is, the activation of Stat1␣ during IFN-␥ signaling does not require the specific participation of Jak2.
It was shown recently that there are two splice variants of the IFNAR2 gene: a short form, Hu-IFN-␣R2b (␣R2b) and a long form, Hu-IFN-␣R2c (␣R2c). They share the same extracellular and transmembrane domains, but the stop codon of the ␣R2b is spliced out in the ␣R2c cDNA resulting in a longer intracellular domain for ␣R2c. Only the ␣R2c variant can complement the mutation in U5A cells defective in IFN-␣ signaling and sustain the IFN-␣ response (Lutfalla et al., 1995); and together with the Hu-IFN-␣R1 chain can reconstitute an active human IFN-␣ receptor complex in mouse cells (Domanski et al., 1995). Since only the ␣R2b variant was known at the time of this work, we used the ␣R2b intracellular domain. After we became aware of the existence of the ␣R2c chain, we constructed chimera ␥R2/␣R2c and expressed it in 16-9 cells. Hu-IFN-␥ induced MHC class I antigens in ␥R2/␣R2c cells to the same extent as in ␥R2 cells. 3 Thus, because we showed that the ␥R2/␣R2b (␥R2/␣R2) chimera was unable to support IFN-␥ signaling (Fig. 4F), we conclude that the intracellular domain of the short form of ␣R2, ␣R2b, is unable to bring PTK activity to the IFN-␥ receptor complex in contrast to the ␣R2c intracellular domain. The box 1 motif (proline-rich sequence) which is required for association with Jaks (Lebrun et al., 1995;Tanner et al., 1995) is not present in the short ␣R2b chain (Domanski et al., 1995). This can explain the failure of ␣R2b to complement U5A cells (cells lacking ␣R2b and c) for IFN-␣ signaling (Lutfalla et al., 1995), as well as the failure to render mouse cells responsive to Hu-IFN-␣ (Domanski et al., 1995).
In addition to demonstrating that other Jaks can substitute for Jak2 in IFN-␥ signal transduction, we provide here evidence that the intracellular domain of CRFB4, a class II cytokine receptor with unknown function (Lutfalla et al., 1993) associates with Tyk2. Therefore, CRFB4 is likely a component of a ligand-receptor system which activates Tyk2 during signal transduction. Tyk2 was shown to be activated by IFN-␣, ciliary neurotrophic factor-related cytokines, IL-10 and IL-12 (Velazquez et al., 1992;Barbieri et al., 1994;Lü tticken et al., 1994;Stahl et al., 1994;Finbloom and Winestock, 1995;Bacon et al., 1995). Since the components of a given cytokine-receptor complex belong to the same class of the cytokine receptor superfamily, it is most likely that CRFB4 is involved in IFN-␣ or IL-10 receptor complexes, as all cloned subunits of these receptors are members of the same class as CRFB4 (Uzé et al., 1990;Liu et al., 1994;Novick et al., 1994) unlike the components of the ciliary neurotrophic factor-related cytokine receptor subunits and the IL-12 receptor. Other evidence supports this hypothesis. The reconstitution of an active IFN-␣ receptor has not been reported . 4 However, the yeast artificial chromosome containing CRFB4, Hu-IFN-␣R1, and Hu-IFN-␣R2 genes (Emanuel, 1995) 5 encodes a functional Hu-IFN-␣ receptor complex (Soh et al., 1994b;Cleary et al., 1994). It is thus enticing to consider that these three genes are all involved in Type I interferon receptor function. In the case of IL-10, only one chain (IL-10R) has been cloned . The activation of two different members of the Jak family during signal transduction usually requires the involve-ment of two different receptor components associated with two distinct Jaks. Since IL-10 activates Tyk2 and Jak1, CRFB4 may be involved in the IL-10 receptor complex as well. Thus, the use of chimeric receptors provides a method to study the properties of intracellular domains of receptors from unknown or incompletely characterized ligand-receptor complexes as illustrated with the chimeric receptors containing the intracellular domains of Hu-IFN-␣R1, Hu-IFN-␣R2, or CRFB4.
Of most importance, we showed that the Jaks are interchangeable for the Jak-Stat signal transduction pathway. The results allow us to expand our model for IFN-␥ signaling , which may be general for class II cytokine receptors (Fig. 7). The signal transducing receptor chains can be divided into two classes: 1) the actual Signal Transducers (ST), containing Stat (or other SH2 domain containing protein) Recruitment Sites (SRS) and Jak Association Sites (JAS); and 2) Helper Receptors (HR), containing only JAS, but no SRS. The primary function of the HR is to bring additional PTK activity to the receptor complex upon ligand binding. They do not contain functionally important Tyr residues. Thus far two receptors fit this HR category: the IL-2R␥ C and IFN-␥R2 chains, as it was shown that the substitution of Tyr residues within their intracellular domains does not change the ability of these receptors to support signal transduction (Lai et al., 1995;Bach et al., 1995). The CRFB4 chain and probably IFN-␣R1 are other candidates for helper receptors. In those cases where homodimerization of a single receptor chain appears sufficient for signal transduction and its intracellular domain contains all the JAS and SRS regions necessary and sufficient for signal transduction (as in the case of EPO-R, GHR, or FIG. 7. Model of the signal transduction by IFN-␥. After oligomerization of the IFN-␥ receptor chains caused by ligand binding, the IFN-␥ homodimer binds to two IFN-␥R1 chains which in turn brings two associated IFN-␥R2 (AF-1) chains and all the associated components (Jaks and Stats) into the complex . The interaction of Jaks with the intracellular chains initiates the cascade of events resulting in activation of specific Stats as described in the text. JAS, represents Jak association site; SRS, Stat recruitment site; ST, signal transducing receptor; HR, helper receptor; PTK, protein tyrosine kinase; ␥R2/X, chimeric receptor with the extracellular domain of the IFN-␥R2 and the intracellular domains of various receptors swapped for the intracellular domain of the Hu-IFN-␥R2 chain. ProR), the activation of a single Jak2 is observed and a separate HR chain is not required (Argetsinger et al., 1993;Witthuhn et al., 1993;Campbell et al., 1994;DaSilva et al., 1994;David et al., 1994;Dusanter-Fourt et al., 1994;Rui et al., 1994;Muthukumaran et al., 1995). We hypothesize that the intracellular domains of the HR can be associated with any Jak and do not provide any specificity for signal transduction. Only their extracellular domains are specific for particular ligand receptor complexes. The Jaks show preferential specificity for association with the receptor intracellular domains just like the Stats (Heim et al., 1995;Stahl et al., 1995), but the kinase domains per se are promiscuous. Finally, we hypothesize that the Jaks do not contribute to the specificity of signal transduction in the Jak-Stat pathway, inasmuch as they do not possess a preferential specificity for Stat activation.