The Carboxyl Terminus of Interferon-γ Contains a Functional Polybasic Nuclear Localization Sequence

Abstract

Cytokines such as interferon-gamma (IFN-γ), which utilize the well studied JAK/STAT pathway for nuclear signal transduction, are themselves translocated to the nucleus. The exact mechanism for the nuclear import of IFN-γ or the functional role of the nuclear translocation of ligand in signal transduction is unknown. We show in this study that nuclear localization of IFN-γ is driven by a simple polybasic nuclear localization sequence (NLS) in its COOH terminus, as verified by its ability to specify nuclear import of a heterologous protein allophycocyanin (APC) in standard import assays in digitonin-permeabilized cells. Similar to other nuclear import signals, we show that a peptide representing amino acids 95–132 of IFN-γ (IFN-γ(95–132)) containing the polybasic sequence126RKRKRSR132 was capable of specifying nuclear uptake of the autofluorescent protein, APC, in an energy-dependent fashion that required both ATP and GTP. Nuclear import was abolished when the above polybasic sequence was deleted. Moreover, deletions immediately NH2-terminal of this sequence did not affect the nuclear import. Thus, the sequence126RKRKRSR132 is necessary and sufficient for nuclear localization. Furthermore, nuclear import was strongly blocked by competition with the cognate peptide IFN-γ(95–132) but not the peptide IFN-γ(95–125), which is deleted in the polybasic sequence, further confirming that the NLS properties were contained in this sequence. A peptide containing the prototypical polybasic NLS sequence of the SV40 large T-antigen was also able to inhibit the nuclear import mediated by IFN-γ(95–132). This observation suggests that the NLS in IFN-γ may function through the components of the Ran/importin pathway utilized by the SV40 T-NLS. Finally, we show that intact IFN-γ, when coupled to APC, was also able to mediate its nuclear import. Again, nuclear import was blocked by the peptide IFN-γ(95–132) and the SV40 T-NLS peptide, suggesting that intact IFN-γ was also transported into the nucleus through the Ran/importin pathway. Previous studies have suggested a direct intracellular role for IFN-γ in the induction of its biological activities. Based on our data in this study, we suggest that a key intracellular site of interaction of IFN-γ is the one with the nuclear transport mechanism that occurs via the NLS in the COOH terminus of IFN-γ. Cytokines such as interferon-gamma (IFN-γ), which utilize the well studied JAK/STAT pathway for nuclear signal transduction, are themselves translocated to the nucleus. The exact mechanism for the nuclear import of IFN-γ or the functional role of the nuclear translocation of ligand in signal transduction is unknown. We show in this study that nuclear localization of IFN-γ is driven by a simple polybasic nuclear localization sequence (NLS) in its COOH terminus, as verified by its ability to specify nuclear import of a heterologous protein allophycocyanin (APC) in standard import assays in digitonin-permeabilized cells. Similar to other nuclear import signals, we show that a peptide representing amino acids 95–132 of IFN-γ (IFN-γ(95–132)) containing the polybasic sequence126RKRKRSR132 was capable of specifying nuclear uptake of the autofluorescent protein, APC, in an energy-dependent fashion that required both ATP and GTP. Nuclear import was abolished when the above polybasic sequence was deleted. Moreover, deletions immediately NH2-terminal of this sequence did not affect the nuclear import. Thus, the sequence126RKRKRSR132 is necessary and sufficient for nuclear localization. Furthermore, nuclear import was strongly blocked by competition with the cognate peptide IFN-γ(95–132) but not the peptide IFN-γ(95–125), which is deleted in the polybasic sequence, further confirming that the NLS properties were contained in this sequence. A peptide containing the prototypical polybasic NLS sequence of the SV40 large T-antigen was also able to inhibit the nuclear import mediated by IFN-γ(95–132). This observation suggests that the NLS in IFN-γ may function through the components of the Ran/importin pathway utilized by the SV40 T-NLS. Finally, we show that intact IFN-γ, when coupled to APC, was also able to mediate its nuclear import. Again, nuclear import was blocked by the peptide IFN-γ(95–132) and the SV40 T-NLS peptide, suggesting that intact IFN-γ was also transported into the nucleus through the Ran/importin pathway. Previous studies have suggested a direct intracellular role for IFN-γ in the induction of its biological activities. Based on our data in this study, we suggest that a key intracellular site of interaction of IFN-γ is the one with the nuclear transport mechanism that occurs via the NLS in the COOH terminus of IFN-γ. interferon(s) nuclear localization sequence allophycocyanin 4-morpholineethanesulfonic acid guanosine 5′-3-O-(thio)triphosphate Janus kinase signal transducer and activator of transcription nucleoprotein interactor-1. The interferons (IFNs)1are cytokines that use the well studied JAK/STAT pathway for signal transduction to the nucleus (1Leaman D.W. Leung S. Li X. Stark G.R. FASEB J. 1996; 10: 1578-1588Crossref PubMed Scopus (271) Google Scholar). This pathway is initiated by the binding of the ligand to the extracellular domain of the appropriate receptor complex followed by the activation of select members of the JAK family of tyrosine kinases at the intracellular cytoplasmic domain of the receptor subunits. These tyrosine kinases in turn phosphorylate appropriate members of the STAT family of transcription factors present in the cytoplasm, thereby targeting these factors, through unknown mechanisms, for translocation to the nucleus to activate transcription. Transcription factors the size of STATs must be taken into the cell nucleus by an active transport process through the nuclear pore complex. Active nuclear import of a large number of nuclear proteins occurs through the Ran/importin pathway (for review, see Ref. 2Gorlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1064) Google Scholar). In this pathway, the nuclear protein initially binds to a heterodimeric nuclear transport protein called importin which contains a α and β subunit. Nuclear proteins interact with the α subunit, importin α, of the transporter via a specific nuclear localization sequence (NLS). These NLSs generally consist of a cluster of basic amino acids or two short clusters separated by a spacer of variable length (a bipartite NLS; 2). The canonical simple polybasic NLS is represented by the sequence of the NLS of the SV40 T-antigen, KKKRK. The β subunit, importin β, in turn mediates the binding of the NLS-importin α complex to the GTPase Ran present at the nuclear pore complex. Nuclear import of this nuclear protein-bearing complex through the nuclear pore is initiated by Ran with the subsequent hydrolysis of GTP and ATP and hence is a strictly energy-dependent process (2Gorlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1064) Google Scholar). As mentioned above, the nuclear import of STAT1 requires active transport. Indeed, it has been shown recently that the IFN-γ-activated STAT1 is transported into the nucleus via the Ran/importin pathway by binding the importin α homolog NPI-1 (3Sekimoto T. Nakajima K. Tachibana T. Hirano T. Yoneda Y. J. Biol. Chem. 1996; 271: 31017-31020Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 4Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar). NPI-1 is known to interact with nuclear proteins via the above polycationic NLSs. However, based on current knowledge, no polybasic NLS has been identified on STAT1 (3Sekimoto T. Nakajima K. Tachibana T. Hirano T. Yoneda Y. J. Biol. Chem. 1996; 271: 31017-31020Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 4Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar). Mutational analysis of STAT1 failed to reveal a conventional NLS on STAT1 which was responsible for mediating the binding to NPI-1 or nuclear translocation of STAT1 (4Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar). These studies are consistent with the concept that the necessary basic NLS required for STAT1 interaction with NPI-1 is provided for by another molecule associated with STAT1. Based on the results of studies from our laboratory on the COOH-terminal domain of IFN-γ, IFN-γ(95–133), and its interaction with the α chain of the receptor, we propose that this candidate chaperone function may be served by the ligand IFN-γ (5Johnson H.M. Torres B.A. Green M.M. Szente B.E. Siler K.I. Larkin III, J. Subramaniam P.S. Biochem. Biophys. Res. Commun. 1998; 244: 607-614Crossref PubMed Scopus (50) Google Scholar). Crucial to this hypothesis, as a first step, is demonstrating that IFN-γ contains a functional NLS. In this report, we have specifically addressed this issue. It has been well documented that human and murine IFN-γ are themselves translocated to the nucleus (6MacDonald H.S. Kushnaryov V.M. Sedmak J.J. Grossberg S.E. Biochem. Biophys. Res. Commun. 1986; 138: 254-260Crossref PubMed Scopus (34) Google Scholar, 7Kushnaryov V.M. MacDonald H.S. Sedmak J.J. Grossberg S.E. Biochem. Biophys. Res. Commun. 1988; 157: 109-114Crossref PubMed Scopus (6) Google Scholar, 8Bader T. Wietzerbin J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11831-11835Crossref PubMed Scopus (70) Google Scholar). The sequence of murine IFN-γ contains a putative polybasic NLS126RKRKRSR132 within the above mentioned COOH-terminal domain. It has been known for some time that this conserved sequence in murine and human IFN-γ is absolutely required for the biological activities of the corresponding IFN-γ (9Szente B.E. Soos J.M. Johnson H.M. Biochem. Biophys. Res. Commun. 1994; 201: 1645-1654Crossref Scopus (52) Google Scholar, 10Szente B.E. Weiner I.J. Jablonsky M.J. Krishna N.R. Torres B.A. Johnson H.M. J. Interferon Cytokine Res. 1996; 16: 813-817Crossref PubMed Scopus (27) Google Scholar, 11Arakawa T. Hsu Y.R. Parker C.G. Lai P.H. J. Biol. Chem. 1986; 261: 8534-8539Abstract Full Text PDF PubMed Google Scholar, 12Dobeli H. Gentz R. Jucker W. Garotta G. Hartmann D.W. Hochuli E. J. Biotechnol. 1988; 7: 199-216Crossref Scopus (65) Google Scholar, 13Arakawa T. Narachi M.A. Hsu Y.-R. Everett R.R. Lai P-H. Fish E.N. Drug Design Deliv. 1989; 4: 217-225PubMed Google Scholar, 14Wetzel R. Perry L.J. Veilleux C. Chang G. Prot. Eng. 1990; 3: 611-623Crossref PubMed Scopus (28) Google Scholar, 15Lundell D. Lunn C. Dalgarno D. Fossetta J. Greenberg R. Reim R. Grace M. Narula S. Prot. Eng. 1991; 4: 335-341Crossref PubMed Scopus (60) Google Scholar, 16Slodowski O. Bohm J. Brigitte S. Otto B. Eur. J. Biochem. 1991; 202: 1133-1140Crossref PubMed Scopus (31) Google Scholar). We have also shown that both human and murine IFN-γ interact with the cytoplasmic domain of the α chain of the corresponding IFN-γ receptor complexes via this COOH-terminal domain containing this putative NLS sequence (17Szente B.E. Johnson H.M. Biochem. Biophys. Res. Commun. 1994; 201: 215-221Crossref PubMed Scopus (34) Google Scholar, 18Szente B.E. Subramaniam P.S. Johnson H.M. J. Immunol. 1995; 155: 5617-5622PubMed Google Scholar, 19Green M.M. Larkin III, J. Subramaniam P.S. Szente B.E. Johnson H.M. Biochem. Biophys. Res. Commun. 1998; 243: 170-176Crossref PubMed Scopus (21) Google Scholar). STAT1 binds to the cytoplasmic domain of the α chain of the receptor at a downstream site. The interaction of the COOH terminus of IFN-γ with the α chain is dependent on the presence of the putative NLS sequence and in turn increases the affinity of the receptor-activated tyrosine kinase JAK2 to an immediately proximal site on the α chain of the receptor (18Szente B.E. Subramaniam P.S. Johnson H.M. J. Immunol. 1995; 155: 5617-5622PubMed Google Scholar). JAK2, along with JAK1, is involved in the receptor recruitment and phosphorylation of STAT1, events that precede the subsequent nuclear translocation of STAT1. Thus, this COOH-terminal domain of IFN-γ has many characteristics that it make a good candidate to be involved in the chaperoning of STAT1α to the nucleus, probably as part of a ligand-receptor-STAT1α complex. Given the properties of this sequence, we tested in this study whether the above polybasic sequence in the COOH-terminal domain of IFN-γ can function as an NLS and facilitate the nuclear translocation of a heterologous protein. We demonstrate that this sequence is, indeed, an NLS and can function to import an associated non-NLS heterologous protein. The NLS functions in the strictly energy-dependent fashion typical of active nuclear import. Competition assays suggest that nuclear import driven by the IFN-γ NLS may functionally overlap with the Ran/importin pathway utilized by the SV40 T-NLS. Cell Culture—Mouse 3T3 cells (Balb/c, clone A31; from ATCC) were grown in ATCC-modified Dulbecco's modified Eagle's medium containing 10% bovine serum. WISH cells were grown in Eagle's minimal essential medium containing 10% fetal bovine serum. Cells were plated onto coverslips for 24 h before use. Peptides used in this study (see Table I) were synthesized on a PerSeptive Biosystems 9050 automated peptide synthesizer using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as detailed previously B.E. Weiner I.J. Jablonsky M.J. Krishna N.R. Torres B.A. Johnson H.M. J. Interferon Cytokine Res. 1996; 16: 813-817Crossref PubMed Scopus (27) Google of used in this was as the first amino acid of peptide sequence to for to The IFN-γ are from the of murine IFN-γ, and in are the first IFN-γ have been to deleted or as the may The NLSs are The of the IFN-γ NLS is from this A was as the first amino acid of peptide sequence to for to The IFN-γ are from the of murine IFN-γ, and in are the first IFN-γ have been to deleted or as the may The NLSs are The of the IFN-γ NLS is from this in a (APC) with the was from and used to the or human IFN-γ with was coupled at a or in containing the of by through an in peptide was by in the through a and the at or and peptide were by assays with cells were based on previously (for review, see PubMed Scopus Google Scholar). Cells and WISH grown on coverslips were at with transport and and Cells were with in transport for at with transport cells were with the transport for at contained and appropriate import and of were in transport containing bovine on and a ATP were first with a of and at for before the of the of the GTP GTP was from the and the was at with the at before the of other peptide competition were at with to APC, in the presence of other components the import for at the was and the with the cells. The of the putative nuclear localization sequence within the COOH-terminal domain of IFN-γ to function in nuclear import was by its ability to mediate the nuclear import of a heterologous protein. This was using the nuclear transport PubMed Scopus Google in digitonin-permeabilized cells. In this study, we used as a peptide corresponding to amino acids 95–132 of murine IFN-γ see coupled to the heterologous autofluorescent protein to a for the As shown in a of the cells the in the nucleus and A and This was at and was dependent on the of factors of a simple of IFN-γ(95–132) with protein did not to nuclear not These data show that the IFN-γ(95–132) was capable of mediating the nuclear import of a heterologous protein when coupled to The transport of proteins the nuclear via the nuclear pore complex is an energy-dependent (2Gorlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1064) Google Scholar). It strictly on the of both ATP and GTP. This binding to the nuclear pore complex to a typical at the nuclear 1988; Full Text PDF PubMed Scopus Google Scholar). As shown in the nuclear import of was strictly of ATP did not the nuclear import of into the nucleus of cells and In the of ATP the as a at the nuclear In a the of GTP coupled with the of the also in of transport into the nucleus and in the of the at the nuclear Thus, these data further demonstrate that the domain represented by IFN-γ(95–132) contains an NLS that functions in a strictly energy-dependent fashion of nuclear import (2Gorlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1064) Google Scholar). further the of this we competition using to the transport of the The used were the (see Table a peptide containing the NLS of the SV40 T-antigen the IFN-γ(95–132) and the peptide IFN-γ(95–125), which is from IFN-γ(95–132) by of the polybasic putative The SV40 T-NLS is one of the NLSs that the well studied Ran/importin pathway for nuclear import (2Gorlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1064) Google Scholar). It contains an NLS that is a simple polybasic cluster to that in IFN-γ(95–132). As can be in transport of was strongly in the presence of IFN-γ(95–132) A and and the SV40 NLS peptide but not by the peptide the putative NLS We in the the were as to transport with nuclear translocation in can be from these the ability of IFN-γ(95–132) to inhibit transport of that the nuclear import of the is specifically driven by the peptide further the specific ability of IFN-γ(95–132) to as an NLS. the ability of the SV40 T-NLS to inhibit transport of the suggests that the nuclear import of occurs through the Ran/importin pathway that is utilized by the prototypical polybasic SV40 T-NLS. the that that is deleted in the polybasic putative NLS is to the import of strongly suggests that the NLS is contained in the deleted This sequence is to that of the polybasic NLS contained in the SV40 T-NLS. Thus, it is consistent that the SV40 T-NLS peptide can the import of We that IFN-γ(95–132) contains a polybasic NLS that functions in nuclear import in a fashion to that of the prototypical SV40 T-NLS. The IFN-γ NLS probably components of the Ran/importin pathway to the nuclear import pathway of the SV40 T-NLS. further that the polybasic sequence126RKRKRSR132 is responsible for the nuclear localization properties of IFN-γ(95–132) we tested the ability of the to function as a in the nuclear import assays with As shown in in to and its in which is deleted in the polybasic NLS was not able to function as a nuclear import We also tested the ability of a peptide from IFN-γ(95–132) the sequence immediately NH2-terminal to the NLS has been (see Table its ability to function as a for nuclear import and as as in nuclear import These data show that this polybasic sequence is necessary and sufficient for the nuclear targeting properties of the COOH-terminal domain IFN-γ(95–132). Finally, we in these assays that intact IFN-γ has the ability to mediate nuclear import of a heterologous protein. As can be in was also transported into the nucleus in a fashion that was strictly dependent on the presence of factors A signal was in these assays with the transport of of the to Nuclear import of was strongly by competition with the IFN-γ(95–132) peptide and the SV40 T-NLS These data show that the nuclear import of IFN-γ is mediated in a fashion to IFN-γ(95–132) and the SV40 T-NLS and probably occurs via the Ran/importin pathway for nuclear import. In this report, we have that the nuclear translocation of IFN-γ occurs via a polybasic NLS in the COOH terminus of the This sequence functions in the nuclear import of IFN-γ via an active energy-dependent Competition studies suggest that the NLS components of the Ran/importin pathway that is to the nuclear import mechanism of the of this of the SV40 T-NLS. We have shown previously that this sequence is a for the biological of the IFN-γ molecule (9Szente B.E. Soos J.M. Johnson H.M. Biochem. Biophys. Res. Commun. 1994; 201: 1645-1654Crossref Scopus (52) Google Scholar, 10Szente B.E. Weiner I.J. Jablonsky M.J. Krishna N.R. Torres B.A. Johnson H.M. J. Interferon Cytokine Res. 1996; 16: 813-817Crossref PubMed Scopus (27) Google Scholar). studies using human IFN-γ have shown that a polybasic within the COOH terminus is absolutely required for biological T. Hsu Y.R. Parker C.G. Lai P.H. J. Biol. Chem. 1986; 261: 8534-8539Abstract Full Text PDF PubMed Google Scholar, 12Dobeli H. Gentz R. Jucker W. Garotta G. Hartmann D.W. Hochuli E. J. Biotechnol. 1988; 7: 199-216Crossref Scopus (65) Google Scholar, 13Arakawa T. Narachi M.A. Hsu Y.-R. Everett R.R. Lai P-H. Fish E.N. Drug Design Deliv. 1989; 4: 217-225PubMed Google Scholar, 14Wetzel R. Perry L.J. Veilleux C. Chang G. Prot. Eng. 1990; 3: 611-623Crossref PubMed Scopus (28) Google Scholar, 15Lundell D. Lunn C. Dalgarno D. Fossetta J. Greenberg R. Reim R. Grace M. Narula S. Prot. Eng. 1991; 4: 335-341Crossref PubMed Scopus (60) Google Scholar, 16Slodowski O. Bohm J. Brigitte S. Otto B. Eur. J. Biochem. 1991; 202: 1133-1140Crossref PubMed Scopus (31) Google Scholar). Thus, in with these studies our data show that the nuclear localization of IFN-γ via this NLS a role in the biological of IFN-γ. Previous studies have suggested a biological role for intracellular IFN-γ and These the that human IFN-γ by a was able to activate murine I.J. J. Immunol. Google human IFN-γ in murine an in these cells J. R. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google IFN-γ can on murine K. J. Immunol. 1990; Google and is also active and can activate the binding of the transcription complex containing STAT1α and A. B. D. J. Interferon Cytokine Res. 1996; 16: PubMed Scopus Google Scholar). Thus, the IFN-γ with intracellular to a biological Based on our data in this study we suggest that a key intracellular interaction is the one with the nuclear transport mechanism which occurs via the NLS in the COOH terminus of IFN-γ. studies in this have identified for the first time the NLS in IFN-γ. it for biological of IFN-γ, the exact of this NLS to biological function is Based on studies from our laboratory on the interaction of this domain of IFN-γ with the receptor and its on we have that a role for the NLS be as a chaperone for the nuclear localization of STAT1α (5Johnson H.M. Torres B.A. Green M.M. Szente B.E. Siler K.I. Larkin III, J. Subramaniam P.S. Biochem. Biophys. Res. Commun. 1998; 244: 607-614Crossref PubMed Scopus (50) Google Scholar). studies have shown that STAT1α is transported to the nucleus via the Ran/importin pathway (3Sekimoto T. Nakajima K. Tachibana T. Hirano T. Yoneda Y. J. Biol. Chem. 1996; 271: 31017-31020Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 4Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar). STAT1α binds to the importin α homolog NPI-1 that mediates the nuclear import of the SV40 in a conventional fashion (4Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar). However, analysis of STAT1α has failed to reveal sequence responsible for nuclear import of STAT1α (4Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar). These studies are consistent with a role for a molecule that binds importin α and the required the interaction of STAT1α with the Ran/importin be We suggest that the NLS required for nuclear import of STAT1α may be provided by the ligand IFN-γ, which at the of the nuclear transport have the presence of putative NLSs in other and FASEB J. 1994; PubMed Scopus Google Scholar, G. 1998; PubMed Scopus Google and have suggested a role for the nuclear targeting of in to the nucleus FASEB J. 1994; PubMed Scopus Google Scholar). In studies have that and functional NLSs S. X. S. T. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L.J. S.E. S. 1997; PubMed Scopus Google Scholar, L.J. S.E. S. 1997; Scopus Google Scholar). We have that when a is such cytokines and a the ability of a to activate the JAK/STAT pathway and the presence of a putative NLS in ligand or receptor (5Johnson H.M. Torres B.A. Green M.M. Szente B.E. Siler K.I. Larkin III, J. Subramaniam P.S. Biochem. Biophys. Res. Commun. 1998; 244: 607-614Crossref PubMed Scopus (50) Google Scholar). This the that this of nuclear translocation of STATs may to other In this as in the we have that the COOH-terminal domain represented by IFN-γ(95–132) specifically with the cytoplasmic domain of the α chain of the IFN-γ receptor (9Szente B.E. Soos J.M. Johnson H.M. Biochem. Biophys. Res. Commun. 1994; 201: 1645-1654Crossref Scopus (52) Google Scholar, B.E. Johnson H.M. Biochem. Biophys. Res. Commun. 1994; 201: 215-221Crossref PubMed Scopus (34) Google Scholar, 18Szente B.E. Subramaniam P.S. Johnson H.M. J. Immunol. 1995; 155: 5617-5622PubMed Google Scholar, 19Green M.M. Larkin III, J. Subramaniam P.S. Szente B.E. Johnson H.M. Biochem. Biophys. Res. Commun. 1998; 243: 170-176Crossref PubMed Scopus (21) Google Scholar). This interaction is also dependent on the presence of the NLS (9Szente B.E. Soos J.M. Johnson H.M. Biochem. Biophys. Res. Commun. 1994; 201: 1645-1654Crossref Scopus (52) Google Scholar, B.E. Johnson H.M. Biochem. Biophys. Res. Commun. 1994; 201: 215-221Crossref PubMed Scopus (34) Google Scholar). The α chain of the IFN-γ receptor is the that binds We have recently shown using and that IFN-γ of intact WISH the cytoplasmic domain of the α chain of the IFN-γ receptor is also translocated to the nucleus. Larkin III, M. R. H. M. and S. in This is specific to the α chain of the receptor the β chain of the receptor which not did not or nuclear Furthermore, the of receptor and STAT1α this process and the of receptor α chain and STAT1α translocation to the nucleus are consistent with the that these events may be a putative NLS sequence be identified in the receptor α a is that translocation of the receptor complex STAT1α activation events is by the NLS in the ligand via the interaction of the domain in the ligand with the cytoplasmic domain of the receptor α This can be tested and is in our