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The Journal of Immunology, 2002, 169: 1959-1969.
Copyright © 2002 by The American Association of Immunologists

Lipid Microdomains Are Required Sites for the Selective Endocytosis and Nuclear Translocation of IFN-{gamma}, Its Receptor Chain IFN-{gamma} Receptor-1, and the Phosphorylation and Nuclear Translocation of STAT1{alpha}1

Prem S. Subramaniam2 and Howard M. Johnson

Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IFN-{gamma} contains a nuclear localization sequence that may play a role in the nuclear transport of activated STAT1{alpha} via a complex of IFN-{gamma}/IFN-{gamma} receptor (IFNGR)-1/STAT1{alpha} with the nuclear importer nucleoprotein interactor 1. In this study, we examine the mechanism of endocytosis of IFNGR-1 and the relationship of its nuclear translocation to that of STAT1{alpha}. In untreated WISH cells, both IFNGR-1 and IFNGR-2 were constitutively localized within caveolae-like microdomains isolated from plasma membrane. However, treatment of cells with IFN-{gamma} resulted in rapid migration of IFNGR-1, but not IFNGR-2, from these microdomains. Filipin pretreatment, which specifically inhibits endocytosis from caveolae-like microdomains, inhibited the nuclear translocation of IFN-{gamma} and IFNGR-1 as well as the tyrosine phosphorylation and nuclear translocation of STAT1{alpha}, but did not affect the binding of IFN-{gamma} to these cells. In the Jurkat T lymphocyte cell line, which does not express caveolin-1, nuclear translocation of IFNGR-1 and STAT1{alpha} were similarly inhibited by filipin pretreatment. Isolation of lipid microdomains from Jurkat cells showed that both IFNGR-1 and IFNGR-2 were associated with lipid microdomains only after stimulation with IFN-{gamma}, suggesting that the IFNGR subunits are recruited to lipid microdomains by IFN-{gamma} binding in lymphocytes (Jurkat) in contrast to their constitutive presence in epithelial (WISH) cells. In contrast, treatments that block clathrin-dependent endocytosis did not inhibit either activation or nuclear translocation of STAT1{alpha} or the nuclear translocation of IFN-{gamma} or IFNGR-1. Thus, membrane lipid microdomains play an important role in IFN-{gamma}-initiated endocytic events involving IFNGR-1, and the nuclear translocation of IFN-{gamma}, IFNGR-1, and STAT1{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The IFN-{gamma} receptor (IFNGR)3 system is a heterodimeric complex consisting of an {alpha}-subunit (IFNGR-1) and a {beta}-subunit (IFNGR-2), both of which are essential for biological activity (reviewed in Refs. 1 and 2). It is well-documented that upon ligand binding, along with the initiation of signal transduction, the ligand is internalized by the process of receptor-mediated endocytosis. Currently, receptor-mediated endocytosis is seen as a mechanism for ligand and receptor recycling and/or attenuation of signaling by an unknown mechanism, with no further contribution to signaling.

However, we have recently shown that both IFN-{gamma} and IFNGR-1 are translocated to the nucleus by a nuclear localization sequence (NLS) in the C terminus of IFN-{gamma} (3, 4). The IFN-{gamma}-activated transcription factor, STAT1{alpha}, by contrast, does not contain an intrinsic NLS (see for example Ref. 5). We have shown that IFN-{gamma} may provide the NLS for nuclear transport of STAT1{alpha}, because IFN-{gamma}-treated cells contain a complex of IFN-{gamma}/IFNGR-1/STAT1{alpha} in the cytosol, which in turn forms a complex with the nuclear importer nucleoprotein interactor 1 (NPI-1) through the NLS of IFN-{gamma} (6). Inhibition of the function of the NLS of IFN-{gamma} disrupts nuclear translocation of IFN-{gamma}, IFNGR-1, and STAT1{alpha} (reviewed in Ref. 7).

To better understand the contribution of specific mechanisms of receptor-endocytosis to STAT1{alpha} nuclear translocation, we have compared the role of two major endocytosis mechanisms in IFN-{gamma}-mediated STAT1{alpha} activation. Based on current knowledge of receptor-mediated endocytosis mechanisms, they fall into two broad groups: clathrin-dependent receptor-mediated endocytosis and receptor-mediated endocytosis from plasma membrane lipid microdomains or "rafts" (reviewed in Refs. 8, 9, 10). The clathrin-dependent processes depend on the protein clathrin to form the framework for vesicle budding (10), while lipid microdomains are exemplified by the endocytosis initiated from caveolae, which are membrane lipid microdomains that express the protein caveolin within the budding vesicle (8, 9). Not all membrane lipid microdomains contain caveolin, but many receptors have been identified in these noncaveolae domains that behave biochemically very similarly to caveolae, and such lipid microdomains are often referred to as caveolae-like microdomains.

The IFNGR complex has recently been identified in caveolae-like microdomains (11, 12). However, no study has been made to examine signaling in the context of endocytic vesicles, or specifically address the behavior of the IFNGR subunits after treatment of cells with IFN-{gamma}. In this study, we show that caveolae-like plasma membrane microdomains (lipid microdomains), and not clathrin-coated pits, play a primary role in IFN-{gamma}-initiated events involved in endocytosis of IFNGR-1 as well as the activation and nuclear translocation of IFNGR-1 and STAT1{alpha}. The data presented address the anatomical aspects of IFN-{gamma} signaling with respect to the plasma membrane-based endocytic domains, STAT1{alpha} activation, and nuclear translocation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Percoll was obtained from Amersham Pharmacia Biotech (Uppsala, Sweden), Ficoll (Histopaque-1077) was obtained from Sigma-Aldrich (St. Louis, MO), and OptiPrep was purchased from Nycomed (Oslo, Norway). Filpin was purchased from Sigma-Aldrich. Recombinant human IFN-{gamma} of specific activity (1 x 107 U/mg) was obtained from BioSource International (Camarillo, CA). AlexaFluor 594-labeled human transferrin (fluoresces red) was purchased from Molecular Probes (Eugene, Oregon). Rabbit Abs to IFNGR-1, IFNGR-2, and STAT1{alpha} were from Santa Cruz Biotechnology (Santa Cruz, CA), while goat anti-STAT1{alpha} Abs were from R&D Systems (Minneapolis, MN). Mouse anti-dynamin-1 Abs and rabbit anti-caveolin-1 Abs were from Transduction Laboratories (Lexington, KY). Secondary Abs were from Jackson ImmunoResearch Laboratories (West Grove, PA) and Molecular Probes.

Cells and cell culture

WISH cells were obtained from American Type Culture Collection (Manassas, VA). WISH cells were grown in Eagle’s MEM with 10% FBS and antibiotics. HeLa clones expressing the dynamin-1 mutant K44A and the wild-type dynamin-1 were kindly provided by Dr. S. Schmid (Scripps Research Institute, La Jolla, CA). These clones have been previously described (13, 14). The cells were grown in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 400 µg/ml geneticin, 200 ng/ml puromycin, and 1 µg/ml tetracycline. Tetracycline repressed dynamin expression. Before use for experiments, cells requiring expression of dynamin (mutant or wild-type) were grown in the absence of tetracycline for 48 h. Initially, dynamin expression was verified in total cell lysates by Western blotting.

Caveolae isolation from WISH cells

WISH cells from three 162-cm2 flasks per sample were used to isolate membrane caveolae using a detergent-free isolation method essentially as previously described (15) with the minor modifications outlined below. Cells, after the appropriate treatments, were washed with ice-cold PBS (2 x 25 ml) and then once with buffer A (described in Ref. 15) and finally scraped into buffer A. Plasma membrane isolation and sonication were performed as described in Ref. 15 . At this stage, 4 ml of the sonicate were placed at the bottom of a Beckmann (Fullerton, CA) UltraClear 25 x 89-mm tube, and overlayed with 32 ml of the 10–20% OptiPrep gradient and centrifuged as described in Ref. 15 . One milliliter fractions were collected from the top of the gradient up to the gradient-sonicate interface and the remaining plasma membrane sonicate was discarded. Every second fraction was analyzed by SDS-PAGE and immunoblotting with the appropriate Abs and detected by ECL.

Triton X-100 extraction of cells

For Jurkat cells, a total of 108 cells were used per sample. Jurkat cells were starved for 1.5 h in RPMI 1640 medium containing 1% BSA at 37°C, before being stimulated for 5 min with 3,000 U/ml of human IFN-{gamma}. Cells were rapidly centrifuged at 14,000 rpm, and washed twice with ice-cold PBS. Lipid microdomains were isolated using previously described procedures with a few modifications (16, 17, 18). Cells were resuspended in Triton X-100 extraction buffer (TNE: 25 mM Tris-HCl, pH 7.5, containing 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 10 µg/ml each of leupeptin, pepstatin and aprotinin, 1 mM sodium-orthovanadate), lysed at 4°C by three passages through a 22-gauge needle, and incubated with rocking at 4°C for 30 min to complete lysis. Cell lysates were equalized for total protein in equal volumes, brought to 40% sucrose using an 80% sucrose solution in TNE, and layered at the bottom of a linear 5–30% sucrose gradient in TNE without Triton X-100. Detergent-insoluble complexes were floated by centrifugation at 260,000 x g in a Beckmann SW41 rotor for 18 h at 4°C. Fractions of 1 ml were collected from the top of the gradient and subjected to further analysis.

For WISH cells, cells from three 162-cm2 flasks per sample were used, lysed in Triton X-100 buffer and lipid microdomains were floated as above for Jurkat cells, except that a discontinuous gradient was used by layering the 40% sucrose lysate with 6 ml of 35% sucrose in TNE without Triton X-100 and then 3 ml of 5% sucrose in the same buffer. Fractions of 1 ml were collected form the top of the gradient for further analysis.

Filipin treatment

Filipin treatment of WISH cells was conducted in complete growth medium by incubating cells with prewarmed (37°C, 5% CO2) medium containing 10 µg/ml filipin from a stock in methanol. After 10 min, the filipin-containing medium was removed and replaced with medium (prewarmed) either with or without IFN-{gamma}, without washing. Reversal of filipin treatment was achieved by washing cells with prewarmed growth medium and incubating cells for 2 h in growth medium without filipin at 37°C, 5% CO2.

For Jurkats, cells at 1 x 106 per ml were starved in RPMI 1640 containing 1% BSA for 1.5 h at 37°C, and then treated with 1 µg/ml of filipin for 1 h at 37°C in the same medium. IFN-{gamma} was then added to the cells at 3000 U/ml, and incubated further for 25 min. Aliquots of the cell suspensions were then cytocentrifuged (5 min) on to microscope slides and immediately fixed in methanol (-20°C) for immunofluorescence staining.

Inhibition of clathrin-dependent endocytosis

Acidification of the cytosol of WISH cells with NH4Cl and potassium-depletion of WISH cells were performed using standard methods (19). Cells were starved in serum free medium for 90 min before use. For acidification experiments, control cells (unacidified cells) were treated identically except that NH4Cl and amiloride were omitted from appropriate buffers, while for potassium-depletion experiments control cells (potassium-replete cells) were treated in buffers containing 10 mM KCl. Following these and other treatments, as indicated in the figure legends, cells were fixed and stained for the appropriate proteins.

Immunofluorescence staining of cells

Immunofluorescence staining of WISH cells for experiments not involving labeled transferrin was performed essentially as described in our previous studies (6, 20). Images were obtained on a Bio-Rad MRC-1024 laser scanning confocal system (Hercules, CA). Multiple image sections 0.2- (WISH) or 0.4-µm (Jurkat) thick were recorded over the depth of the cells, and then deconvolved using the constrained iterative algorithm of the Microtome software (Vaytek, Fairfield, IA) to remove out-of-focus haze for each section. Images were then displayed as two-dimensional (2D)-stacked projections of the deconvolved sections through appropriate regions of the cell. Quantitation of images was performed using the IPLab software (Scanalytics, Fairfax, VA) by measuring fluorescence densities from the 2D-stacked images in the nuclear (Fn) and non-nuclear regions (Fc) of the cells across three fields and the Fn-Fc ratio was determined for each cell. The average Fn-Fc for all cells was then plotted.

For experiments involving inhibition of clathrin-dependent processes (acidification of cytosol and potassium depletion), cells were fixed as follows. Cells were washed in ice-cold growth medium to remove unbound labeled transferrin, and then fixed for 5 min in ice-cold PBS (without Ca2+ or Mg2+) containing 2% formaldehyde at 4°C, before returning the cells, covered with fixative, to room temperature to continue fixing for an additional 25 min. Cells were washed in PBS (three times), and then incubated for 5 min each in three changes of PBS containing 50 mM NH4Cl to quench residual formaldehyde. Permeabilization and staining were then performed as described previously (6, 20).

Radiolabeling of IFN-{gamma} and binding studies

Human IFN-{gamma} was radiolabeled with Na125I using the chloramine T procedure as previously described (21), except that a solution of potassium iodide (70 mg/ml) and tyrosine (2 mg/ml) in PBS/0.1 N NaOH was used to stop the reaction, and separation columns were run in PBS containing 0.3% gelatin. Specific activity of the labeled product was ~70 µCi/µg.

For analysis of the effects of filipin on receptor binding, WISH cells were cultured to confluence in 24-well plates. Cells at 37°C were treated for 8 min at 37°C with 10 µg/µl of filipin in 1 ml complete growth medium, and the medium was replaced with ice-cold growth medium (2 ml) containing filipin and cells moved to 4°C. After 2 min at 4°C, the cells were incubated at 4°C with 1 ml of ice-cold growth medium without filipin containing appropriate dilutions of 125I-labeled IFN-{gamma} (125I-IFN-{gamma}) for a further 30 min, with or without a 100-fold excess of unlabeled IFN-{gamma} to control for nonspecific binding. These times were chosen to mimic the functional experiments performed with filipin. Cells were then washed three times with cold growth medium, once with cold PBS, lysed with 1% SDS in water and lysates were counted. Samples were run in triplicate.

Nuclear translocation of IFN-{gamma}

Nuclei from cells grown in 162-cm2 flasks were isolated using a centrifugation method as previously described (22). Nuclei were washed in centrifugation buffer, lysed, and analyzed as described previously (20). Equal amounts of protein from nuclear lysates were loaded for SDS-PAGE and 125I-IFN-{gamma} was detected by autoradiography following Western transfer to nitrocellulose membranes.

Cells were treated with 3000 U/ml of 125I-IFN-{gamma}. To monitor background, one flask of cells was cooled to 4°C and incubated with 125I-IFN-{gamma} at 4°C for 30 min before isolation of nuclei. For isolation of nuclei, one flask of cells for each sample was washed with 3 x 25 ml of ice-cold growth medium, and then once with cold PBS. Cell surface-bound IFN-{gamma} was removed by acid-washing at 4°C for 5 min with 0.25 M cold acetic acid, pH 2.5, containing 140 mM NaCl. Cells were then washed sequentially with 1 x 25 ml cold PBS and 2 x 25 ml cold growth medium before being scraped into 13 ml of cold growth medium. Cells were pelleted at 4°C and used to isolate nuclei as described above, which were either frozen at -80°C or used immediately.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Recent studies in unstimulated fibroblast cells localized the IFNGR complex to caveolae-like microdomains at the plasma membrane (11, 12). However, nothing is known about the behavior of the receptor complex upon binding IFN-{gamma}, or the role of caveolae-like microdomains/lipid microdomains vs clathrin-dependent events in the signaling mechanisms of IFN-{gamma}. Thus, we first used well-characterized detergent-free density gradient centrifugation techniques to isolate caveolae-like microdomains from the plasma membrane of WISH cells (15) and to compare the localization of both receptor subunits, IFNGR-1 and IFNGR-2, in untreated cells vs cells treated with IFN-{gamma} for 5 min. Fig. 1GoA shows the results of fractionation of extracts from plasma membranes of untreated WISH cells (left panels) or cells treated with IFN-{gamma} (right panels). Fractions from the respective gradients were subjected to SDS-PAGE and then immunoblotted with anti-caveolin Abs to identify the positions of the caveolae-like fractions and with Abs for the IFNGR subunits. Untreated cells showed constitutive localization of both subunits IFNGR-1 (Fig. 1GoA, upper left panel) and IFNGR-2 (lower left panel) with the caveolin-marked microdomain fractions. This is consistent with previous studies that showed that these subunits are localized to these domains in untreated cells (11, 12). In marked contrast, following IFN-{gamma} treatment the IFNGR-1 subunit migrated out of these domains (Fig. 1GoA, upper right panel), while the IFNGR-2 subunit remained within these caveolae-like microdomains (lower right panel). Thus, IFN-{gamma} treatment induced the selective migration of IFNGR-1 out of caveolae-like domains and its selective endocytosis compared with the IFNGR-2 subunit. This result is consistent with our earlier studies comparing the nuclear translocation of IFNGR-1 and IFNGR-2 where we found that only IFNGR-1 was translocated to the nucleus after IFN-{gamma} treatment along with the transcription factor STAT1{alpha}, while IFNGR-2 remained on the cell surface (20).



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  		FIGURE 1. Selective migration of IFNGR-1 from caveolae-like microdomains following IFN-{gamma} treatment of WISH cells. A, Isolation of caveolae-like microdomains from plasma membranes of untreated WISH cells (left panels) or cells treated with 3000 U/ml of IFN-{gamma} at 37°C for 5 min (right panels) was performed using a standard detergent-free method as described in Materials and Methods. Every second fraction from the OptiPrep gradient was subjected to SDS-PAGE and immunoblotting detection (ECL) with Abs to IFNGR-1 and caveolin-1 (upper panels), and then reprobed with Abs to IFNGR-2 and caveolin-1 (lower panels). Gradient fractionation was stopped at the interphase of the OptiPrep gradient and the plasma membrane extract at the bottom of the tube, which was then discarded. B, Localization of IFNGR-1 in total cellular lipid microdomain fraction from WISH cells as determined by cold Triton X-100 extraction. Triton X-100 lysates were prepared from untreated (left panels) or IFN-{gamma}-treated (3000 U/ml, 5 min; right panels) WISH cells, and fractionated by floatation through a discontinuous sucrose density gradient as described in Materials and Methods. Fractions of 1 ml were collected from the top of the gradient and aliquots subjected to SDS-PAGE and immunoblotting with Abs to IFNGR-1 and caveolin-1.

 
To compare the compartmentation of IFNGR-1 in the isolated plasma membrane, as described above, with the general distribution within total lipid microdomains from all cell compartments, we used established cold detergent (Triton X-100) extraction with sucrose gradient fractionation procedures to isolate total lipid microdomains (16, 17, 18). Fig. 1GoB shows the profile for IFNGR-1. Again, lipid microdomain fractions were marked by caveolin-1. Quantitation of the bands within the lipid microdomain fractions and normalizing for the total caveolin-1 in all fractions outside the detergent soluble fractions (the nonspecific fractions at the bottom of the tube) showed that IFNGR-1 levels were reduced by up to 40% following IFN-{gamma} treatment for 5 min. Note that the detergent-insoluble fractions also represent lipid microdomains from regions other than the cell surface plasma membrane (in contrast to Fig. 1GoA). Thus, a large portion of the IFNGR-1 chain continues to be associated with lipid microdomains. This includes the portion of IFNGR-1 that is endocytosed in lipid microdomain derived-vesicles after IFN-{gamma} stimulation. Overall, we can conclude that IFN-{gamma} stimulation induces the endocytosis of IFNGR-1 from lipid microdomains. However, the internalized IFNGR-1, continues to be associated with lipid microdomains.

We have previously shown that one destination of the selectively endocytosed IFNGR-1 is the nucleus (20). We next determined whether the selective endocytosis of IFNGR-1 from caveolae-like/lipid raft domains following IFN-{gamma} treatment is related to the selective nuclear translocation of IFNGR-1. Endocytosis from such membrane microdomains is specifically inhibited by the drug filipin (23, 24), a cholesterol-binding agent that inhibits the function of caveolae-like microdomains and other related lipid microdomains. We thus examined the effect of filipin pretreatment on WISH cells that were subsequently treated with IFN-{gamma}. Fig. 2GoA shows the results of immunofluorescence studies on the nuclear localization of IFNGR-1 and STAT1{alpha} in WISH cells that were treated with filipin for 10 min before treatment with IFN-{gamma}. Filipin significantly inhibited the ability of IFN-{gamma} to induce the nuclear translocation of both IFNGR-1 and STAT1{alpha} (Fig. 2GoB). We next determined the ability of filipin to inhibit IFN-{gamma} activation of STAT1{alpha} in WISH cells. As can be seen in Fig. 2GoC, filipin treatment of cells followed by IFN-{gamma} resulted in inhibition of STAT1{alpha} tyrosine phosphorylation (compare lanes 1 and 2), consistent with the immunofluorescence data in Fig. 2GoA. Filipin inhibition of IFN-{gamma}-induced STAT1{alpha} phosphorylation was completely reversible (lane 3), because filipin-treated cells subsequently incubated for 2 h in the absence of filipin responded to IFN-{gamma} similarly to cells not treated with filipin (compare lanes 1 and 3). These results indicate that functional lipid microdomains are required for IFN-{gamma} signaling.



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  		FIGURE 2. Nuclear translocation of IFNGR-1 and STAT1{alpha}, and tyrosine phosphorylation of STAT1{alpha} is inhibited in filipin-treated cells. A, WISH cells were pretreated with 10 µg/ml of filipin for 10 min at 37°C, and then medium replaced with prewarmed (37°C) medium containing 3000 U/ml of IFN-{gamma}. Cells were then incubated for 30 min before being fixed and stained with Abs to IFNGR-1 and STAT1{alpha}, as indicated. Images were acquired and analyzed using a confocal fluorescence microscope system as described in Materials and Methods. B, Quantitation of images from A. The ratio of nuclear fluorescence intensity (Fn) to cytoplasmic fluorescence intensity (Fc), Fn/Fc, in all cells in the field was measured and the average Fn/Fc plotted (see Materials and Methods). Error bars represent SD of Fn/Fc values determined across three independent fields of cells. Differences were found to be significant to p < 0.001. C, Tyrosine phosphorylation is blocked by pretreatment of cells with filipin. Treatment with filipin and IFN-{gamma} was as in A. Upper panel, Cells were lysed and STAT1{alpha} was immunoprecipitated from whole cell extracts, immunoblotted, and probed with an Ab specific for Tyr701-phosphorylated STAT1{alpha} to monitor tyrosine phosphorylation using an ECL protein detection system. Lower panel, The blot was reprobed with anti-STAT1{alpha} Abs to follow total STAT1{alpha} immunoprecipitated. D, Filipin pretreatment of WISH cells does not inhibit 125I-IFN-{gamma} binding. WISH cells were pretreated with filipin for 10 min at 37°C before binding of 125I-IFN-{gamma} was determined at 4°C as described in Materials and Methods. Labeled IFN-{gamma} was added at the indicated concentrations and nonspecific binding was determined at each concentration in the presence of a 100-fold excess of unlabeled IFN-{gamma}. Samples were run in triplicate and error bars represent mean ± SEM.

 
In contrast to these functional effects, filipin pretreatment did not affect the binding of 125I-IFN-{gamma} to IFNGR on WISH cells (Fig. 2GoD). However, filipin did inhibit the subsequent internalization of 125I-IFN-{gamma} in these cells (data not shown; also see below). These data are similar to studies on the binding of cholera toxin, a well-established marker for lipid microdomains, to its receptor where it was shown that although filipin inhibited cholera toxin internalization and function, it did not block the binding of cholera toxin to its receptor (24).

We also examined the effect of filipin treatment on the nuclear translocation of IFN-{gamma} (Fig. 3GoA). WISH cells pretreated with filipin or left untreated were examined for their ability to endocytose and translocate extracellular 125I-IFN-{gamma} to the nucleus by SDS-PAGE analysis of nuclear extracts. Control cells were incubated at 4°C without filipin treatment. As can be seen, cells treated with filipin were strongly inhibited in their ability to import IFN-{gamma} into the nucleus. The levels of the constitutively expressed basal transcription factor, TAFII, were used as internal controls for protein loading. Treatment to block clathrin-dependent endocytic events by potassium-depletion of cells did not affect nuclear translocation of 125I-IFN-{gamma} (Fig. 3GoB). TAFII levels were again used as a control. Additional experiments are presented later in this manuscript that further examine the role of clathrin-dependent events in STAT1{alpha} nuclear translocation. Taken together, the above data show that IFN-{gamma} induces the activation (phosphorylation) and nuclear translocation of STAT1{alpha}, the nuclear translocation of IFN-{gamma}, as well as the selective endocytosis and nuclear translocation of the IFNGR-1 subunit of the receptor complex from within caveolae-like plasma membrane microdomains. Thus, these domains appear to be a primary site for initiation of IFN-{gamma}-dependent intracellular signaling pathways.



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  		FIGURE 3. Nuclear translocation of IFN-{gamma} is inhibited in cells blocked in caveolae-related endocytic processes, but not in cells inhibited in clathrin-dependent endocytic processes. A, Cells were pretreated with filipin as in Fig. 2Go, and incubated with 3000 U/ml of 125I-IFN-{gamma} for 10 min at 37°C. After washing to remove unbound 125I-IFN-{gamma}, cells were acid-washed to remove cell surface-bound IFN-{gamma} before isolating nuclei. Control cells were not treated with filipin but were incubated with 125I-IFN-{gamma} at 4°C before processing as above. B, Clathrin-dependent processes were blocked by potassium-depletion of cells as in described in Materials and Methods (K+-free, 37°C) at 37°C. Potassium-replete cells (K+, 37°C) were treated identically except that 10 mM KCl was included in all buffers. Control cells (K+, 4°C) were incubated at 4°C and then processed as above. All cells were acid-washed to remove surface-bound 125I-IFN-{gamma}. 125I-IFN-{gamma} was followed by SDS-PAGE, Western transfer, and autoradiography. Protein levels were monitored by immunoblotting for the nuclear protein TAFII.

 
Lymphocytes have been shown to lack caveolin-1, and hence, do not contain caveolae (29). However, these cells contain plasma membrane lipid microdomains or lipid "rafts" that are biochemically similar to caveolae (29), and have been shown to be the primary sites for signaling by receptors like the B cell and TCR complexes in lymphocytes (Refs. 18 and 25 ; reviewed in Refs. 26 , 27 , and 28). Thus, we examined the Jurkat T cell line that is a prototype for caveolin-negative lymphocytes (25) for the role of the caveolae-related lipid microdomains in IFNGR localization and IFN-{gamma} signaling. Isolation of lipid microdomains was performed using a standard cold detergent (Triton X-100) extraction procedure and fractionation on a sucrose gradient (16, 17, 18). Fractions were then assessed for presence of IFNGR-1 and IFNGR-2 (Fig. 4Go). Lipid microdomain fractions were marked by the presence of the tyrosine kinase p56lck, which is constitutively present in lipid microdomains (17, 26). Caveolin-1 was not detected in these fractions (data not shown). In contrast to WISH cells, in untreated Jurkat cells IFNGR-1 and IFNGR-2 were not associated with lipid microdomain fractions. However, treatment of Jurkat cells with IFN-{gamma} for 5 min showed the presence of IFNGR-1 and IFNGR-2 in lipid microdomain fractions. This suggests that in Jurkat T cells, IFN-{gamma} binding recruits IFNGR subunits into lipid microdomains. This recruitment of IFNGR chains into lipid microdomains in Jurkat cells is very similar to the established recruitment into lipid microdomains of other receptors in lymphocytes like the B cell receptor and the TCR, where a similar profile for these receptor complexes is seen after appropriate receptor stimulation (18, 25, 26). Further, a significant portion of IFNGR-1 appears to be associated with fractions of intermediate density, in comparison to the localization of IFNGR-2. This is analogous for what has been observed with the B cell receptor (18), and probably indicates the egress of IFNGR-1 out of the lipid microdomains over the time of stimulation, when compared with the relatively static distribution of IFNGR-2.



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  		FIGURE 4. Localization of IFNGR-1 and IFNGR-2 in lipid microdomains of lymphocytes. Cold Triton X-100 extraction of Jurkat cells treated with 3000 U/ml of IFN-{gamma} at 37°C for 5 min (right panels) or left untreated (left panels) was performed as described in Material and Methods and lipid microdomains were floated through a linear 5–30% sucrose gradient. Fractions of 1 ml were collected from the top of the gradient and equal aliquots were analyzed by SDS-PAGE and immunoblotting with Abs IFNGR-1, IFNGR-2, and the constitutive lipid microdomain kinase p56lck as marker.

 
To examine the functional significance for this localization, we tested the inhibitory effect of filipin on Jurkat cells with respect to the IFN-{gamma}-induced nuclear translocation of IFNGR-1 and STAT1{alpha}, as was done for WISH cells above. As can be seen in Fig. 5GoA, IFN-{gamma} treatment of Jurkat cells caused the nuclear translocation of IFNGR-1 and STAT1{alpha}, but not of IFNGR-2 (Fig. 5GoB). Although not as dramatic as in WISH cells, the movement of IFNGR-1 into the nucleus is verified by the deconvolved 2D projections of multiple confocal image planes. This selective translocation of IFNGR-1, but not IFNGR-2, in Jurkat cells is similar to the effects in WISH cells (Fig. 2GoA), and to our previous studies on the nuclear translocation of these subunits (20). Pretreatment of Jurkat cells with filipin strongly inhibited the IFN-{gamma}-inducible nuclear translocation of IFNGR-1 and STAT1{alpha} (Fig. 5Go, A and B). Quantitation of the data is presented in Fig. 5GoC (differences were found to be significant to p < 0.005). These data show that lipid microdomains are important sites for IFN-{gamma} signaling in Jurkat cells, and are consistent with the localization of IFNGR subunits in lipid microdomains after IFN-{gamma} stimulation (Fig. 4Go). Thus, in Jurkat T lymphocytes, IFN-{gamma} treatment causes the recruitment of IFNGR subunits into lipid microdomains from where signaling leading to the nuclear translocation of IFNGR-1 and STAT1{alpha} is initiated. Also, though the dynamics of the specific events leading to lipid microdomain-dependent signaling may differ in fibroblast cells (WISH) and lymphocytes (Jurkat), our data show that lipid microdomains ultimately play an important role in IFN-{gamma} signaling in both cell types.



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  		FIGURE 5. IFN-{gamma}-induced nuclear translocation of IFNGR-1 and STAT1{alpha} in Jurkat T cells is inhibited by filipin pretreatment. Jurkat cells were starved and then pretreated, where indicated, with 1 µg/ml of filipin for 1 h at 37°C, before the addition of 3000 U/ml of IFN-{gamma} for 30 min in the presence of filipin. Cells were fixed and immunofluorescently stained simultaneously with Abs to IFNGR-1 and STAT1{alpha} (A), or IFNGR-2 and STAT1{alpha} (B). Multiple confocal image planes were acquired and deconvolved and 2D projections were displayed as described in Materials and Methods. The light white outline indicates the extent of the nucleus in respective cells. Data are representative of two separate experiments. (C). Quantitation of images presented in A. Pixel quantitation was performed as described in Materials and Methods. The average of the ratios of the nuclear fluorescence (Fn) to fluorescence in non-nuclear regions (Fc) for individual cells within two fields has been plotted. Differences in values for IFNGR-1 in treated and untreated cells were found to be significant to p < 0.005. D, Quantitation for images presented in B, performed as in C.

 
To confirm the primary nature of the caveolae-like domains in signaling, we also determined whether the alternative clathrin-dependent endocytic pathway had any contribution in IFN-{gamma} signaling. We used two standard methods for inhibition of clathrin-dependent processes: acidification of the cytosol and potassium depletion of cells (19). Fig. 6Go shows the results of immunofluorescence experiments involving acidification of the cytosol of IFN-{gamma}-treated and untreated cells using NH4Cl. As a control for clathrin-mediated uptake, we used Texas Red-labeled transferrin, which has been well-characterized as a marker for the clathrin-dependent pathway (reviewed in Ref. 30). For activation of IFN-{gamma} signal transduction, we followed the nuclear translocation of STAT1{alpha} that occurs with IFN-{gamma} treatment. In untreated cells (no cytosol acidification), transferrin (Fig. 6Go, middle panel, red) is internalized and accumulates in a typical perinuclear fashion. Treatment of these cells with IFN-{gamma} also showed nuclear translocation of STAT1{alpha} (upper panel, green). Acidification of the cytosol of the cells (Fig. 6Go, lower panel) inhibited endocytosis and perinuclear accumulation of transferrin, but did not affect the IFN-{gamma} induction of the nuclear translocation of STAT1{alpha}, suggesting that nuclear translocation of STAT1{alpha} is a clathrin-independent process.



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  		FIGURE 6. Inhibition of clathrin-dependent endocytosis by cytosol acidification does not inhibit IFN-{gamma}-induced STAT1{alpha} nuclear translocation. WISH cells subjected to cytosol acidification as described under Materials and Methods. Control cells (Not Acidified) were treated similarly to acidified cells except that NH4Cl and amiloride were omitted from buffers at the appropriate steps. Cells (middle and lower panels) were then treated with 5 µg/ml of AlexaFluor 594-labeled human transferrin alone (No IFN), or a mixture of AlexaFluor-labeled transferrin (5 µg/ml) and 3000 U/ml of IFN-{gamma} (IFN-{gamma} treated) for 30 min at 37°C. Upper panel, Control cells were prepared as above, except that they were left without treatment with transferrin or IFN-{gamma}. Cells were then washed to remove unbound transferrin and then fixed and stained with Abs to STAT1{alpha}. Images were acquired and analyzed using a deconvolution fluorescence microscope system described in Ref. 20 .

 
Growth of cells in a potassium-depleted medium similarly inhibits clathrin-dependent endocytosis (19). In the absence of IFN-{gamma}, cells grown in potassium-containing medium (Fig. 7Go, upper panel, K+-plus) again showed perinuclear accumulation of transferrin after its endocytosis (red), and uniform distribution of unactivated STAT1{alpha} (green). Potassium-depletion of cells (Fig. 7Go, middle panel, K+-depleted) in the absence of IFN-{gamma} blocked the internalization and perinuclear accumulation of transferrin, while STAT1{alpha} was diffuse in these cells. When the same cells were treated with IFN-{gamma} (Fig. 7Go, lower panel, K+-depleted), STAT1{alpha} nuclear translocation proceeded as expected, further supporting the conclusion that signal transduction from the IFNGR complex that leads to STAT1{alpha} nuclear translocation is not initiated via clathrin-dependent processes.



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  		FIGURE 7. Inhibition of clathrin-dependent endocytosis by potassium depletion of cells does not inhibit IFN-{gamma}-induced nuclear translocation of STAT1{alpha}. WISH cells were subjected to potassium depletion as described in Materials and Methods. Control cells (K+ Plus) were treated similarly to acidified cells except that 10 mM KCl was added to all buffers. Cells (middle and lower panels) were then treated with 5 µg/ml of AlexaFluor 594-labeled transferrin alone (No IFN-{gamma}), or a mixture of Alexa-labeled transferrin (5 µg/ml) and 3000 U/ml of IFN-{gamma} (IFN-{gamma} treated) for 30 min at 37°C. Cells were then washed to remove unbound transferrin and then fixed and stained with Abs to STAT1{alpha}. Images were acquired and analyzed using a deconvolution fluorescence microscope system described in Materials and Methods.

 
Further support that clathrin-dependent events are not primary for IFN-{gamma} signal transduction comes from the study of HeLa cells that can be induced to overexpress a dominant-negative mutant of the GTPase dynamin-1, a GTPase that is required for clathrin-dependent endocytosis (13, 14). In these HeLa cell clones, expression of a K44A dominant-negative mutant of dynamin-1 strongly and specifically inhibits clathrin-dependent endocytosis, without affecting processes from lipid microdomains (31). This system uses a tetracycline-repressible expression construct allowing one to study the same clone in the presence and absence of the mutant dynamin. Fig. 8GoA shows the results from K44A dynamin mutant cells under induced (upper and middle panels) and noninduced (lower panel) conditions. As can be seen, overexpression of the mutant K44A dynamin-1 does not affect the ability of IFN-{gamma} to induce the nuclear translocation of STAT1{alpha} (Fig. 8A, compare upper panel with middle panel). STAT1{alpha} nuclear translocation in IFN-{gamma} cells that were induced to express mutant dynamin-1 were indistinguishable from similar cells in which mutant expression was repressed (Fig. 8GoA, compare middle and lower panels). Cells expressing dynamin K44A were found to be inhibited in transferrin uptake, consistent with defective clathrin-dependent endocytosis (data not shown). HeLa cells similarly transfected to overexpress wild-type dynamin-1 gave results identical to those seen in the dynamin mutant cells with respect to nuclear translocation of STAT1{alpha} (Fig. 8GoB). Thus, the above data together demonstrate that the IFNGR signal transduction that leads to STAT1{alpha} nuclear translocation is a clathrin-independent process. These results are consistent with our previous conclusion that the caveolae-like membrane microdomains are primary sites for the initiation of IFN-{gamma} signaling transduction pathways.



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  		FIGURE 8. Expression of a dominant-negative dynamin mutant, dynamin K44A, or wild-type dynamin, had no effect on IFN-{gamma}-induced nuclear translocation of STAT1{alpha}. A, HeLa cells transfected with the dominant-negative dynamin K44A mutant were induced to express the mutant (upper and middle panels) by tetracycline withdrawal, or left uninduced (lower panel) by retaining tetracycline in the medium as described in Materials and Methods. Cells were then treated with 3000 U/ml IFN-{gamma} at 37°C, or left untreated, as indicated. Cells were then fixed and stained with Abs to dynamin-1 and STAT1{alpha}. In uninduced cells, staining for dynamin-1 is from the endogenous protein. B, HeLa cells similarly transfected to overexpress wild-type dynamin-1 were treated as in A. Images were acquired and analyzed using a deconvolution fluorescence microscope system as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The IFNGR complex has been localized to caveolae-like plasma membrane domains/lipid microdomains in unstimulated cells (11, 12), but the significance of this to IFN-{gamma} signal transduction is unknown. In this study, we demonstrate for the first time that following IFN-{gamma} stimulation, IFNGR-1, but not IFNGR2, is selectively endocytosed and translocated to the nucleus from these lipid microdomains. These domains are also the primary sites for the endocytosis and nuclear translocation of the ligand IFN-{gamma} and for the initiation of signaling events that lead to the phosphorylation and nuclear translocation of STAT1{alpha}, the transcription factor that drives many of the IFN-{gamma}-activated genes. We have also shown that lymphocytes, which do not express the prototypical caveolae lipid microdomains, use other noncaveolae lipid microdomains as the primary sites for IFN-{gamma} signaling. The pattern of IFNGR-1 migration into lipid microdomains following IFN-{gamma} binding in the Jurkat T cell line is remarkably similar to the behavior of the B cell and TCR complexes following ligand interaction (18, 25, 26). In contrast, the clathrin-dependent pathway does not contribute significantly to these IFN-{gamma} signaling events.

The selective endocytosis of IFNGR-1 and its nuclear translocation from lipid microdomains following IFN-{gamma} binding is consistent with our previous studies showing the selective nuclear translocation of IFNGR-1, but not IFNGR-2, in WISH cells after stimulation with IFN-{gamma} (20). This "passive" role of IFNGR-2 is in keeping with the characterization of its role as a simple framework for the attachment and presentation of Janus kinase (JAK)2 (32), which is required for the activation of receptor signaling events that lead in part to the phosphorylation and activation of STAT1{alpha} (32). In this regard, we have shown that IFN-{gamma}, through its C terminus, interacts with the cytoplasmic domain of IFNGR-1 at a site immediately adjacent to that for JAK2 binding, and that this binding of IFN-{gamma} enhances the affinity of JAK2 for IFNGR-1 severalfold (33). Others have shown that JAK2, which in the unactivated receptor complex resides on IFNGR-2, is recovered as a complex with IFNGR-1 only after activation by IFN-{gamma} (34). This would suggest that the interaction of IFN-{gamma} with IFNGR-1 shifts the equilibrium of JAK2 from IFNGR-2 to IFNGR-1. The above may also explain why IFNGR-2 remains associated with membrane lipid microdomains and is "removed" from further intracellular signaling events.

Of the three molecules studied–IFN-{gamma}, IFNGR-1, and STAT1{alpha}–whose translocation to the nucleus is initiated from caveolae-related domains, only IFN-{gamma} contains a known NLS (3). Although an intrinsic NLS is implicit in the current dogma of STAT signaling, to date STATs have not been shown to possess an NLS demonstrable in a simple nuclear localization assay as has been shown for other NLSs including the IFN-{gamma} NLS. Others have recently acknowledged the absence of an intrinsic NLS in STAT1{alpha} (35). We have shown previously that following activation and recruitment of phosphorylated STAT1{alpha} to IFNGR-1, the NLS of IFN-{gamma} mediates the formation of a complex of IFN-{gamma}/IFNGR-1/STAT1{alpha} with NPI-1, which has been specifically implicated in the nuclear import of STAT1{alpha}. Thus, IFN-{gamma} in effect chaperones the nuclear entry of STAT1{alpha}. In these events, IFNGR-1 may act as the "adapter" on which STAT1{alpha} and IFN-{gamma} are brought together.

Recent studies with the angiotensin II receptor (AT1) provide support for the need for STAT1{alpha} to be chaperoned to the nucleus. In the case of the AT1 receptor system, it has been shown that tyrosine phosphorylation of STAT1{alpha} by AT1 alone is not sufficient to induce its nuclear translocation (36). The ability of STAT1{alpha} to bind to the AT1 receptor appears to be required for STAT1{alpha} nuclear translocation by AT1 (36). STAT1{alpha} that is not attached to the receptor can be phosphorylated and dimerized in the cytoplasm but is not translocated to the nucleus. This is contradictory to current dogma that suggests that STAT1{alpha}, once phosphorylated and dimerized, leaves the receptor and translocates as a dimer to the nucleus. An answer to this dilemma is apparent from earlier studies on the AT1 receptor that showed that the receptor itself is translocated to the nucleus upon AT1 stimulation through a NLS in AT1 (37). Thus, the attachment of STAT1{alpha} to the receptor now provides a NLS that is required to transport it to the nucleus, explaining the previous observations with the AT1 receptor. These studies are consistent with our model that suggests that STAT1{alpha} needs to be chaperoned into the nucleus by components of the ligand/receptor system that activates it.

Similar to the AT1 receptor system, studies with other STATs have also suggested that phosphorylation may neither be a neces-sary nor sufficient condition for nuclear translocation of STAT1{alpha} and gene activation (38, 39, 40). For example, mutant STAT1{alpha}, where the phosphorylation-targeted Tyr701 is replaced by Phe, is still competent to activate transcription, suggesting that tyrosine phosphorylation of STAT1{alpha} is not necessary to activate nuclear genes (38). Phosphorylation of Tyr701 is not required for binding of STAT1{alpha} to IFNGR-1. Similarly, epidermal growth factor can induce the nuclear translocation of STAT2 in the absence of tyrosine phosphorylation (39). It is of interest that the epidermal growth factor receptor undergoes endocytosis from caveolae (41). Studies with chimeric STAT1, STAT2, and STAT5 further demonstrate a disconnect between the processes of phosphorylation/dimerization and nuclear translocation since, for example, chimerics between STAT2 and STAT1 that were apparently otherwise capable of binding DNA remained phosphorylated and dimerized in the cytoplasm but did not translocate to the nucleus (40). Thus, phosphorylated and dimerized STAT1{alpha} depends on additional molecules, which our previous data identify as components of the activating ligand/receptor complex, to chaperone it to the nucleus. Tyrosine phosphorylation of STAT1{alpha} most likely promotes dimer formation that is required for DNA binding, but does not per se facilitate the nuclear translocation of these molecules. This may explain why EMSA assays using cytoplasmic extracts of activated STATs can detect DNA complexes.

The question of why the IFNGR complex segregates within caveolae-like domains, at least during the processes of early signaling that we have studied, is not entirely understood. However, some insight may be gleaned from the current report and other recent studies on certain protein toxins such as cholera toxin, nonenvelope viruses like SV40 (42, 43), and intracellular pathogens like Chlamydia species among others (44) that use the caveolae pathway, which showed that these agents traffic in caveolin-containing vesicles that are separate from the recently characterized Rab family-associated endocytic pathways. Interestingly, these caveolin-containing vesicles, or caveosomes, traffic along a pathway independent of the clathrin pathway to perinuclear compartments (including Golgi and endoplasmic reticulum) that bypass the lysosomes and hence degradation (42). Caveosomes also retain a neutral pH throughout, and these properties ensure the viability and integrity of trafficking proteins and pathogens (42, 43, 44). In contrast, endocytosis via clathrin-dependent pathways is well-established to connect with endosomes that use the Rab family of adapter molecules, have an acidic pH, and feed into the lysosomal pathway (30, 45). Thus, caveolae-related vesicles may not be used for degradation/recycling, a function possibly performed preferentially by the clathrin-dependent pathway, but may be used for targeted delivery of intact cargo to specific cellular compartments and possibly aid in extending intracellular signal transduction mechanisms.

In the context of the nuclear translocation of the IFN-{gamma}/IFNGR-1/STAT1{alpha} complex, where IFNGR-1 and STAT1{alpha} are probably translocated to the nucleus intact via the IFN-{gamma} NLS, the acidic nature of clathrin-dependent endosomes and their connection to the lysosomal pathway would disrupt such complexes. The choice of a caveosome pathway may thus be selected to ensure undisrupted delivery of the ligand-receptor complex and any attached cargo away from acidic environments and the lysosomal pathway to intracellular compartments like the nucleus, where their function in their intact state may be required. The fate of IFN-{gamma} or IFNGR-1 once they exit the nucleus remains to be determined with respect to endocytic vesicles. The above scenario does not preclude other signaling pathways that originate from the IFNGR complex that do not depend directly on IFN-{gamma} or IFNGR-1 trafficking, or on STAT1{alpha}, and may involve other endocytic pathways. Thus, further study into the mechanisms of endocytosis of IFNGR-1 along the caveolae-related and other lipid microdomain pathways should provide insight into any such segregation of IFN-{gamma} signaling mechanisms.


   Acknowledgments
 
We thank Dr. Sandra Schmid (Scripps Institute, La Jolla, CA) for providing dynamin-expressing HeLa clones. This manuscript is Florida Agriculture Experiment Station Journal Series R-08929.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant CA38587 (to H.M.J.). Back

2 Address correspondence and reprint requests to Dr. Prem S. Subramaniam, Department of Microbiology and Cell Science, University of Florida, Room 1052, Building 981, Gainesville, FL 32611. E-mail address: prem{at}ufl.edu Back

3 Abbreviations used in this paper: IFNGR, IFN-{gamma} receptor; NLS, nuclear localization sequence; NPI-1, nucleoprotein interactor 1; JAK2, Janus kinase 2; AT1, type 1 angiotensin II receptor; 2D, two-dimensional; 125I-IFN-{gamma}, 125I-labeled IFN-{gamma}. Back

Received for publication March 25, 2002. Accepted for publication May 31, 2002.


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 Materials and Methods
 Results
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