Interferon signaling is dependent on specific tyrosines located within the intracellular domain of IFNAR2c. Expression of IFNAR2c tyrosine mutants in U5A cells.

Type I interferons (IFNs) are cytokines that play a central role in mediating antiviral, antiproliferative, and immunomodulatory activities in virtually all cells. These activities are entirely dependent on the interaction of IFNs with their particular cell surface receptor. In this report, we identify two specific tyrosine residues located within the cytoplasmic domain of IFNAR2c that are obligatory for IFN-dependent signaling. Various IFNAR2c tyrosine mutants were expressed in a human lung fibroscarcoma cell line lacking IFNAR2c (U5A). Stable clones expressing these mutants were analyzed for their ability to induce STAT1 and STAT2 activation, ISGF3 transcriptional complex formation, gene expression, and cell growth regulation in response to stimulation with type I IFNs. The replacement of all seven cytoplasmic tyrosine residues of IFNAR2c with phenylalanine resulted in a receptor unable to respond to IFN stimulation. Substitution of single tyrosines at amino acid residue 269, 316, 318, 337, or 512 with phenylalanine had no effect on IFN-dependent signaling, suggesting that no single tyrosine is essential for IFN receptor-mediated signaling. In addition, IFNAR2c retaining five proximal tyrosines residues (269, 306, 316, 318, and 337) or either two distal tyrosine residues (411 or 512) continued to be responsive to IFN stimulation. Surprisingly, the presence of only a single tyrosine at either position 337 or 512 was sufficient to restore a complete IFN response. These results indicate that IFN-dependent signaling proceeds through the redundant usage of two tyrosine residues in the cytoplasmic domain of IFNAR2c.

Many cell surface receptors contain tyrosine residues that are phosphorylated upon ligand-induced receptor activation. These phosphorylated tyrosines are specifically recognized by Src homology domains (SH2 or SH3) that are present in many transcription factors, phosphatases, and other mediators of downstream signaling (1). The binding of SH2 or SH3 domains to phosphorylated tyrosines is, in many cases, an absolute requirement for the activation of transcription factors (2). In addition, these phosphotyrosine residues are essential mediators of the interactions of specific phosphatases with certain cell surface receptors (3)(4)(5)(6)(7). For some receptors, the mutation or deletion of specific tyrosines completely abrogates ligand-dependent signaling, whereas other cell surface receptors, such as the erythropoietin receptor, do not appear to depend upon any one particular tyrosine but instead are capable of utilizing any one of several tyrosines for signaling (8). Overall, such observations demonstrate the importance of tyrosine residues in signaling and suggest that in certain pathways particular tyrosine residues can mediate the activation of specific transcription factors that may in turn induce the expression of a distinct subset of genes.
One example of how intracellular tyrosines are utilized in signaling is the type I interferon (IFN) 1 pathway. Human type I IFNs (␣, ␤, and ) have been shown to induce the expression of a large number of genes involved in regulating a variety of important biological responses, including antiviral, antiproliferative, and immunomodulatory activities. The mechanisms by which type I IFNs initiate such a broad spectrum of biological activities is only beginning to emerge. Type I IFN-dependent signaling requires both type I IFN receptor chains, IFNAR1 (human type I interferon ␣ receptor chain 1) and IFNAR2c (human type I interferon ␣ receptor chain 2) (9 -12). Binding of type I IFNs induces the assembly of these receptor chains, which leads to the phosphorylation of tyrosine residues located in the intracellular domain of each receptor chain. These tyrosine phosphorylation events are thought to be carried out by the Janus kinases TYK2 and JAK1, which are themselves activated by tyrosine phosphorylation (13,14). IFN receptor activation also leads to the phosphorylation and activation of the transcription factors STAT1 and STAT2. Additional transcription factors, such as STAT3 (15), phosphatidylinositol 3-kinase (16), mitogen-activated protein kinase (17), and CrkL/ STAT5 (18,19), also become activated in response to IFN. It is clear that phosphorylation of the cytoplasmic tyrosines of IFNAR1 and IFNAR2c is involved in type I IFN signaling; however, the role that specific tyrosines play in this signal transduction pathway is unclear.
It is thought that the phosphorylation of tyrosine residues on IFNAR1 and IFNAR2c plays a critical role in the activation of STAT1 or STAT2. The proposed STAT2-binding site on IFNAR1 includes two phosphorylated residues, Tyr 466 and Tyr 481 (20). STAT-binding sites on IFNAR2c have also been proposed from studies using IFNAR2c truncation mutants expressed in human (21) or mouse cell lines (22). In human cells, truncation of the cytoplasmic domain of IFNAR2c to amino acid 417 produces a receptor that is unable to mediate the phosphorylation of STAT1 or STAT2. Expression of a specific tyrosine mutant of human IFNAR2c in mouse L929 cells suggests that the tyrosine phosphorylation of at least one of the proximal tyrosines (Tyr 269 , Tyr 306 , Tyr 316 , Tyr 318 , and Tyr 337 ) of IFNAR2c is required but not sufficient by itself, for STAT2 activation (22). Thus, in both human and mouse cells, IFN-dependent signaling requires both the presence of specific tyrosines within the cytoplasmic domains of the receptor chains and the presence of STATs competent to interact with these domains.
To examine the role of tyrosines present in the cytoplasmic domain of IFNAR2c, we have expressed tyrosine mutants of IFNAR2c in U5A cells, which do not express IFNAR2c, and have analyzed their ability to respond to IFN (23). This approach has allowed us to analyze, for the first time, the functional importance of specific tyrosine residues of IFNAR2c in a human cell background.
Selection of U5A Cell Lines Stably Expressing IFNAR2c Tyrosine Mutants-The mutants analyzed in this study are shown in Fig. 1. A vector expressing IFNAR2c and a form of IFNAR2c in which all the intracellular tyrosines were substituted with phenylalanine, pZIFN␣R␤LYF (7F) (21,22), was kindly provided by Dr. Oscar Colamonici (Department of Pharmacology, University of Chicago, Chicago, IL). The 7F construct was subjected to site-directed mutagenesis to add back single tyrosines creating plasmids F269Y, F306Y, F316Y, F318Y, and F337Y. The 7F plasmid was also used as a PCR template and cloned into the vector pCDEF1, kindly supplied by Dr. Jerome A. Langer (Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Piscataway, NJ) using HindIII and SpeI. This construct was then mutagenized using the QuikChange site-directed mutagenesis kit (Stratagene) to add back single tyrosines at positions 411 and 512. A double tyrosine add-back at residues 411 and 512 (4Y5Y) was also constructed in this vector. Plasmids containing single tyrosine to phenylalanine substitutions (Y269F, Y316F, Y318F, Y337F, and Y512F) were kindly provided by Dr. M. Rubinstein (Department of Molecular Genetics and Virology, The Weizmann Institute of Science, Rehovot, Israel). The Y512F plasmid was mutagenized to create the double tyrosine knock-out at residues 411 and 512 (4F5F). U5A cells (1 ϫ 10 6 cells/35-mm well) were transfected using Superfect (Qiagen) as previously described (21). Multiple stable cell populations for each IFNAR2c mutant were selected in growth medium containing G418 (1 mg/ml). After drug selection, cell populations were selected for single clones expressing IFNAR2c by fluorescence-activated cell sorting (FACS) and cloned by limiting dilution (FACS Vantage TM SE, Becton Dickinson Bioscience, San Jose, CA). For FACS analysis, 1 ϫ 10 7 cells were incubated with a purified monoclonal antibody (27D11), recognizing the extracellular domain of IFNAR2c, for 1 h at 4°C, washed with ice-cold phosphate-buffered saline and then incubated with a phycoerythrin-conjugated anti-mouse Fab antibody (Dako, Glostrup, Denmark) for 1 h at 4°C. The resulting individual clones were then screened by FACS, and only clones having a FACS signal equal to or greater than the parental HT1080 cells were picked and expanded. IFNAR2c expression for all clones was further confirmed by using a radioligand binding assay (see below).
Ligand Binding Assay-EZ-Label TM human IFN␣2 (PBL Biomedical Laboratories) was labeled with ␥-33 P-labeled ATP as previously described (21). Radiolabeled IFN␣2 (278 pM; specific activity of 60 -62 Ci/g) was added to 5 ϫ 10 5 cells and allowed to bind for 90 min at room temperature before the unbound IFN was washed away with cell culture medium. Nonspecific binding was determined by adding a 100fold excess of unlabeled IFN␤1b to compete for binding during the 90-min incubation. The cells were lysed in phosphate-buffered saline containing 1% SDS, and the radioactivity was determined by scintillation counting.
Electrophoretic Mobility Shift Assay-Gel shift assays were performed using the 32 P-labeled double-stranded IFN-stimulated response elements oligonucleotide 5Ј-GCTTCAGTTTCGGTTTCCCTTTCCCGA-G-3Ј and its complement (25). Briefly, the cells were stimulated with IFN␣2 (1000 units/10 6 cells) or IFN␤1b (1000 units/10 6 cells) for 30 min, and the cell pellets were collected and processed for electrophoretic mobility shift assay as previously described (25). The resulting DNAbinding complexes were separated by electrophoresis through a 6% polyacrylamide gel and analyzed by autoradiography.
RNase Protection Assay-The cells were stimulated with either human IFN␣2 (1000 units/10 6 cells) or IFN␤1b (1000 units/10 6 cells) for 17 h before RNA was isolated, and gene expression was analyzed by an RNase protection assay as previously described (21,26).
Cell Growth Assay-The cells were seeded (5 ϫ 10 4 cells/well) in a 6-well 35-mm cell culture plate and allowed to attach overnight. Complete medium alone or containing IFN␤1b (1500 units/10 6 cells, IFN␤1b) was added every day for the duration of the experiment. Cell growth was determined using the Alamar Blue method as previously described (27). Alamar Blue TM (BIOSOURCE International) dye reduction was measured after incubation with cells for 40 min at 37°C using a Millipore fluorescence measurement system Cyberfluor 2300 ( emission ϭ 590; excitation ϭ 530).

RESULTS
The cell surface expression of IFNAR2c for all the stable IFNAR2c tyrosine mutant clones ( Fig. 1) was confirmed by FACS ( Fig. 2A) and radioligand binding (Fig. 2B). FACS and radioligand binding analysis correlated closely with each other (Fig. 2) and demonstrated that the cell surface expression levels of IFNAR2c on all the clones analyzed was equal to or greater than that observed in the parental HT 1080 cell line. The only cell line in which IFNAR2c could not be detected on the cell surface was the U5A cell line that was previously shown to lack IFNAR2c (Fig. 2).
Using these clones, we analyzed STAT1 and STAT2 activation by phosphotyrosine immunoblotting after 15 min of stimulation with either IFN␣2 or IFN␤1b (1000 units/ml/10 7 cells). For all clones in which only a single tyrosine had been substituted with phenylalanine, STAT1 and STAT2 activation were efficiently induced by both IFN␣2 and IFN␤1b (Fig. 3A). IFNdependent STAT activation was also observed in the two cluster mutants of IFNAR2c, one having both the distal tyrosines at amino acid positions 411 and 512 substituted to phenylalanine (4F5F) and the other having all five proximal tyrosines (Tyr 269 , Tyr 306 , Tyr 316 , Tyr 318 , and Tyr 337 ) substituted by phe-nylalanine (4Y5Y) (Fig. 3B). However, STAT activation was not observed when all seven tyrosines were absent (clone 7F) (Fig. 3B). Next, IFNAR2c mutants in which each of the seven tyrosines were individually added back were assessed for their ability to induce STAT activation in response to IFN stimulation (Fig. 3C). Surprisingly, a single tyrosine at either amino acid position 337 or 512 (clones F337Y or F512Y, respectively) was sufficient to mediate IFN-dependent activation of STAT1 and STAT2. All of the remaining single tyrosine add-back clones (269Y, 306Y, 316Y, 318Y and 411Y) were unable to sustain any detectable IFN-dependent STAT activation.
After receptor activation, STAT1 and STAT2 are phosphorylated and form a complex with the DNA-binding protein p48 to make up the IFN-stimulated growth factor complex, ISGF3. The ISGF3 transcription factor then translocates to the nucleus and binds to IFN-stimulated response elements, located within the promoter regions of IFN-responsive genes, to induce gene expression. Therefore, it was important to determine whether the IFN-dependent tyrosine phosphorylation of STAT1 and STAT2, mediated by the various IFNAR2c tyrosine mutants, resulted in the assembly of a functional ISGF3 (Fig.  4). Cell lines expressing IFNAR2c tyrosine mutants were stimulated with 1000 units/10 6 cells IFN␤1b or IFN␣2 for 30 min, cells were harvested, and ISGF3 complex formation was determined as described under "Materials and Methods." In all cases, formation of an ISGF3 complex was detected in IFNAR2c tyrosine mutants that were previously shown (Fig. 3) to be capable of inducing tyrosine phosphorylation of STAT1 and STAT2 in response to IFN stimulation.
IFN stimulation results in the expression of a plethora of genes that mediate a variety of important biological effects. To further characterize IFNAR2c tyrosine mutants, we investigated the differential expression of a number of IFN-inducible genes using an RNase protection assay. The I-TAC, ISG 54, and TRAIL genes 2 are preferentially induced by IFN␤1b, as compared with various IFN␣ species, whereas the ISG 6-16 gene was induced by either IFN (21,26). Briefly, 1 ϫ 10 6 cells were stimulated with 1000 units/ml of either IFN␤1b or IFN␣, cells were harvested, RNA was isolated, and the RNase protection assay was performed as described under "Materials and Methods." Induction of I-TAC, ISG 54, ISG 6-16, and TRAIL by IFN appeared to be unaffected by any of the single tyrosine-tophenylalanine substitutions (Fig. 5A). As expected, both the U5A cells and the 7F clone did not demonstrate IFN-mediated gene expression, whereas the 4F5F and 4Y5Y cluster mutants were fully functional (Fig. 5B). Furthermore, single tyrosine add-backs, except for 337Y and 512Y, failed to exhibit IFNinducible expression of I-TAC, ISG 54, and TRAIL. Surprisingly, IFN-dependent stimulation of the 337Y and 512Y clones resulted in the full expression of all of the genes analyzed. IFN-inducible gene expression in 337Y and 512Y was at levels equal to or greater than those observed for the parental HT1080 cells (Fig. 5C). It is also of interest to note that the expression of ISG 6-16, especially in response to IFN␤1b, was not completely lost in the single tyrosine add-back clones 269Y, 306Y, 316Y, and 318Y (Fig. 5C).
The relative levels of gene expression did vary somewhat among the different IFNAR2c tyrosine mutants with the highest level observed in a cell line expressing wild type IFNAR2c (Fig. 5). Finally, the preferential induction of I-TAC, ISG 54, and TRAIL by IFN␤1b that was observed in the parental HT1080 cell line was still present in both the 337Y and 512Y clones.
IFNs have potent antiproliferative effects on many different cell types. Therefore, to evaluate the IFNAR2c mutants in terms of a functional response, we measured the IFN-dependent antiproliferative effects for each clone (Fig. 6). The results are expressed as the percentages of growth compared with untreated controls. Cell growth rates were measured for up to 6 days using Alamar Blue, as previously described (27). As expected, in U5A cells expressing wild type IFNAR2c, all the single tyrosine knock-out mutants and the proximal and distal cluster mutants were able to express clear antiproliferative effects in response to IFN␤1b stimulation (Fig. 6). Among the single tyrosine add-backs, only the 337Y and 512Y IFNAR2c clones were able to elicit an antiproliferative effect in response to IFN stimulation. The magnitude of the effect was equal to or greater that that observed for HT1080 or U5A cells expressing wild type IFNAR2c (R2c). This result further illustrates the importance of Tyr 337 and Tyr 512 in IFN-dependent signaling and extends this observation to include their roles in the mediation of important biological effects such as antiproliferative responses.

DISCUSSION
The regulation of many cellular activities is mediated through the control of phosphorylation and dephosphorylation events. Many different cytokine and growth factor receptors contain specific tyrosine residues within their cytoplasmic domains that are required for signaling. In the case of the type I IFN receptor, tyrosine phosphorylation events have been shown to involve the JAK-STAT signaling pathway (28). The role of phosphorylation and dephosphorylation events in type I IFN signaling appears to be tightly controlled by the interplay of a complex system of different tyrosine kinases and phosphatases, which is not yet understood.
In terms of IFN signaling, STAT1 and STAT2 play a major role in type I IFN signaling, although STAT3 (15), STAT4 (29), and STAT5 (19) can also be activated in response to IFN in some cells. In all cases, ligand-dependent activation of STATs require that they become phosphorylated on specific tyrosines located in the C-terminal portions of the proteins. The phosphorylation of these tyrosines involves the receptor-associated Janus kinases JAK1 and TYK2, which themselves become activated by tyrosine phosphorylation. Activation of JAK1 and TYK2 requires the heterodimeric assembly of the two type I IFN receptor chains, IFNAR1 and IFNAR2c. It is thought that one of the first phosphorylation events carried out by activated Janus kinases is the specific phosphorylation of tyrosine residues on IFNAR1 and IFNAR2c.
Because it is known that the Janus kinases can phosphorylate various STATs, other mechanisms must be involved that confer signaling specificity. One of these mechanisms may involve the specific phosphorylation of either IFN receptor chain. Indeed, a number of studies have identified important tyrosines located within the cytoplasmic domain of many cytokine and growth factor receptors. It has been shown that these tyrosines can mediate the specific docking of adapter proteins, STATs, and phosphatases to the receptors (30). Indeed, specific tyrosines in IFNAR1 have been suggested to be involved in the regulation of IFNAR1-associated phosphatase activity (31) as well as STAT2 binding (20). Further studies have suggested that STAT2 and STAT1 preassociate with IFNAR2c and that phosphorylation of Tyr 466 on IFNAR1 acts as an additional docking site and/or activation step for STAT2 that leads to STAT1 activation and the subsequent formation of a STAT1-STAT2 heterodimer (32). Recent studies using mutants of IFNAR2c having a truncated (21,22) or substituted (20,(33)(34)(35) cytoplasmic domain lend support to the hypothesis that the cytoplasmic tyrosines of IFNAR2c play a role in the activation of STAT1 and STAT2. Furthermore, such studies suggest that tyrosines located in the cytoplasmic domain of IFNAR2c play an important role in STAT activation leading to IFN-mediated gene activation and antiproliferative effects. This assumption is supported by recent studies carried out using mouse L929 cells expressing a tyrosine mutant of human IFNAR2c in which it was shown that tyrosines in the cytoplasmic domain of IFNAR2c are important for IFN-dependent activation of ISGF3 (22). However, other studies demonstrate that when a form of human IFNAR1, in which all the tyrosines are mutated to phenylalanines, was expressed in a mouse background, it retained its ability to respond to human IFN (36). This observation calls into question the essential role of IFNAR1 tyrosines in IFN signaling and explains the complicated nature of analyzing the effects of human receptor chains expressed in a mouse cell background.
A summary of the functional properties of all the IFNAR2c tyrosine mutants analyzed in the study is presented in Table I. We have shown that replacing all of the cytoplasmic tyrosines of IFNAR2c with phenylalanines results in a receptor unable to induce JAK-STAT-mediated signaling in response to IFN stimulation, thereby demonstrating the general importance of these tyrosines in type I IFN-dependent signaling. Tyrosine residues within the cytoplasmic domain of IFNAR2c are physically clustered into two groups, both proximal (Tyr 269 , Tyr 306 , Tyr 316 , Tyr 318 , and Tyr 377 ) and distal (Tyr 411 and Tyr 512 ) to the cell membrane. Of the seven cytoplasmic tyrosines of IFNAR2c, Tyr 306 and Tyr 411 are not conserved across species and are therefore not likely to be crucial to signaling. Complete tyrosine-to-phenylalanine substitution of either tyrosine cluster (4F5F or 4Y5Y) resulted in clones fully responsive to IFN stimulation, suggesting that important tyrosines reside in both the proximal and distal tyrosine clusters. Furthermore, the single substitution of each of the remaining tyrosines-to-phenylalanine had no effect on IFN signaling, which also suggests Cell growth was measured in all mutants cell lines in the absence or presence of IFN␤1b (1000 units/ml) as described under "Materials and Methods." Cell growth was determined using the Alamar Blue TM method and expressed on the y axis as a percentage of cell growth relative to untreated cells. The data represent the mean values (n ϭ 3), and variations between replicates were less than 15%. Similar results were obtained in a 6-day assay (data not shown). HT1080, parental cell line; U5A, U5A cells. All other cell lines are as described for Fig. 1. that no single tyrosine is absolutely essential for IFN-dependent STAT activation, gene regulation, and biological effects.
When single IFNAR2c tyrosine add-backs were analyzed for their ability to mediate IFN-dependent signaling, Tyr 337 and Tyr 512 were both capable of mediating signaling on their own. Surprisingly, only the individual substitution of these two tyrosines was able to restore STAT1 and STAT2 activation, ISGF3 complex formation, gene expression, and antiproliferative responses in U5A cells. Detailed studies are ongoing to determine the roles of Tyr 337 and Tyr 512 in IFN-dependent antiviral responses.
Previous studies using IFNAR2c truncation mutants expressed in U5A cells suggested that the distal region of IFNAR2c (amino acids 462-515) was not necessary for receptor function. However, truncation of IFNAR2c at amino acid residues 417 or 346 resulted in a completely inactive receptor (21). Both of these mutants contain Tyr 337 , which we have now shown is sufficient to mediate signaling in the context of an otherwise functional receptor. This apparent discrepancy could be explained by the fact that a deletion of the cytoplasmic domain of IFNAR2c might disrupt the tertiary structure around Tyr 337 in such a way that it is no longer able to play a role in IFN-dependent signaling.
It is interesting to note that the induction by IFN␤1b of the ISG 6-16 gene could be observed at low levels in some IFNAR2c mutants having only a single tyrosine. These clones show no detectable STAT1/STAT2 activation, ISGF3 formation, antiproliferative response, or ISG 54, TRAIL, or I-TAC gene induction, suggesting that the IFN receptor may utilize other means of gene induction independent of JAK-STAT signaling.
The redundant usage of different tyrosines in the regulation of receptor-mediated signaling has been previously documented for other receptor systems. However, this is the first demonstration of a similar signaling mechanism in the type I IFN system. It remains unclear why these two specific tyrosines can mediate similar IFN-dependent signaling events. It is possible that the proximal location or microenvironment of Tyr 337 and Tyr 512 within the cytoplasmic domain of IFNAR2c presents these two residues as excellent substrates for JAK kinases, redundant docking sites for SH2 domain-containing proteins, or important docking sites for as yet unknown signaling components.
The tyrosine phosphorylation of IFNAR1 has also been demonstrated to be important in IFN-mediated signaling. The importance of the interaction between both phosphorylated IFN receptor chains with each other remains to be determined. The proposed interactions with other receptor chains, such as IFNGR2, also needs further investigation (37). It is likely that these interactions help to regulate the efficiency of IFN-medi-ated signaling and the mechanism by which proteins involved in signaling, such as STATs, phosphatases, adapter molecules, and docking proteins, interact with the assembled receptor complex. There is currently no human cell line available in which IFNAR1 is not expressed; therefore, most IFNAR1 studies have made use of human receptor chains expressed in mouse cells or by using competing phosphopeptides. Using human U5A cells that lack IFNAR2c but stably express specific tyrosine mutants of IFNAR2c, we have been able to demonstrate the direct role of Tyr 337 and Tyr 512 in IFN-dependent signaling. It is possible that phosphorylated tyrosines within either IFNAR2c or IFNAR1 interact with SH2 domains present in proteins, such as STATs, functionally bridging the two receptor chains. In the case of STATs, this interaction could then result in the presentation of the functionally important STAT1 and STAT2 tyrosines (Tyr 701 and Tyr 690 , respectively) as high affinity substrates for either JAK1 or Tyk2. In this way, both IFNAR1 and IFNAR2c receptor chains would work in concert to achieve a maximum response to IFN stimulation. We conclude, based on this study and others, that the tyrosine residues critical for carrying out this function are Tyr 466 on IFNAR1 and Tyr 337 or Tyr 512 on IFNAR2c.
In this report, we have focused on characterizing the role of tyrosine phosphorylation in the JAK-STAT signaling pathway by using IFNAR2c tyrosine mutants expressed in U5A cells. Further work is needed to clarify the mechanism by which IFN-mediated signaling pathways utilize specific phosphorylated tyrosines of either IFNAR1 or IFNAR2c to mediate downstream signaling events. In addition to the JAK-STAT signaling pathway, other proposed IFN-mediated signaling pathways that need further study include the activation of the insulin receptor substrate/phosphatidylinositol 3-kinase, Vav protooncogene, CBL/Crk-protein family, and mitogen-activated protein kinase. Furthermore, the regulation of phosphorylation events by phosphatases such as SHP-1 or SHP-2, both of which contain SH2 domains, remains unclear. The identification of two critical tyrosines in IFNAR2c that are essential for efficient JAK-STAT signaling and the use of IFNAR2c tyrosine mutants characterized within this study will help to further elucidate the type I IFN signaling pathway.