©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Expression of a Long Form of the Subunit of the Interferon Receptor That Is Required for Signaling (*)

(Received for publication, June 21, 1995; and in revised form, July 14, 1995)

Paul Domanski (1) Michael Witte (1) Merril Kellum (2) Menachem Rubinstein (3) Rebecca Hackett (4) Paula Pitha (2) Oscar R. Colamonici (1)(§)

From the  (1)Department of Pathology, University of Tennessee, Memphis Tennessee 38163, (2)The Johns Hopkins School of Medicine, Baltimore, Maryland 21287, the (3)Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel 76100, and the (4)Food and Drug Administration, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The interferon alphabeta receptor (IFNalphaR) or type I IFN-R is formed by a 110-kDa alpha subunit or IFNAR and by a beta subunit, which has short and long forms (molecular masses of 55 and 95-100 kDa, respectively). In this report, we demonstrate that the IFNalpha/betaR cDNA recently cloned corresponds to the 55-kDa or short form of the beta subunit, while the 95-100-kDa species reported here corresponds to a longer form of the IFNalpha/betaR cDNA that is probably produced by alternative splicing of the same gene. Stable transfection of the alpha subunit with either form of the beta subunit results in the expression of low and high affinity receptors, while expression of either form of the beta subunit alone only produces low affinity receptors. More important, only expression of the alpha and long form of the human beta subunits in mouse L-929 cells reconstitutes the activation of the Jak kinases and the Stat factors, as well as the antiviral response to human type I IFNs.


INTRODUCTION

Characterization of the interferon alphabeta receptor or type I interferon receptor (IFNalphaR or IFN-R) (^1)with monoclonal antibodies (mAb) has revealed that this receptor is composed of at least two chains: the alpha and beta subunits (recognized by the IFNaR3 and IFNaRbeta1 monoclonal antibodies, respectively)(1, 2) . Using mAbs and affinity cross-linking methods, we described two forms of the type I IFN-R: normal and variant(2, 3) . The ``normal'' receptor is expressed in most cells and is composed of alpha and beta subunits with molecular masses of 110 and 100 kDa, respectively. The ``variant'' receptor is expressed in monocytic cell lines and normal bone marrow cells, and in contrast to the ``normal'' receptor, its beta subunit has a molecular mass of 55 kDa(2, 3) . Two cDNAs encoding IFNAR (4) and IFNalphabetaR (5) subunits of the type I IFN-R have been cloned. We have recently demonstrated that the alpha subunit is encoded by the IFNAR cDNA(6) , and its cytoplasmic domain directly interacts with Tyk-2 tyrosine kinase.

The IFNalphabetaR cDNA product (5) has a similar M(r) as the variant form of the beta subunit (2) and is proposed to form a disulfide-bonded dimer that it is only cleaved by high concentrations of reducing agents(5) . The finding that the IFNalpha/betaR cDNA was cloned from a monocytic cDNA library led us to postulate that a longer form of this protein may be expressed in other cell types. In this report, we demonstrate that the IFNalpha/betaR cDNA encodes what was previously designated as the variant or short form of the beta subunit (beta(S))(2) . A long form of the beta subunit (beta(L)) was cloned from a U-266 cDNA library. The beta(L) subunit and the IFNalpha/betaR protein (beta(S)) have identical extracellular and transmembrane domains but only share identitity in the first 15 amino acids of the cytoplasmic domain. Expression of the beta(L) subunit in mouse L-929 cells resulted in a protein with a molecular mass of 100 kDa, while L-929 cells transfected with the beta(S) subunit expressed a protein with a molecular mass of 55 kDa. L-929 cells coexpressing the alpha and beta(L) subunits displayed the affinity cross-linking pattern described for normal receptors, while coexpression of the alpha and beta(S) subunits resulted in the expression of variant receptors. Interestingly, only cells transfected with the long form of the beta subunit were able to tyrosine phosphorylate the Tyk-2 and Jak-1 kinases, activate the ISGF3 and FcRF1/2 transcription factors, and induce an antiviral state in response to human type I IFNs.


EXPERIMENTAL PROCEDURES

Cloning of the Long Form of the beta Subunit

A cDNA library was made with poly(A) RNA obtained from the human myeloma U-266 cell line using a commercial cDNA kit, followed by subcloning into the pcDNA II vector (Invitrogen). Screening was performed using a IFNalpha/betaR cDNA (5) probe. Positive colonies were obtained after three rounds of screening, and the inserts were mapped using restriction endonucleases and polymerase chain reaction. Both strands of the longest cDNA clone (4A1) were sequenced using an automated sequencing machine (373A DNA sequencer, Applied Science Laboratories) by the method of DyeDeoxy Terminator, Cycle Sequencing Kit (Applied). Partial sequences of shorter cDNA clones were also obtained using the same method.

Expression of the Different Type I IFN-R Subunits in Mouse L-929 Cells

Mouse L-929 cells were transfected using Lipofectamine Reagent (Life Technologies, Inc.) with the beta(S), beta(L), and alpha subunits of the human type I IFN-R using the pZ.IFNalphaRbeta(S), pZ.IFNalphaRbeta(L) (corresponding to pZipNeoSVX vector containing the beta(S) and beta(L) subunits, respectively), pR4.IFNalphaRalpha (pREP4 vector containing the alpha subunit), and pcDNAalpha (pcDNA I vector containing the alpha subunit) constructs and selected in medium containing G-418 (500 µg/ml) alone or in combination with hygromycin B (500 µg/ml). Clones were isolated and screened for receptor expression using affinity cross-linking and binding methods (see below). The LpZalpha cell line corresponds to the SVX.2 cell line previously reported(7, 8, 9) , which has been renamed.

IFNs and Antibodies

Human recombinant IFNalpha2 and IFNbeta were kindly provided by Drs. M. Brunda (Hoffman-La Roche) and L. Ling (Biogen Corp, Cambridge, MA), respectively. The antiphosphotyrosine and anti-Jak-1 antibodies were obtained from UBI (Lake Placid, NY). Mouse IFNalpha/beta was purchased from Lee Biochemicals. The mAbs against the alpha and beta subunits of the type I IFN-R (IFNaR3 and IFNaRbeta1) and the rabbit sera anti-Tyk2 have been previously described(1, 2, 6) .

Radioiodination of Type I IFNs, Competitive Displacements, and Affinity Cross-linking

Radioiodination of IFNalpha2, competitive displacement assays, and affinity cross-linking procedures were performed as previously described(1, 2, 3) .

Immunoblotting

Cells were treated with different concentrations of the indicated IFNs for 15 min, rapidly centrifuged at 2000 times g for 30 s in an Eppendorf microfuge, 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.). The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene dilfluoride filters (Immobilon, Millipore). 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 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).

Electrophoretic Mobility Shift Assay (EMSA)

Whole cell extracts were prepared as described (10) and analyzed by EMSA using end-labeled oligonucleotides encoding ISRE (5`-GATCGGGAAAGGGAAACCGAAACTGAAGCC-3`) (11) and GRR (5`-AGCATGTTTCAAGGATTTGAGATGTATTTCCAGAAAAG-3`(12) .


RESULTS AND DISCUSSION

Cloning of the Long Form of the beta Subunit

To determine whether the IFNalphabetaR/pCEV-m6 cDNA (5) product was recognized by the IFNaRbeta1 mAb(2) , we subcloned this cDNA into the retroviral vector pZipNeoSV(X), and the resulting construct (pZ.IFNalphaRbeta(S)) was transfected into mouse L-929 cells. Stable transfectants were selected in G-418 (500 µg/ml) and tested for reactivity with the IFNaRbeta1 mAb using flow cytometric analysis and binding studies with radioiodinated IFNalpha2. Almost 90% of the clones studied showed strong positivity for the IFNaRbeta1 mAb. This mAb also blocked binding of radioiodinated human IFNalpha2 to these transfectants, demonstrating that the protein encoded by the IFNalphabetaR cDNA corresponds to the previously designated variant form of the beta subunit of the type I IFN-R (data not shown). It was also reported that the IFNalphabetaR protein forms a dimer with an approximate molecular mass of 100 kDa that is only cleaved by high concentrations of reducing agents (100 mM dithiothreitol)(5) . To determine if the 100-kDa form of the beta subunit corresponds to a dimer, lysates obtained from surface-iodinated U-266 cells were immunoprecipitated with the IFNaRbeta1 mAb, and the samples were treated with high concentrations of reducing agents. Even after treatment with 250 mM dithiothreitol, the IFNaRbeta1 mAb recognized a 100-kDa protein (data not shown). These results suggested that the 100-kDa form of the beta subunit does not correspond to a dimer and that there may be long and short forms of this subunit as we previously proposed(2) . Our hypothesis was also supported by the fact that monocytic cell lines express this form of the beta subunit (2, 3) and that the IFBalphabetaR cDNA was obtained from a monocytic library(5) .

To search for a putative long form of the beta subunit, we screened a human myeloma U-266 cDNA library using the IFNalphabetaR cDNA as the probe. We initially screened 200,000 colonies from which six positive clones were obtained. Restriction endonuclease mapping revealed that some inserts contained a BamHI site not present in the IFNalphabetaR cDNA. Thus, a BamHI-XbaI fragment (the XbaI restriction site is contained in the polylinker of the pcDNA II vector) was used for a second round of screening where 13 additional clones were obtained. Sequencing of the longest clone (4A1, 2636 bp) revealed an open reading frame of 515 amino acids (nucleotides 67-1611), a polyadenylation signal (nucleotides 2295-2299), and a poly(A) tail (nucleotide 2628) (Fig. 1A). Alignment of the predicted amino acid sequence from clone 4A1 with the IFNalphabetaR protein revealed identical amino acid sequences from residues 1-280, resulting in identical extracellular and transmembrane domains but sharing identity in only the first 15 amino acids of the intracellular region (amino acids 265-280). Thus, the open reading frame encoded by clone 4A1 predicts a cytoplasmic domain of 251 amino acids (residues 265-515), while the form of the beta subunit encoded by the IFNalphabetaR cDNA has a cytoplasmic region of 67 amino acids (265-331). For simplicity and to follow the designation with Greek letters used for other cytokine receptors, we will refer to the IFNalphabetaR and 4A1 cDNAs as short (beta(S)) and long (beta(L)) forms of the beta subunit, respectively. Northern blot analysis (Fig. 1C) with a probe corresponding to the cytoplasmic domain of beta(L) detected mRNAs of 1.85 and 4.4 kilobases similar to what it was previously reported for beta(S) (Fig. 1C and (5) ). Partial sequencing of another clone (clone 1A1) demonstrated that this cDNA had the same coding region as the beta(S) form previously reported(5) ; however, 1A1 was preceded by a slightly longer 5`-untranslated region. The majority of the clones sequenced were similar to the 4A1 clone, suggesting that beta(S)-coding transcripts may be only a fraction of the beta receptors transcribed in U-266 cells. The differences in the size of the 4A1 clone (2636 base pairs) and the mRNA observed in Northern blots (4.4 kilobases) suggest that the coding region may be preceded by a longer 5`-untranslated region than that present in the 4A1 clone. Fig. 1C also shows that a probe encoding the cytoplasmic region of clone 4A1 recognizes the mouse homolog (Fig. 1C, 3T3T cells). This is not surprising, since the human beta(L) has the ability to interact with the mouse signaling proteins (see below). It is likely that the different forms of the beta subunit correspond to alternatively spliced transcripts from the same gene. Searching of the data bases using the BLAST computer program did not show significant similarities between the distinct region of the cytoplasmic domain of beta(L) and any known proteins. However, multiple alignment with other members of the cytokine receptor superfamily, using the computer program MACAW(13) , revealed that beta(L) but not beta(S) contains a cytoplasmic sequence proximal to the transmembrane domain that resembles the BOX 1 motif observed in most cytokine receptors (Fig. 1A, boxedsequence). We also found six regions with high content of acidic residues whose significance is under investigation (Fig. 1A, shadedsequences).



Figure 1: Sequence of the long form of the beta subunit of the type I IFN-R. A, the nucleotide (top) and amino acid (bottom, three-letter code) sequences for the long form of the beta subunit are shown. The polyadenylation signal (doubleunderlined), the point where the beta(S) and beta(L) sequences are similar (between arrows), the transmembrane region (thickunderline), the box 1 motif (boxed), and the acidic domains (shadedareas) are indicated. B, schematic representation of the IFNalphabetaR (homologous to clone 1A1) and 4A1 clones. The identical (thicksolidline) and different parts of the 5`- and 3`-untranslated regions (UTR) of beta(S) (thinsolidline) and beta(L) (dashedline) as well as the differences in the cytoplasmic domain are shown. C, Northern blot analysis using probes encoding the specific cytoplasmic regions of clone 4A1 and IFNalphabetaR cDNA. Total RNA (20 µg) was used for Northern blot analysis. kb, kilobases.



Expression of the Different Forms of the Human beta Subunit in Mouse L-929 Cells

The predicted molecular mass of the protein encoded by clone 4A1 is 57,674 Da, while the long form of the beta subunit expressed in most cell types has a molecular mass of 95-100 kDa. To determine whether the 4A1 clone encodes the 100-kDa form of the beta subunit and to study the contribution of each form of the beta subunit to the formation of the type I IFN-R, we stably expressed beta(S) and beta(L) alone and in association with the alpha subunit in mouse L-929 cells. Fig. 2A shows that transfection of beta(L) alone (LpZbeta(L) cells) results in the expression of a 95-100-kDa IFNalpha2 binding protein (these molecular masses do not include 20 kDa corresponding to one molecule of I-IFNalpha2 cross-linked to the receptor). Therefore, the 4A1 clone encodes the beta(L) subunit of the type I IFN-R, and 40-50 kDa of the molecular mass of this subunit correspond to post-translational modifications, i.e. glycosylation. For reasons that remain to be elucidated, cross-linking of radioiodinated IFNalpha2 to either beta(L) or beta(S) (data not shown) has reproducibly resulted in weaker signals than when these subunits are coexpressed with the alpha subunit (see below). Coexpression of beta(L) with the alpha subunit results in the affinity cross-linking pattern observed in human U-266 cells (``normal'' receptor) characterized by alpha and beta(L) subunits with molecular masses of 110 and 100 kDa, respectively (Fig. 2, A and B, alpha + beta(L) and LpZRalphabeta(L).10, respectively), and complexes that correspond to the association of the alpha and beta(L) subunits (Fig. 2B, alpha + beta(L)). Expression of the alpha and beta(S) subunits resulted in proteins with molecular masses of 110 (alpha subunit) and 55 kDa (beta(S)) and a complex that corresponds to the association of alpha and beta(S) subunits similar to those previously described for ``variant'' receptors (3) (Fig. 2B, LpZCalphabeta(S).4 and LpZRalphabeta(S).11). Fig. 2B also shows that neither the beta(S) nor beta(L) protein is recognized by the anti-alpha subunit antibody IFNaR3 (Fig. 2, LpZCalphabeta(S).4, LpZRalphabeta(S)11, LpZRbeta(L)). U-266 cells express predominantly the normal form of the receptor, although variant beta(S) alone and the association of the alpha and beta(S) subunits can be observed in long exposures of the autoradiograms (Fig. 2B).


Figure 2: Expression of the different forms of the beta subunit of the type I IFN-R. A, mouse L-929 transfectants were produced as described under ``Experimental Procedures.'' LpZbeta(L) cells correspond to L-929 cells transfected with the beta(L) subunit. LpZCalphabeta(S).4 and LpZRalphabeta(S).11 correspond to mouse L cells transfected with the alpha and beta(S) subunits, while LpZRalphabeta(L).10 cells were transfected with the alpha and beta(L) subunits. Affinity cross-linking was performed as previously described(9) . The high molecular weight complexes observed in U-266 cells and in L-929 transfected with the alpha and either beta(S) or beta(L) subunits correspond to associations of these subunits. The human alpha subunit is specifically detected by the IFNaR3 mAb in cells cotransfected with the human alpha and either beta(S) or beta(L) subunits. Cross-linking of I-IFNalpha2 to beta(S) in cells transfected with beta(S) alone always resulted in faint bands even after long exposures of the autoradiograms (data not shown).



We next studied the contribution of the different subunits to the formation of the low and high affinity receptors. Table 1shows that transfection of either beta(S) or beta(L) results in the expression of only low affinity IFNalphaR (K(d) of approximately 450 pM). Cotransfection of either form of the beta subunit with the alpha subunit results in low and high affinity receptors (K(d) values of 1.4-6.5 nM and 26-114 pM for low and high affinity receptors, respectively) similar to those observed in U-266 cells(14) . As previously reported, the sole expression of the alpha subunit did not produce detectable IFNalpha binding(7) . Thus, either form of the beta subunit determines the low affinity binding, while the alpha subunit, unable to bind ligand independently, joins together with the beta subunit in the formation of high affinity receptor complexes.



Role of the alpha, beta, and beta Subunits in Type I IFN Signaling

To determine the contribution of each subunit to IFNalpha signaling, we analyzed activation of the Jak-1 and Tyk-2 kinases, binding of Stat factors to the ISRE and GRR sequences present in the ISGs, and induction of an antiviral state by human type I IFNs. Human IFNalpha2 treatment of murine L-929 cells expressing the alpha or beta(S) subunits alone does not induce tyrosine phosphorylation of the Jak kinases above basal levels (Fig. 3A, lanes6 and 12, (9) , and data not shown), while these kinases can be activated by murine IFNalphabeta (lanes5 and 11). Transfection of beta(L) alone into mouse L-929 cells results in an increase in human IFNalpha2-induced tyrosine phosphorylation of the Tyk-2 and Jak-1 kinases in two independent clones (Fig. 3A, clones pZbeta(L) and pZRbeta(L).5, lanes3, 9, 15, and 21). The levels of human IFNalpha2-induced tyrosine phosphorylation observed in pZbeta(L) and pZRbeta(L).5 cells are similar to those observed with murine IFNalphabeta (lanes2, 8, 14, 20). However, human IFNalpha2-induced tyrosine phosphorylation of the Jak kinases reaches higher levels than murine IFNalphabeta when beta(L) is coexpressed with the alpha subunit (lanes18 and 24). In contrast, only low levels of activation of the Jak kinases (not reproducible in all experiments) are achieved in human IFNalpha2-treated L-929 cells cotransfected with the alpha and beta(S) subunits (lanes27 and 30). The lower levels of response are not due to an alteration in IFNalpha signaling since these cells respond to murine IFNalphabeta (lanes26 and 29).


Figure 3: Contribution of the different subunits of the type I IFN-R to signaling. A, anti-phosphotyrosine immunoblotting. Mouse L-929 cells transfected with the different subunits of the type I IFN-R were treated for 15 min with human IFNalpha2, murine IFNalphabeta, or left untreated. Immunoblotting was performed with the antiphosphotyrosine mAb 4G10 (UBI). Low levels of basal Jak-1 kinase activation are observed in most transfectants. EMSA with GRR (B) and ISGF3 (C) probes is shown. Whole cell extracts were obtained from mouse L-929 transfectants treated with 6,000 units/ml of IFNalpha2 or murine IFNalphabeta or left untreated. D, EMSA was performed using GRR and ISRE probes (12) in the presence of a 50-fold excess of unlabeled GRR or ISRE oligonucleotides. A dilution of the indicated anti-Stat sera was used for supershifts. The positions of FcRF1, FcRF2, ISGF3, and free GRR and ISRE probes are indicated.



We have recently reported that the cytoplasmic domain of beta(L) but not beta(S) interacts with the Jak-1 tyrosine kinase, as well as the Stat1 and Stat2 transcription factors. (^2)To determine whether activation of the Jak kinases was accompanied by formation of the FcRF1/2 and ISGF3 complexes, whole cellular extracts were prepared from the various L-929 transfectants treated with human IFNalpha2, murine IFNalphabeta, or left untreated. The IFNalpha-dependent activation of the Stat1 and Stat2 transcriptional regulators was assessed by EMSA with probes encoding the GRR present in the Fc1 receptor gene (Fc receptor for IgG) (12, 16) and the ISRE(17, 18, 19) . Fig. 3B shows that high levels of FcRF1/2 and ISGF3 complexes are observed in mouse L-929 cells transfected with beta(L) alone or cotransfected with the alpha and beta(L) subunits (Fig. 3, B and C, lanes6, 12, and 15). No retardation of the GRR or ISRE probes in response to human IFNalpha2 treatment was observed in cells transfected with the alpha or beta(S) subunits alone or in combination (Fig. 3, B and C, lanes3 and 9, and data not shown). However, these cells did show retardation of the GRR and ISRE probes after treatment with murine IFNalphabeta (Fig. 3, B and C,lanes2 and 8). FcRF1/2 and ISGF3 DNA binding activity were blocked by an excess of unlabeled GRR and ISRE oligonucleotides (Fig. 3D, lanes6 and 12), respectively. The FcRF1/2 complexes were supershifted by anti-Stat1 sera, indicating the presence of Stat1 in these complexes. Similarly, the ISGF3 complex was supershifted by the anti-Stat1 sera and to a lesser extent by the anti-Stat2 sera (Fig. 3D, lanes4, 10, and 11). These results demonstrate that the presence of beta(L) is required for the formation of the FcRF1/2 and ISGF3 complexes and confirms our previous observation indicating that only beta(L) docks the Stat1 and Stat2 transcription factors.^2

Since the corollary of the IFNalpha response is the induction of an antiviral state, we tested the ability of human type I IFNs to induce the antiviral response in different transfectants. Table 2shows that only the mouse L-929 cells expressing human beta(L) subunit (alone or in combinantion with the alpha subunit) respond to the antiviral effect of human IFNalpha2 and human IFNbeta. The lack of response to the antiviral effects of human IFNalpha2 or human IFNbeta in L-929 cells transfected with either alpha or beta(S) subunit alone or cotransfected with the alpha and beta(S) subunits is not due to defects in the initial steps of the signaling pathway, since both the activation of the Jak kinases and DNA binding activity were detected in cells treated with murine IFNalphabeta.



Our results clearly demonstrate that expression of the beta(L) subunit is required for type I IFN-induced antiviral activity. However, activation of the alpha subunit must be required as indicated by the finding that knockout mice lacking the alpha subunit fail to elicit antiviral responses(20) . Thus, we are cautious in the interpretation of the results obtained in mouse cells expressing only the human beta(L) subunit, since we cannot rule out that after binding of human IFNalpha2 and IFNbeta to the beta(L) subunit these IFNs may also interact and activate the mouse alpha subunit. This is supported by the finding that in cells transfected with either beta(S) or beta(L) alone, a weak band with electrophoretic mobility similar to the alpha subunit, but not recognized by the IFNaR3 mAb (specific for the human alpha subunit), is cross-linked to human IFNalpha2 (data not shown).

The results presented here provide novel information concerning the structure and function of the type I IFN-R. First, we demonstrate that the product of the IFNalphabetaR cDNA corresponds to the previously defined beta subunit of the type I IFN-R. Second, there are two forms of the beta subunit probably derived from an alternative splicing of the same gene. Thus, the 100-kDa form of the beta subunit is encoded by its own mRNA, and it is not a result of the dimerization of the IFNalphabetaR (beta(S)) protein as suggested by Novick et al.(5) . Both forms of the beta subunit have identical extracellular and transmembrane domains but share only the first 15 amino acids of the cytoplasmic domains. The long form of the beta subunit also contains the box 1 motif present in other cytokine receptors (Fig. 1A) and has the ability to interact with Jak-1, Stat1, and Stat2.^2 We propose to designate the different forms of the beta subunit according to their size as beta(L) (515 amino acids) and beta(S) (331 amino acids). Third, expression of the different forms of the beta subunit alone or in association with the alpha subunit demonstrated that the beta subunit is the binding subunit, while the alpha subunit is necessary to form high affinity receptors. Fourth, beta(L) is absolutely required for the IFNalphabeta response. However, it should be noted that despite the fact that beta(L) alone can activate the signaling pathway and elicit an antiviral state in mouse cells, both the alpha and beta(L) subunits are probably required for IFNalpha and IFNbeta signaling as indicated by knockout experiments(20) .

Although expression of beta(S) alone or in association with the alpha subunit is not sufficient to induce an antiviral state in response to type I IFNs, this does not rule out the possibility that beta(S) plays a role in type I IFN signaling by forming a complex with the alpha and beta(L) subunits. In this scenario, it is possible that L-929 cells cotransfected with the human alpha and beta(L) subunits may use the mouse beta(S) chain in the formation of the receptor complex. Coexpression of the human alpha, beta(S), and beta(L) subunits in mouse L-929 cells and reconstitution of the human type I IFN-R in knockout mice for the different receptor subunits will help to address these questions.

The formation of the receptor complex is likely to involve not only the receptor subunits but also the Jak kinases and Stat factors. Several lines of evidence support this concept. First, mutant cell lines that lack the expression of the Tyk-2 (21, 22) and Jak-1 (23) kinases showed impaired binding for Type I IFNs. Second, it has been demonstrated that there is a direct interaction between the Tyk-2 and Jak-1 kinases with the alpha and beta(L) subunits, respectively ( (5) and (6) and data not shown). Finally, the beta(L) subunit constitutively docks the Stat1 and Stat2 transcription factors.^2 Mutational analysis of the intracellular domains of the different receptor subunits will define the domains required for this interactions and type I IFN signaling.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA55079 (to O. R. C.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U29584[GenBank].

§
To whom correspondence should be addressed: Dept. of Pathology, University of Tennessee, 899 Madison Ave. M-576, Memphis, TN 38163. Tel.: 901-448-6173; Fax: 901-448-6979.

(^1)
The abbreviations used are: IFNalphaR, interferon alpha receptor; IFN, interferon; Stat, signal transducer and activator of transcription; mAb, monoclonal antibody; ISRE, interferon-stimulated response element; GRR, IFN response region; EMSA, electrophoretic mobility shift assay.

(^2)
P. Domanski, H. Yan, M. M. Witte, J. J. Krolewski, and O. R. Colamonici, submitted for publication.


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