Identification of a Domain in the beta  Subunit of the Type I Interferon (IFN) Receptor That Exhibits a Negative Regulatory Effect in the Growth Inhibitory Action of Type I IFNs*

Leonidas C. PlataniasDagger , Paul Domanski§, Owen W. Nadeau§, Taolin Yi, Shahab UddinDagger , Eleanor Fishpar , Benjamin G. Neel**, and Oscar R. Colamonici§Dagger Dagger

From the Dagger  Section of Hematology/Oncology, University of Illinois at Chicago, and West Side Veterans Affairs Hospital, Chicago, Illinois 60607, the § Department of Pathology, University of Tennessee, Memphis, Tennessee 38163, the  Department of Cancer Biology, The Cleveland Clinic Foundation Research Institute, Cleveland, Ohio 44195, the par  Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada, and the ** Cancer Biology Program, Division of Hematology-Oncology, Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02215

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of human alpha  and long form of the beta  (beta L) subunits of type I interferon receptor (IFN-R) in mouse cells is sufficient to activate the Jak-Stat pathway and to elicit an antiviral state in response to human IFNalpha 2 and IFNbeta . We demonstrate herein, however, that these cells respond to the antiproliferative effects of murine IFNalpha beta but not human type I IFNs. These results suggest that an unknown species-specific component is required for the antiproliferative effect of human type I IFNs. The absence of this component can be complemented by expressing the human beta L chain truncated at amino acid 346. Thus, the distal region of beta L appears to function as a negative regulator of the growth inhibitory effects of type I IFNs. Further studies looking for possible targets of the beta L regulatory domain demonstrated that this region associates with a tyrosine phosphatase. These results suggest that a protein associated with the negative regulatory domain of beta L, likely a tyrosine phosphatase, plays a role in regulating the growth inhibitory effects of human type I IFNs.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The most prominent effects of type I interferons (IFN)1 are the antiviral and antiproliferative actions (1). These effects are mediated through binding to the type I interferon receptor (IFN-R or IFNalpha R), which is composed of two subunits termed alpha , or IFNAR1, and beta , or IFNAR2 (2-10). The genes encoding the different subunits of the type I IFN-R are clustered in the q22.1 region of human chromosome 21 (6, 7, 11-16). This region also harbors an orphan class II cytokine receptor, the CRFB-4 gene, which is encoded on human chromosome 21 between the genes for the beta  chain of the IFNalpha R and the beta  subunit of the IFNgamma R (10, 17). Expression of the human alpha  and long form of the beta  chain (beta L) subunits in mouse L-929 cells fully reconstitutes the activation of the Jak-Stat pathway and the induction of an antiviral state in response to HuIFNalpha 2 and HuIFNbeta (9). Furthermore, only the first 82 amino acids of the cytoplasmic domain of the beta L chain are required to activate the Jak-Stat pathway and induce the antiviral effect in response to IFNalpha 2 (18).

The alpha  and beta  subunits of the type I IFN-R associate with protein tyrosine kinases of the Jak family (4, 8, 18). The alpha  subunit interacts with Tyk2 (4, 19, 20) while the cytoplasmic domain of the beta L contains a docking site for Jak1 (18). Binding of type I IFNs to their receptor triggers rapid tyrosine phosphorylation of Tyk2 and Jak1 kinases, type I IFN-R subunits (21-25), and Stat factors (reviewed in Refs. 26-28). Regulation of tyrosine kinase activity is mediated in most cytokine systems by protein tyrosine phosphatases (PTPs). For example, SHP1 (also named SHP, SHPTP1, HCP; PTP1C, Ref. 29), a predominantly hematopoietic tyrosine phosphatase that regulates the activity of the erythropoietin and IL-3 systems (30-35), has also been implicated in IFNalpha signaling in hematopoietic cells (36, 37). However, the role of SHP1 in other cell types is not clear since this PTP is mainly expressed in hematopoietic cells, whereas the IFN system functions in almost all, if not all, cell lineages.

Mouse L-929 cells that coexpress wild-type human alpha  and beta  subunits respond to the antiviral effects of human type I IFNs, demonstrating the presence of functional human type I IFN-R (9). We therefore decided to test these cells for their ability to respond to the antiproliferative effects of type I IFNs. Human IFNalpha and IFNbeta induced only a minimal antiproliferative response, whereas murine type I IFNs produced a marked inhibition of cell proliferation. These data indicate that (i) induction of the antiproliferative and antiviral responses occurs through partially divergent pathways and (ii) that a novel species-specific signaling component is required, in addition to the alpha  and beta L chains, for the growth inhibitory effect. Surprisingly, the antiproliferative response was observed in cells that express the human beta L chain truncated at amino acid 346. Thus, the distal part of beta L apparently contains a negative regulatory domain that controls the growth inhibitory effects of type I IFNs. Further characterization of this negative regulatory domain revealed that it interacts with a PTP that appears to be distinct from SHP1 and SHP2. The data herein suggest that a novel species-specific component is required for the growth inhibitory effect and that this effect is regulated by a distal region corresponding to amino acids 346-417 on the beta  subunit.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

IFNs and Antibodies-- Human recombinant IFNalpha 2 was kindly provided by Drs. M. Brunda (Hoffmann-La Roche) and Paul Trotta (Schering-Plough). The anti-phosphotyrosine antibody (4G10) was obtained from UBI (Lake Placid, NY). The polyclonal antibodies against the alpha  (alpha 511-557, IFNaRC-1), beta S (IFNaRC-2), and beta L (beta L265-515, and beta L375-515) subunits of the type I IFN-R, as well as the rabbit sera anti-Tyk2 have been previously described (4, 38, 39). The anti-SHP1 and -SHP2 sera used for immunoprecipitations and Western blotting were purchased from UBI, Santa Cruz Biotechnologies, and Transduction Laboratories. The anti-JAK1 sera was kindly provided by Dr. J. N. Ihle (St. Jude's Children's Hospital, Memphis, TN).

Constructs and Expression of the Human Type I IFN-R Subunits in Mouse L-929 Cells-- Mouse L-929 cell lines coexpressing different constructs of the human alpha  and beta L chains LpZRalpha beta L, LpRZalpha beta L462, LpRZalpha beta L417, LpRZalpha beta L346, and LpZRalpha beta L300 were described previously (9, 18). The L-929 transfectants stably coexpressing mutations of tyrosine 466 and truncation at amino acid 511 of the alpha  subunit (designated as alpha Y1F511) with either wild type or truncation 346 of the beta L subunit (LpZRalpha Y1F(511)beta Lwt and LpZRalpha Y1F(511)beta L346, respectively), as well as coexpressing wild-type alpha  chain and beta L subunit carrying a mutation of tyrosine 411 to phenylalanine, are described elsewhere.2

Cell Proliferation Assays-- Cell proliferation was assessed by performing MTT assays (7, 40) and cell counts after treatment with the indicated amount of human and mouse IFNs. Briefly, cells were seeded at 6,000 cells/well in 24-well plates in a final volume of 0.6 ml and treated with the indicated concentrations of IFNs. The numbers of cells per well were determined by trypsinization and counting of duplicate wells in a hemocytometer. Experiments were performed at least twice with two independent clones carrying the same mutation.

Immunoblotting-- Cells were treated with different concentrations of the indicated IFNs for 15 min, rapidly centrifuged at 2000 × g for 30 s in an Eppendorf microfuge, and subsequently solubilized in lysis buffer. Immunoprecipitation and immunoblotting were performed as described previously (4).

Phosphatase Assays-- For protein phosphatase assays, cells expressing wild-type alpha  chain and the beta L subunit truncated at the indicated amino acids were treated with or without IFNalpha 2 for 10 min at 37 °C and lysed in lysis buffer as described previously (4). The beta L subunit was precipitated using a polyclonal serum raised against a GST fusion protein encoding the entire cytoplasmic domain (beta L265-515) (39), immunoprecipitates were washed three times in cold phosphatase buffer to remove phosphatase inhibitors. The phosphatase activity of the immunocomplexes was determined using pNPP (Sigma) as a substrate. The phosphatase assay was carried out at 37 °C for 0.5 h in 50 µl of reaction mixture (100 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10 mM pNPP). The reaction was terminated by adding 950 µl of 1 M NaOH. The reaction product, p-nitrophenolate, was quantified by measuring absorbance at 405 nm.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of the Human alpha  and beta L Subunits of the Type I IFN-R Is Not Sufficient to Reconstitute the Antiproliferative Response-- Mouse L-929 cells transfected with the human alpha  and beta L subunits, LpZRalpha beta L.10, activate the Jak-Stat pathway and are highly responsive to the antiviral effects of HuIFNalpha 2 and HuIFNbeta (9). To characterize the antiproliferative effect of type I IFNs in these cells, we first performed MTT proliferation assays using L-929 cells stably transfected with wild-type human alpha  and beta L subunits (LpZRalpha beta L.10). Fig. 1A shows that high doses of HuIFNalpha 2 (100,000 units/ml) had little effect on cell proliferation. However, treatment with MuIFNalpha beta or MuIFNbeta at doses between 1,000 and 10,000 units/ml induced a significant antiproliferative effect. To confirm the results observed with MTT assays, we performed similar experiments in which cell numbers were assessed. Fig. 1B shows that treatment of LpZRalpha beta L.10 cells (Fig. 1B, alpha beta L cells) with MuIFNalpha beta reduced cell proliferation more than 95% over a period of 6 days, whereas HuIFNalpha 2 produced a minimal response (20%, from 545,000 cells/well to 445,000 cells/well in control and HuIFNalpha 2-treated cells, respectively). Similar results were observed when MuIFNbeta and HuIFNbeta were used (data not shown). These results indicate that the endogenous mouse type I IFN-R expressed in L-929 cells can trigger a complete antiproliferative effect in response to mouse IFNs. Thus, in contrast to the antiviral effect, reconstitution of the human receptor with the alpha  and beta L chains in these cells is not sufficient to trigger an antiproliferative effect in response to human type I IFNs, suggesting that an additional human signaling component is required for this effect.


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Fig. 1.   The human alpha  and beta L subunits are not sufficient to restore the antiproliferative effect of type I IFNs. Cells expressing wild-type alpha  and beta L chains (alpha beta L cells) of the type I IFN-R were treated with the indicated doses of HuIFNalpha 2, MuIFNalpha beta , or MuIFNbeta . A, cell proliferation was assessed using MTT assays. B, a similar experiment assessing cell numbers (see "Materials and Methods"). Cells were treated with 10,000 units/ml of HuIFNalpha 2 or 3,000 units/ml of MuIFNalpha beta . C, effect of MuIFNalpha beta and HuIFNalpha 2 in the proliferation rate of A9+21 cells. Cells were treated as described in panel B.

It has been reported that human/rodent somatic cell hybrids carrying human chromosome 21 acquire the ability to respond to the antiviral effect and induction of HLA class I antigens by human type I IFNs (7, 11-16, 41-45). This is in part due to the fact that the alpha , beta S, and beta L subunits of the receptor are encoded by genes on this chromosome. However, it has also been reported that additional IFN signaling components may also reside on the distal part of human chromosome 21q (46, 47). Therefore, we tested mouse A9+21 cells, which carry several copies of human chromosome 21, for their ability to respond to the antiproliferative effects of HuIFNalpha 2 and HuIFNbeta . Fig. 1C shows a proliferation assay performed with HuIFNalpha 2 and MuIFNalpha beta . An 85% reduction in the growth occurred for A9+21 cells in response to MuIFNalpha beta , whereas only a 40% decrease (from 165,000 cells/well to 100,000 cells/well in controls and HuIFNalpha 2-treated cells, respectively) in cell proliferation was induced by HuIFNalpha 2. These data show that binding of human type I IFNs by human alpha  and beta L chains expressed in mouse cells (either by transfection in L-929 cells or by incorporation of human chromosome 21 in A9+21 cells) induces only a partial growth inhibitory response. Since human/rodent somatic cell hybrids carrying human chromosome 21, including A9+21 cells, have been shown to develop an antiviral state and induce HLA class I antigens in response to human type I IFNs (7, 11-16, 41-45), the inability of these cells to respond to the antiproliferative effects of human type I IFNs further suggests that an additional species-specific signaling component is required. Moreover, it is unlikely that this signaling element is encoded by any genes present on human chromosome 21.

Deletion of a Negative Regulatory Region of the beta L Chain Promotes the Growth Inhibitory Effects of Human Type I IFNs-- Since deletion of the distal region of the alpha  chain produces an increase in HLA class I response induced by human IFNalpha 2 (48), we wished to determine whether the antiproliferative response could be induced by human IFNs in L-929 cells that express alpha  and beta L subunits with deletions of various regions of their cytoplasmic domains. Cells that express the beta L chain truncated at amino acid 417 are more responsive to MuIFNalpha beta than to HuIFNalpha 2 (Fig. 2A, panel A). These results parallel our findings for L-929 or A9+21 cells, which express wild-type receptor subunits (Fig. 1, B and C). By direct contrast, L-929 cells coexpressing both the wild-type alpha and beta L truncated at amino acid 346 (Fig. 2, panels B, alpha beta L346.2 cells), responded to the growth inhibitory effects of human type I IFNs. Truncation of the cytoplasmic domain of beta L at amino acid 300, which removes the Jak1 binding site (18), abolished the antiproliferative response to human type I IFNs but did not affect the antiproliferative response to MuIFNalpha beta (Fig. 2C), demonstrating that the mouse signaling machinery is intact in these cells. We also studied L-929 transfectants expressing wild-type beta L and an alpha  chain with a deletion of the negative regulatory domain (truncation at amino acid 511 and tyrosine 466 mutated to phenylalanine; Fig. 2, panel D, alpha Y1Fbeta L.11 cells) (48). These cells showed a significant response to MuIFNalpha beta , whereas human type I IFNs induced only a partial antiproliferative effect, which was similar to that observed in cells expressing wild-type receptors. This result suggests that the negative regulatory region of the alpha  subunit does not control cell proliferation (48). However, L-929 cells coexpressing the same mutations of the alpha  subunit and beta L truncated at amino acid 346 (Fig. 2, panel E, alpha Y1F511beta L346.3) were extremely sensitive to the antiproliferative effect of human and mouse type I IFNs. Similar results were obtained with two independent clones carrying the same mutation (data not shown). These data strongly suggest that a region corresponding to amino acids 346-417 in the beta L chain contains a negative regulatory domain and may be a possible target for mouse regulatory proteins. Thus, removal of this negative regulatory domain appears to complement the absence of an unknown species-specific component required for the antiproliferative effect (see "Discussion").


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Fig. 2.   Deletion of the distal region of the human beta L chain restores the ability of mouse cells to respond to the antiproliferative effect of human type I IFNs. Mouse L-929 cells coexpressing different constructs of the alpha  and beta L subunits were evaluated for their response to the antiproliferative effect of human and murine IFNs. alpha beta L417, alpha beta L346, and alpha beta L300 correspond to clones expressing wild-type alpha  subunit and truncations at residues 417, 346, or 300 of the beta L chain, respectively. alpha Y1Fbeta L clones express wild-type beta  chain and alpha  subunit with a deletion of the negative regulatory domain (truncated at amino acid 511 and mutation of Tyr-466 to phenylalanine). alpha Y1Fbeta L346 clones express the same mutations of the alpha  subunit (truncation 511 and mutation of Tyr-466 to phenylalanine) and beta L truncation at amino acid 346. alpha beta LY411F cells correspond to L-929 cells expressing wild-type alpha  chain and beta L with a mutation of Tyr-411 to phenylalanine. Proliferation was assessed as described in Fig. 1.

The beta L Subunit of the Type I IFN-R Associates with a Phosphatase-- Tyrosine phosphorylation plays a central role in IFN and cytokine signaling. Therefore, to determine if the negative regulatory domain of beta L was associated with a PTP, we performed in vitro phosphatase assays after immunoprecipitation with anti-beta L sera. As a source of beta L chain, we used cell lysates obtained from mouse L-929 cells cotransfected with the wild-type alpha  subunit and truncations of the beta L chain at amino acids 346, 417, or 462, respectively. Cells were treated with IFNalpha 2 for 10 min, cell lysates were immunoprecipitated with an antibody that recognizes all truncated forms of the beta L chain, and in vitro phosphatase assays were performed on the immunoprecipitates. Fig. 3 shows that significant phosphatase activity is associated with beta L462 and beta L417, but not with beta L346 after IFNalpha 2 treatment, indicating that the 346-417 region of beta L associates with a PTP. Since the increase in phosphatase activity associated with the 346-417 region of beta L is observed only after IFNalpha treatment, we could not elucidate whether the beta L-associated PTP is recruited to the receptor complex after IFNalpha stimulation or is constitutively associated with the beta L chain and activated by IFNalpha 2 stimulation.


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Fig. 3.   IFNalpha 2-induced phosphatase activity associated with the beta L subunit of the type I IFN-R. Phosphatase activity associated with the indicated truncations of the beta L subunit was assessed in immunocomplexes after immunoprecipitation with the anti-beta L subunit antisera beta L265-515 as described under "Materials and Methods." The -fold increase in receptor-associated phosphatase activity is shown. The data represent the mean value of duplicate samples. The levels of phosphatase activity in the absence of IFNalpha treatment were 0.022, 0.043, and 0.024 for alpha beta L346, alpha beta L417, and alpha beta L462, respectively.

Deletion of the Negative Regulatory Domain of the beta L Subunit at Amino Acid 346 Results in Strong and Prolonged Tyrosine Phosphorylation of Jak1-- We next sought to test if deletion of the 346-417 region of the beta L chain and, consequently, removal of the phosphatase interaction site had an effect on tyrosine phosphorylation. We performed time course and dose response experiments with mouse L-929 cells stably cotransfected with wild-type alpha  chain and beta L truncated at amino acids 346 or 417, respectively. Fig. 4A shows that more intense phosphorylation of Jak1 was observed at lower doses of IFNalpha 2 in cells expressing the beta L subunit truncated at amino acid 346, as compared with the beta L chain truncated at residue 417. Moreover, deletions distal to amino acid 346, but not at amino acid 417, prolonged the period of time that Jak1 was phosphorylated (Fig. 4B). The increase in tyrosine phosphorylation observed was not due to different amounts of immunoprecipitated Jak1 protein since stripping and reblotting of the same membranes with an anti-Jak1 mAb showed a similar amount of Jak1 in all lanes (Fig. 4, A and B, lower panels). Two independent clones expressing beta L truncated at amino acid 346, alpha beta L346.2 and alpha beta L346.4, produced equivalent results. The intensity of Tyk2 phosphorylation, however, was unaffected by truncation of beta L at residue 346 as revealed by immunoprecipitation experiments with anti-Tyk2 sera followed by immunoblotting with antiphosphotyrosine antibodies (data not shown). Altogether, these data suggest that the 346-417 region of beta L interacts with a phosphatase that regulates Jak1 phosphorylation.


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Fig. 4.   Truncation of the beta L subunit of the type I IFN-R at amino acid 346 results in increased tyrosine phosphorylation of Jak1. A, dose response experiment. Cells coexpressing the alpha  chain and truncation mutants of the beta L chain at amino acids 346 (alpha beta L346) or 417 (alpha beta L417) were treated with the indicated doses of IFNalpha 2 for 8 min at 37 °C. Cells were lysed and immunoprecipitated with anti-Jak1 sera as described under "Materials and Methods." Immunoblotting was first performed with anti-phosphotyrosine antibody (top panel) followed by stripping and reprobing of the filters with an anti-Jak1 monoclonal antibody (bottom panel) to demonstrate that similar amounts of proteins were present in all lanes. B, kinetics of Jak1 phosphorylation. alpha beta L346 and alpha beta L417 cells were treated with 20,000 units/ml of IFNalpha 2 for the indicated periods of time. Cells were lysed, and lysates were immunoprecipitated with an anti-Jak1 sera followed by sequential immunoblotting with anti-phosphotyrosine (top panel) and -Jak1 (bottom panel) antibodies.

Mutation of Tyrosine 411 in the beta L Chain of Type I IFN-R Does Not Alter the Response of Mouse Cells to Human Type I IFNs-- It has been previously reported that SH2-containing phosphatases (SHP1 and SHP2) interact with the alpha  subunit of type I IFNR (37, 49). Since the negative regulatory region of beta L contains only one tyrosine (Tyr-411), which (if phosphorylated) may serve as docking site for SH2-containing tyrosine phosphatases, we studied the effect of a phenylalanine mutation of tyrosine 411 on cell proliferation. Panel F (Fig. 2) shows that mutation of tyrosine 411 to phenylalanine does not reconstitute the antiproliferative effect of type I IFNs. Similar results were also obtained with different clones carrying the same mutations (data not shown). Therefore, tyrosine 411 is not critical for induction of the negative regulatory effect and is presumably not a docking site for SH2-containing phosphatases.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The type I IFNs have multiple biological actions, including antiviral and antiproliferative effects, which are the most prominent of these cellular responses (1). Binding of human type I IFNs to mouse L-929 cells that coexpress human alpha  and beta L chains is sufficient to trigger activation of the Jak-Stat pathway and to produce a full antiviral response, which demonstrates the presence of functional human type I IFN-R subunits (9). We therefore tested these cells for their ability to respond to the antiproliferative actions of human type I IFNs. Although these cells were able to respond to the full growth inhibitory effects of MuIFNalpha beta , only a minimal response was observed for human type I IFNs. Thus, the antiproliferative pathway is intact in these cells but not fully activated via the human receptors. Similar results were also observed for different clones of mouse A9+21 cells that carry several copies of human chromosome 21, which is thought to contain the type I IFN-R cluster (7, 11-16, 41-45) and an uncharacterized signaling component (46, 47). Altogether, these data indicate that (i) induction of the antiproliferative and antiviral responses occurs through partially divergent pathways and (ii) that a novel species-specific signaling component is required, in addition to the alpha  and beta L chains, for the growth inhibitory effect.

It has been reported that elements of the cytoplasmic domains of cytokine receptors have a negative regulatory role in signaling. For example, removal of the docking site for SHP1 in EPO-R results in hypersensitivity to EPO and prolonged phosphorylation of Jak2 (31). Similarly, deletion of the distal region of the alpha  subunit of the IFNalpha R results in increased sensitivity to induction of HLA class I antigens by IFNalpha 2 (48). Correspondingly, removal of the distal region (346-417) of the cytoplasmic domain of beta L resulted in a gain in response to the growth inhibitory effects of IFNalpha , indicating that this region encodes a negative regulatory domain. The effect of the negative regulatory domain of beta L is specific, as demonstrated by the finding that deletion of a homologous region of the alpha  chain did not have an effect on cell proliferation (Fig. 2, alpha Y1F511beta L cells). The negative regulatory domain of beta L interacts with a PTP as indicated by detection of phosphatase activity associated with the 346-417 region of beta L and the finding that deletion of this region resulted in prolonged phosphorylation of Jak1 in mouse cells. Mutation of the only tyrosine in the negative regulatory domain (Tyr-411) did not have the same effect on the antiproliferative response as deletion of the 346-417 region, indicating that the putative phosphatase is not docked to beta L through an SH2 domain. Moreover, immunoprecipitation with antibodies against the alpha  and beta L chains, and pull-down experiments with GST fusion proteins encoding the cytoplasmic domain of the alpha  and beta L chains failed to precipitate SHP1 or SHP2 (data not shown). Taken together, these data indicate that the 346-417 region of beta L functions as a negative regulator for the antiproliferative effect of IFNs, possibly by recruiting a regulatory phosphatase through an SH2-independent mechanism. Consistently with the finding in mouse cells, immunoprecipitations with anti-beta L antibodies also revealed phosphatase activity specifically associated with the beta L subunit expressed in human cells (data not shown). It should be noted, however, that in human cells no conclusive data have been obtained using GSTbeta L fusion proteins.

Since mouse IFNs completely inhibit proliferation of cells expressing human alpha  and beta L chains, we can conclude that the mouse receptor couples the signal induced by murine IFNs with the intracellular proteins responsible for antiproliferative pathway in these cells. Consequently, the inability of LpZRalpha beta L and A9+21 cells to respond to the growth inhibitory effect of human IFNs centers the defect at the level of the human receptor. One possibility is that the intracellular domains of the human alpha  and/or beta L chains, which are responsible for activation of the antiproliferative pathway, are not homologous to their murine counterparts. However, this possibility is highly unlikely, based on two primary observations. First, deletion of the negative regulatory domain of beta L allows a full human IFNalpha -induced antiproliferative effect, indicating that the human receptor subunits are capable of interacting with the appropriate mouse signaling proteins. Second, these transfectants respond to the antiviral effect of IFNs. Another possibility is that the missing species-specific component corresponds to a third receptor subunit. In this scenario, the antiproliferative response requires the assembly of a receptor composed of three subunits; in this complex, the third receptor subunit regulates the activity of a protein associated with the negative regulatory domain of beta L, presumably a PTP. If, in fact, a phosphatase is involved, the third receptor subunit may delay activation of the PTP or releases the PTP from the complex, resulting in prolonged activation of Jak1 and generation of the antiproliferative response. Thus, deletion of the negative regulatory domain of beta L has the same outcome as activating the third receptor subunit, blocking the action of the PTP or an unknown protein that associates with this region.

    ACKNOWLEDGEMENTS

We thank Dr. J. N. Ihle for providing the Jak1 antisera.

    FOOTNOTES

* This work was supported by National lnstitutes of Health Grants CA73381 (to L. C. P.) and GM54709 (to O. R. C.), and by Grants 96-24 (to L. C. P.) and DB-74554 (to T. Y.) from the American Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger 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; E-mail: OColamonici{at}utmem1.utmem.edu.

1 The abbreviations used are: IFN, interferon; IFN-R, interferon receptor; PTP, protein tyrosine phosphatase; beta L, long form of the beta  chain; HuIFN, human interferon; MuIFN, mouse interferon; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GST, glutathione S-transferase; pNPP, p-nitrophenyl phosphate.

2 Domanski and Colamonici, manuscript in preparation.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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