©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transmembrane Signaling by the Subunit of the Type I Interferon Receptor Is Essential for Activation of the Jak Kinases and the Transcriptional Factor ISGF3 (*)

(Received for publication, December 1, 1994)

Oscar R. Colamonici (1)(§) Leonidas C. Platanias (2)(¶) Paul Domanski (1) Raj Handa (1) Kimberly C. Gilmour (3) Manuel O. Diaz (2) Nancy Reich (3) Paula Pitha-Rowe (4)

From the  (1)Department of Pathology, University of Tennessee, Memphis, Tennessee 38163, the (2)Division of Hematology/Oncology, Loyola University of Chicago and Edward Hines Jr. Veterans Administration Medical Center, Maywood, Illinois 60153, the (3)Department of Pathology, State University of New York, Stony Brook, New York 11794, and the (4)Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Type I interferon (IFN) receptor has a multisubunit structure. The component of the receptor that has been most thoroughly studied is the alpha subunit. Expression of the alpha subunit in mouse L-929 cells confers antiviral response to human IFNalpha8, but not to human IFNalpha2 or IFNbeta. This antiviral effect is observed without a significant increase in IFN binding. It has not been determined why mouse cells expressing the human alpha subunit show different response to the antiviral activity of distinct human Type I IFNs. In this report, we demonstrate that the response to human Type I IFNs in mouse cells expressing the alpha subunit is dependent on cross-binding to the mouse receptor. This is supported by the finding that human IFNalpha8, but not human IFNalpha2, cross-binds to the mouse receptor even in the absence of expression of the human alpha subunit. We also demonstrate that only mouse cells expressing the human alpha subunit are able to tyrosine-phosphorylate p135 and JAK-1 and to form the ISGF3 complex in response to human IFNalpha8. These results demonstrate that the alpha subunit is essential for IFNalpha signaling through the JAK kinases and ISGF3.


INTRODUCTION

Type I Interferons (IFNs) (^1)exert a wide variety of effects that include antiviral and antiproliferative effects, histocompatibility leukocyte antigen induction, boosting of natural killer activity, etc.(1, 2) . Type I IFNs specifically bind to a multisubunit cell surface receptor(3, 4, 5, 6) . To follow the designation of other receptor subunits of the cytokine receptor superfamily, the different subunits of the Type I IFN-R are designated with Greek letters. Experiments performed with specific anti-Type I IFN receptor (IFN-R) monoclonal antibodies (mAb) have demonstrated that there are at least two receptor subunits designated as the alpha (110 kDa) and beta (100 kDa) subunits(5, 6) . Two subunits of the Type I IFN-R have also been cloned(7, 8) . The subunit cloned by Uzéet al.(7) corresponds to the alpha subunit and is recognized by the IFNaR3 mAb (9) . Thus, we will refer to the cDNA cloned by Uzéet al.(7) as the alpha subunit of the Type I IFN-R(9) . The receptor subunit cloned by Novick et al.(8) corresponds to the beta subunit. (^2)

The alpha subunit was cloned by its ability to confer antiviral response to human IFNalpha8 in mouse cells(7) . Interestingly, expression of this subunit in mouse L-929 cells makes them responsive to human IFNalpha8 without a significant increase in human IFN binding(10, 11, 12) . More importantly, knockout mice for the alpha subunit fail to respond to different viral agents underscoring the role of this subunit in the antiviral response(13) . However, the alpha subunit should also play a role in Type I IFN binding as indicated by cross-linking of this subunit to radioiodinated human IFNalpha2 (6) and a slight increase in binding observed in Xenopus laevis oocytes transfected with the cDNA encoding this subunit(14) .

We have demonstrated that the alpha subunit of the Type I IFN-R is rapidly phosphorylated on tyrosine after IFN binding to the receptor (15, 16) . Furthermore, one of the Janus tyrosine kinases involved in IFNalpha signaling, the p135 kinase, associates with and tyrosine phosphorylates the alpha subunit in vitro(9, 17) . We have mapped the p135 binding domain to the 47 juxtamembrane amino acids of the alpha subunit. This region contains two tyrosine residues (tyrosines 466 and 481) that are the putative targets for p135 phosphorylation(9) .

In this report, we show that the alpha subunit binds human IFNalpha8, as well as IFNalpha2, IFNalpha7, IFNbeta, and IFN, when is part of the Type I IFN-R complex expressed in human cells. However, binding of human IFNalpha8, but not human IFNalpha2, can be detected in mouse cells even in the absence of the human alpha subunit. In this context, the response to human IFNalpha8 in mouse L-929 cells transfected with the alpha subunit is dependent on cross-binding of human IFNalpha8 to the mouse receptor. Since the alpha subunit is associated with and tyrosine phosphorylated by p135, we tested if this receptor subunit was able to activate this kinase in mouse cells. Our data indicate that activation of the p135 and JAK-1 tyrosine kinases(20, 21, 22, 23) , and the transcriptional factor ISGF3alpha(21, 24, 25, 26, 27) , occurs in mouse cells transfected with the alpha subunit in response to IFNalpha8. Since the JAK-1 tyrosine kinase does not associate with the alpha subunit, and it is not activated in cells transfected with vector alone, these data raise the possibility that the JAK-1 tyrosine kinase may be downstream of p135.


EXPERIMENTAL PROCEDURES

Materials

Human recombinant IFNalpha2 was kindly provided by Drs. M. Brunda and P. Trotta (Hoffman-La Roche and Schering-Plough, respectively). IFNbeta, IFN, and IFNalpha8 were provided by Drs. Colby (Triton Biosciences, currently Berlex Laboratories), G. Adolf (Ernst Boehringer Institute fur Arzneimittelforschung), and H. Hochkeppel and D. Gangemi (Ciba-Geigy and Clemson University, respectively). The anti-phosphotyrosine and anti-JAK-1 antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse IFNalpha/beta were purchased from Amgen and Lee Biochemicals. The anti-alpha subunit mAb IFNaR3 and the rabbit sera anti-Tyk2 and 411-557 (against the cytoplasmic domain of the alpha subunit) have been described previously (6, 9) .

Cell Lines and Antiviral Assays

The human myeloma U-266 and H-929 cell lines were obtained from the American Tissue Culture Collection (ATCC) and Dr. A. Gazdar, respectively. Both cell lines were grown in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics (Life Technologies, Inc.). The cell lines SV.1, SVX.1, and SVX.2 have been described previously (10) and correspond to L-929 cells transfected with pZipNeo vector alone and vector containing the cDNA for the Type I IFN-R subunit cloned by Uzéet al.(7) , respectively. We will refer to this subunit as the alpha subunit(9) .

Surface Iodination, Radioiodination of Type I IFNs, and Affinity Cross-linking

These procedures were performed as described previously(4, 5, 6) .

Immunoblotting

Cells were treated with different concentrations of IFNalpha2 for the indicated periods of time, rapidly centrifuged at 2000 times g for 30 s in an Eppendorf microcentrifuge, and subsequently solubilized in lysis buffer (1% Triton X-100, 150 mM NaCl, 25 mM HEPES (pH 7.5), 1 mM EDTA, 200 µM sodium orthovanadate, 100 mM NaF, 1 mM MgCl(2), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin) at 4 °C for 30 min. Protein complexes were precipitated from cell lysates with the indicated antibodies and protein G-Sepharose (Pharmacia Biotech Inc.). Incubations ranged from 1 h to overnight, at 4 °C. After three to five washes with lysis buffer containing 0.1% Triton X-100, the immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters (Schleicher and Schuell). Nonspecific binding sites on the filter were blocked with 10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% Tween 20 containing 5-8% bovine serum albumin or 5% non-fat dry milk for 1 h at room temperature. Immunoblots were subsequently incubated with the indicated primary antibodies and appropriate secondary antibodies (linked to horseradish peroxidase) and developed using an enhanced chemiluminescence kit (Amersham Corp.).

Electrophoretic Mobility Shift Assay

Nuclear and cytoplasmic extracts were prepared as described and analyzed by electrophoretic mobility shift assays using an end labeled oligonucleotide (5`-GATCGGGAAAGGGAAACCGAAACTGAAGCC-3` and 3`-CCCTTTCCCTTTGGCTTTGACTTCGGAG-5`) to detect ISGF3(25, 26) .


RESULTS

Differences in the Antiviral Response to Human Type I IFN in Mouse Cells Transfected with the alpha Subunit of the Type I IFN-R

We have reported previously that the cDNA cloned by Uzéet al.(7) corresponds to what we have previously designated as the alpha subunit of the Type I IFN-R(9) . However, differences in the molecular mass of this subunit have been reported. For instance, in human cells the alpha subunit has an molecular mass 110-120 kDa, whereas expression of the extracellular domain of this subunit in Sf9 or COS7 cells produces a protein with an molecular mass of 58 and 75 kDa, respectively(5, 9, 19) . To determine the molecular mass of the mature human alpha subunit protein expressed in mouse L-929 cells, we performed surface iodination experiments. Fig. 1shows an experiment performed with mouse L-929 cells transfected with the alpha subunit cDNA (SVX.2 cells) or with retroviral vector alone (SV.1 cells). We selected the SVX.2 cell line to perform most experiments, because it expresses higher levels of the alpha subunit(9) . Both the IFNaR3 monoclonal antibody and a polyclonal serum(411-557) raised against the cytoplasmic domain of the alpha subunit detect a protein with an molecular mass of approximately 120 kDa in SVX.2 cells, but not in SV.1 cells. This result indicates that in SVX.2 cells the molecular mass of the alpha subunit is similar to that reported for the fully glycosylated form expressed in human cells(6) .


Figure 1: Expression of the alpha subunit in mouse L-929 cells. L-929 cells transfected with the pZipNeo/IFNR construct (SVX.2 cells) or pZipNeo vector (SV.1 cells) were surface-iodinated, and labeled proteins were immunoprecipitated with the anti-alpha subunit mAb IFNaR3 and the rabbit serum 411-557 (against the cytoplasmic domain of the alpha subunit).



It has been reported previously that mouse cells that express the alpha subunit of the Type I IFN-R show an exclusive or more significant antiviral response to human IFNalpha8 than to other Type I IFN subtypes (7, 10, 18) . Table 1and Table 2show that SVX.1 and SVX.2 cells (transfected with the alpha subunit cDNA), but not SV.1 cells (transfected with vector alone), display a more significant antiviral response with human IFNalpha8 than with human IFNalpha2 for either encephalomyocarditis virus or vesicular stomatitis virus.





In order to explain the differences observed with these two human Type I IFNs in mouse cells expressing the alpha subunit, we considered the following possibilities: 1) the alpha subunit is specific for human IFNalpha8; 2) since expression of this subunit in mouse cells does not increase human Type I IFN binding(10) , the ability of a given human Type I IFN to trigger an antiviral response was conditioned by its ability to cross-bind to the mouse receptor.

To test these possibilities, we first studied the ability of the alpha subunit to bind different Type I IFNs in human cells. We affinity-cross-linked radioiodinated IFNalpha2, IFNalpha7, IFNalpha8, IFNbeta, and IFN to the Type I IFN-R expressed in human cells. Fig. 2(A and B) shows that the IFNaR3 mAb precipitates an IFN-receptor complex with an molecular mass of 130-150 kDa (alpha subunit), as well as the high molecular mass complex corresponding to the association of the alpha and beta subunits previously reported(4, 6) . The complexes were more prominent with radioiodinated IFNalpha2, IFNalpha8, and IFNbeta. IFNalpha7 and IFN were cross-linked to the receptor with lower efficiency than other IFNs. However, a weak band corresponding to the alpha subunit cross-linked to radioiodinated IFNalpha7 and IFN is detected by the IFNaR3 mAb. These results suggest that the alpha subunit is not specific for human IFNalpha8 and that it binds different Type I IFNs, in association with other receptor components, when expressed in human cells. Although the alpha subunit-radioiodinated IFN complex (130-150 kDa) resolved in most experiments as a wide band ( Fig. 2IFNalpha2, IFNalpha8, and IFNbeta), in some experiments the alpha subunit also resolved as a doublet (Fig. 3, U-266 cells, and data not shown).


Figure 2: Different radioiodinated Type I IFNs are cross-linked to the alpha subunit. Type I IFNs were iodinated and cross-linked to the Type I IFN-R expressed in the human myeloma cell lines U-266 and H-929. In A two different concentrations of IFNalpha2, IFNalpha7, and IFNalpha8 were used for affinity cross-linking. Immunoprecipitation with an anti-IFNalpha serum was used as a positive control. In B an aliquot of the cross-linked lysate was directly analyzed as positive control for IFNalpha and IFN (left side). The same lysates were used for immunoprecipitation with the anti-alpha subunit mAb IFNaR3 (center). Immunoprecipitations of IFNbeta and IFNalpha (control for that experiment) cross-linked to the alpha subunit are also shown (right side).




Figure 3: Human IFNalpha8 binds to the mouse Type I IFN-R. Radioiodinated human IFNalpha2 and human IFNalpha8 were cross-linked to the receptor expressed in mouse SV.1 and SVX.2 cells. Lysates were precipitated with an anti-IFNalpha serum or with the anti-alpha subunit mAb IFNaR3. Cross-linking of radioiodinated IFNalpha8 to U-266 cell was used as a positive control. The bands corresponding to the alpha and beta subunits are indicated. The difference in electrophoretic mobility observed in SV.1 and SVX.2 for the IFNalpha8-receptor complex are due to an artifact produced when the gel was dried.



We next studied the ability of mouse SV.1 and SVX.2 cells to bind human IFNalpha2 and human IFNalpha8. Since it is not possible to measure human IFNalpha2 or human IFNalpha8 binding in these mouse cell lines(10) , we performed affinity cross-linking experiments. Fig. 3shows that radiolabeled human IFNalpha8 can be cross-linked to the mouse receptor expressed in both SV.1 and SVX.2 cells even in the absence of the human alpha subunit. This is supported by the finding that the IFNalpha8-receptor complexes are precipitated by an antibody against the ligand (anti-IFNalpha serum) and not by the anti-alpha subunit antibody IFNaR3. It should be noticed that very weak precipitation of the human alpha subunit cross-linked to human IFNalpha8 was detected by the IFNaR3 mAb in SVX.2 cells in long exposures of the autoradiograms (data not shown). Interestingly, no detectable amounts of IFNalpha2 cross-linked to the receptor were detected using this method. The detection of the alpha subunit as a doublet in human U-266 cells (positive control) could correspond to the different electrophoretic mobility species previously reported for IFNalpha8 (28) or to the presence of two binding sites in this chain of the receptor as previously proposed by Bazan(29) . The binding of IFNalpha8, but not IFNalpha2, to the mouse cells indicates that the mouse receptor is involved in human IFNalpha8 binding. Thus, the human IFNalpha-induced antiviral response observed in L-929 mouse cells expressing the human alpha subunit is dependent on cross-binding of a particular human Type I IFN to the mouse receptor. In this context, the human alpha subunit provides for species specificity.

Expression of the alpha Subunit and Transmembrane Activation of the JAK Kinases and ISGF3

Binding of Type I IFNs to the receptor triggers tyrosine phosphorylation and activation of the p135 and JAK-1 tyrosine kinases, as well as the transcriptional factor ISGF3. Moreover, p135 directly associates with and tyrosine phosphorylates the alpha subunit(9) . Since the alpha subunit can transduce the signal for an antiviral response, we studied whether this subunit is able to activate the different components of the IFN signaling pathway such as the p135 and JAK-1 tyrosine kinases and consequently the transcriptional factor ISGF3. The SV.1 and SVX.2 cells were treated for 10 min at 37 °C with human IFNalpha8, murine IFNalphabeta, or left untreated. Cell lysates were immunoprecipitated with anti-tyk2 and -JAK-1 sera, the immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis, and tyrosine-phosphorylated proteins were detected by Western blotting with an anti-phosphotyrosine mAb. Fig. 4(upper panel) shows that murine IFNalphabeta can induce rapid tyrosine phosphorylation of the p135 and JAK-1 tyrosine kinases in both L-929 cells transfected with vector alone (SV.1) and with vector containing the alpha subunit cDNA (SVX.2). More importantly, human IFNalpha8 exclusively induces tyrosine phosphorylation of p135 and JAK-1 in SVX.2 cells transfected with the human alpha subunit cDNA. The absence of IFNalpha8-induced tyrosine phosphorylation of the JAK kinases in SV.1 cells indicates that binding of this human IFN to the mouse receptor is not sufficient to activate these kinases. No tyrosine phosphorylation of the JAK tyrosine kinases was observed when human IFNalpha2 was used (data not shown).


Figure 4: Human IFNalpha8 activates p135 and JAK-1 in mouse cells expressing the human alpha subunit. SV.1 and SVX.2 cells were stimulated (10 min. at 37 °C) with murine IFNalphabeta, human IFNalpha8, or left untreated. Cells were lysed, and the lysates immunoprecipitated with the indicated antibodies. Western blots were performed using anti-phosphotyrosine antibodies (anti-pTyr). The position of the p135 protein in the anti-phosphotyrosine immunoblot is indicated by a dash. Blots were then stripped and reprobed with anti-p135 or anti-JAK-1 antibodies (lower panel). Note that in the anti-p135 immunoblot the heavily phosphorylated bands that correspond to p135 in the upper panel show as a ``negative'' image (arrow) probably due to incomplete stripping of the filter.



We also determined if transmembrane signaling by the alpha subunit was enough to activate the transcriptional factor ISGF3. We obtained nuclear extracts from SV.1 and SVX.2 cells after treatment with murine IFNalphabeta, human IFNalpha2, and human IFNalpha8 for 1 h. Human IFNalpha8 specifically induced formation of the ISGF3 complex in SVX.2 cells, but not in SV.1 cells (Fig. 5A), whereas murine IFNalphabeta induced ISGF3 formation in both SV.1 and SVX.2 cells. Fig. 5B shows that the formation of the ISGF3 complex is blocked by a 50-fold excess unlabeled oligonucleotide (Fig. 5B, lane 3), and by polyclonal serum against human Stat1alpha (38) (lane 6), but not by normal rabbit serum (lane 7), further indicating that the complex corresponds to ISGF3. A weak band observed with human IFNalpha2 in SVX.2 cells is a background band that comigrates with ISGF3, but it does not react with anti-ISGF3 antibodies (data not shown).


Figure 5: Human IFNalpha8 induces formation of the ISGF3 complex in mouse cells that express the human alpha subunit of the Type I IFN-R (A). SV.1 and SVX.2 cells were treated as described in the legend to Fig. 4. Nuclear extracts were used for electrophoretic mobility shift assay. The migration of the ISGF3 complex is indicated. B, the formation of the ISGF3 complex is specifically blocked a 50-fold molar excess of unlabeled (Unl.) oligonucleotide, and by an anti-Stat1alpha antiserum (2 µl). Only partial blocking was achieved with the anti-Stat1 serum, because it was raised against human Stat1, and it has a lower affinity for the mouse counterpart. This is supported by the finding that this serum completely inhibits ISGF3 formation in human extracts(38) . Lanes 1 and 4 correspond to nuclear extract from untreated SVX.2 cells; lanes 2, 3, 5, 6, and 7 correspond to nuclear extracts from IFNalpha8-treated SVX.2 cells in the presence of a 50-fold molar excess of unlabeled ISRE (lane 3), 2 µl of anti-Stat1 serum (lane 6), or 2 µl of normal rabbit serum (lane 7).




DISCUSSION

Mouse cells expressing the subunit of the Type I IFN-R cloned by Uzéet al.(7) are protected against vesicular stomatitis virus challenge by human IFNalpha8. This subunit of the receptor corresponds to a receptor component recognized by the IFNaR1, IFNaR2, and IFNaR3 mAbs that we previously designated as the alpha subunit(9) . Expression of this subunit in mouse cells confers an antiviral response to human IFNalpha8, but not to human IFNalpha2 or IFNbeta (7, 10, 18) . However, the increase in antiviral activity is not accompanied by a corresponding increase in human IFN binding(10) . Unlike rodent cells transfected with the alpha subunit, rodent cells carrying human chromosome 21 are able to bind and respond to different human Type I IFNs(30, 31, 32, 33, 34, 35) . As expected, the gene for the alpha subunit is localized on human chromosome 21(36) . The finding that rodent cells transfected with the alpha subunit do not show an increase in Type I IFN binding and only respond to IFNalpha8 suggested that there should be a second receptor component in this region of human chromosome 21. With the help of a new specific monoclonal antibody against what it was designated as the beta subunit of the receptor, we demonstrated that two receptor components are localized on the same area of human chromosome 21(5) . The presence of a second Type I IFN-R component in this human chromosome has been recently confirmed using hamster cells transfected with a yeast artificial chromosome that contains the q22.1 region of human chromosome 21(11, 12) . A cDNA different than the alpha subunit has been recently cloned by Novick et al.(8) . It remains to be confirmed if this cDNA maps to human chromosome 21.

It has not been elucidated why there are differences in the antiviral response to human IFNalpha2, IFNbeta, and IFNalpha8 in cells transfected with the alpha subunit of the Type I IFN-R. One possibility would be that the alpha subunit is specific for IFNalpha8. This idea has been challenged by two findings: 1) a monoclonal antibody produced against the recombinant protein produced in COS7 cells is able to block the biological activity of different Type I IFNs(19) , and 2) the alpha subunit can be cross-linked to IFNalpha2 in human cells(4, 6) . Furthermore, when we performed affinity cross-linking experiments with different radioiodinated Type I IFNs followed by immunoprecipitation with the specific anti-alpha subunit mAb IFNaR3, our results indicated that the alpha subunit can be cross-linked to different Type I IFNs when expressed in human cells. However, similar experiments performed in mouse L-929 cells demonstrated that human IFNalpha8, but not human IFNalpha2, was able to bind to the mouse Type I IFN-R even in the absence of the human alpha subunit. Mouse cells expressing the alpha subunit respond to human IFNalpha8, because this IFNalpha, unlike IFNalpha2, binds to the mouse receptor. In this context the human alpha subunit is necessary for the species specificity of the antiviral response. Our results also indicate that the alpha subunit expressed in mouse L-929 cells has an molecular mass similar to that observed in human cells. This result confirms a previous report (37) that indicated that the alpha subunit is heavily glycosylated.

Since expression of the human alpha subunit in mouse L-929 cells suffices to trigger an antiviral effect in response to IFNalpha8, we sought to determine which components of the IFNalpha signaling pathway were activated by transmembrane signaling through this subunit. We previously reported that p135 associates and tyrosine-phosphorylates the alpha subunit(9, 17) , but we have not detected association of this subunit with JAK-1. Moreover, it has been reported that the JAK-1 kinase associates with a different receptor component recently cloned by Novick et al.(8) . Our results indicate that IFNalpha8 specifically triggers the transmembrane signaling by the alpha subunit with the consequent activation of the p135 and JAK-1 tyrosine kinases. The activation of these kinases was accompanied by formation of ISGF3 as detected by electrophoretic mobility shift assay. Our data further support a direct relationship between activation of the JAK tyrosine kinases, induction of the ISGF3 complex, and antiviral response in mouse cells transfected with the alpha subunit. The presence of functional JAK-1 and p135 kinases appears to be necessary for tyrosine phosphorylation of p135 and JAK-1 in response to IFNalpha, suggesting that these tyrosine kinases may cross-activate each other simultaneously or in a cascade(20) . Since p135 associates with the alpha subunit, the activation by tyrosine phosphorylation of this JAK tyrosine kinase is not surprising. However, JAK-1 does not seem to be activated through the mouse beta subunit as indicated by the lack of tyrosine phosphorylation of JAK-1 in response to IFNalpha8 in cells transfected with vector alone. Thus, tyrosine phosphorylation of the JAK-1 tyrosine kinase should be explained by a different mechanism: 1) JAK-1 is downstream of p135 in the IFNalpha pathway. In this case the activation of p135 leads to the direct or indirect activation of JAK-1. 2) JAK-1 is activated through the alpha subunit in an p135-independent pathway. In this case, the JAK-1 tyrosine kinase may associate to the alpha subunit through an intermediary protein such as the adapters associated to other cytokine receptors. Since binding of human IFNalpha8 to the mouse receptor is involved in the antiviral effect of cells expressing the human alpha subunit, the contribution of each receptor to the IFN response cannot be determined. To answer these questions, we are developing hybrid cytokine receptors in which the binding domain of alpha subunit of the IL2R is linked to the transmembrane and cytoplasmic domain of the alpha subunit of the IFN-R. In this system, IL2 should induce transmembrane signaling exclusively through the cytoplasmic domain of the alpha subunit of the Type I IFN-R.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA55079 (to O. R. C.), R29CA507703 (to N. R.), AI19737 (to P. M. P.), and CA49133 (to M. O. D.) and by a grant from the Department of Veterans Affairs (to L. C. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: University of Tennessee, Dept. of Pathology, 899 Madison Ave., Memphis, TN 38163. Tel.: 901-448-6173; Fax: 901-448-6979.

Recipient of a Career Development award from the American Cancer Society.

(^1)
The abbreviations used are: IFN, interferon; IFN-R, Type I Interferon receptor; ISGF, interferon-stimulated gene factor; mAb, monoclonal antibody.

(^2)
P. Domanski and O. R. Colamonici, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Merrill Kellum for performing the antiviral assays and Drs. P. Trotta (Schering-Plough), M. Brunda (Hoffman-LaRoche), H. Hochkeppel (Ciba-Geigy), D. Gangemi (Clemson University), and G. Adolf (Ernst Boehringer Institute fur Arzneimittelforschung) for different IFN preparations.


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