auberate (DSS Pierce Chemical Co.) freshly prepared in dimethyl 15,000 X g for .... 78,3-14. Pestka. S.. Lamer. J. A.. Zoon. K. C., and Samuel, C. E. (1987) Annu.
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 30, Ieaue of October 25,pp. 19875-19877,1991 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Communication Human Interferon Omega ( w ) Binds to the a/@Receptor*
EXPERIMENTAL PROCEDURES
Interferons-Human IFN-a, -0, and "y were prepared as reported (Tarnowski et al., 1986; Moschera et al., 1986; Kung et al., 1986). HuIFN-w waskindly provided by Dr. G. Bodo (Ernst-Boehringer Institut (Received for publication, June 17,1991) fim Arzneimittelforschung, Austria). Hu-IFN-aA-P1, amodified IFNa that contains a phosphorylation site, was prepared as described (Li Idhaliz FloresS, ThomasM. Mariano, and et al., 1989). The antiviral activity of IFN-fi and -7 was measured on Sidney Pestka WISH cells, and of Hu-IFN-a and-w on MDBK cells, by a cytopathic From the Department of Molecular Genetics and effect inhibition assay with vesicular stomatitis virus as thechallenge Microbiology, University of Medicine and Dentistry of virus (Familletti et al., 1981). New Jersey-Robert Wood Johnson Medical School, Cells-Daudi (human lymphoblastoid cells) and WISH cells (huPiscataway, New Jersey 08854-5635 man amnioncells) were grownin RPMI-1640 medium and Dulbecco's modified Eagle's Medium (Sigma), respectively, supplemented with 10% heat-inactivated fetal bovine serum and 50 pg/ml gentamicin It was proposed that human interferon omega (w) bindstotheinterferon a/fl receptorbutnot to the sulfate. Cells were incubated in ahumidified incubator supplemented 5% COz at 37 "C. For binding and cross-linking assays, WISH interferon y receptor. However,since no studies were with cell monolayers were washed with phosphate-buffered saline (PBS), performed to provide directevidence for this hypoth- released with 1 X trypsin in PBS (1min at 37 "C) and resuspended esis, we carried outcross-linkingexperiments andsat- in 10 ml of medium. Both Daudi, a suspension cell line, and WISH uration binding assays between a s2P-labeledhuman cells were counted with a hemocytometer, pelleted by centrifugation at 1500 rpm for 6 min, and resuspended in fresh medium a t the interferon-a (Hu-IFN-a)andunlabeledHu-IFN-aA, -8, -7, and -w. These assays demonstrated that Hu-IFN- indicated densities. Labeling of Hu-IFN-d-P1 and Hu-IFN-y with 32P-The proceaA,-8, and -a,but not Hu-IFN-y, were able to block for labeling Hu-IFN-aA-P1 and Hu-IFN-y with 32Phas been bindingofs2P-labeledHu-IFN-aA to human cells. dure reported (Rashidbaigi et al., 1985; Kung and Bekesi, 1986; Mariano These results indicate thatHu-IFN-w binds to the a/@ et al., 1987; Mariano and Pestka, 1991; Kumar et al., 1988, Li et al., receptor. 1989). Briefly, 1 pg of interferon was incubated at 30 "C for 60 min with 1 mCiof [y-32P]ATP(6000 Ci/mmol; Du Pont-New England Nuclear) and 30 units of the catalytic subunit of CAMP-dependent protein kinase (15 unitslpl in 324 mM dithiothreitol; Sigma) in a reaction mixture of 30 p1 containing 100 mM NaCl, 1 2 mM MgCl,, Interferons (IFNs)' are potent antiviral and antiprolifera- and 20 mM Tris. HCl, pH 7.4. The reaction was stopped by the tive proteins secreted by cells in response to viruses and other addition of 0.4 ml of a cold (4 "C) solution of 5 mg/ml bovine serum agents. Three classes of interferons have been differentiated albumin (radioimmunoassay grade; Sigma) in 10 mM sodium pyroon the basis of antigenicity, biochemicalproperties, and pro- phosphate buffer, pH 6.7. The 32P-labeledinterferon was dialyzed 4 ducer cell: IFN-a, IFN-@,and IFN-7 (Pestka and Baron, 1980; times against 250 ml of 10 mM sodium pyrophosphate, pH 6.7, at 4 "C. The specific activities of the phosphorylated interferons after Pestka, 1986; Pestka et al., 1987). IFN-a and -8, which have dialysis ranged from 26 to 34 pCi/pg for Hu-IFN-aA-P1 and 73 to been grouped together as type I interferons (Pestka and 123 pCi/Ng for Hu-IFN-y. Labeled interferons were aliquoted in small Baron, 1980), bind to the same receptor on the cell surface: volumes, then frozen and stored in a liquid nitrogen freezer until use. the a//3 receptor. On the other hand, IFN-7, known as type Prior to their use, labeled interferons were thawed at room temperaI1 interferon, binds t o a different receptor, the receptor ture and centrifuged a t 14,000 X g for 2 min in a microcentrifuge. Competition between p2P]Hu-ZFN-d-P1 or r2P]Hu-IFN-y and (Adolf, 1987; Pestka et al., 1987). Unlabeled Interferons-Daudi cells were used for assaying the alp In 1985, three groups independently reported the discovery receptor, while WISH cells were used for the y receptor. Cells were of a novel type of interferon (Capon et a l , 1985; Feinstein et centrifuged and resuspended at a density of 1 X 10' cells/ml. Twoal., 1985; Hauptmann and Swetly, 1985)which is now termed fold dilutions of [32P]IFN-aA-P1(28 or 34 pCi/pg) or [32P]IFN-y IFN-omega ( w ) (Pitha-Rowe et aZ., 1990). Due to a sequence (73.3 pCi/pg), starting with 5 X lo6 cpm/ml cells, were added to 0.1 ml of the cell suspensions (1 X lo6 cells) in the presence or absence homology of about 60-70% between IFN-w and IFN-a (Feinof an excess of competing ligands (unlabeled Hu-IFN-aA, $3,-7, or stein et al., 1985; Hauptmann and Swetly, 1985), IFN-w was w ) , and incubated a t room temperature for 60 min on a rocking device. originally considered a subfamily of IFN-a (thus its old des- Then, 40 pl of the reaction mixture were layered on top of 350 p1 of ignation as IFN-aII) (Shirono et al., 1990; Capon et aL, 1985). 10% sucrose in PBS in 0.4-ml polypropylene tubes and centrifuged 2 Although it was speculated that IFN-w binds to the a//3 min a t 14,000 X g. Tubes were frozen in liquid nitrogen, and thetips, receptor, no definitive experiments were carried out to deter- which contained the cell pellets, were cut off and placed in scintillation vials to measure the radioactivity bound to the cells with a mine whether it binds to that receptor, t o the IFN-./ receptor, Beckman LS3801 liquid scintillation counter. The radioactivity presor to a novel receptor. The experiments reported here were ent in the remainder of the tube was also counted to determine counts of unbound ligand. Total radioactivity present in each reaction was aimed at answering this question. the sum of the bound and the unbound counts/min (cpm). Specific * This study was supported by United States Public Health Service binding was defined as the quantity of the labeled ligand bound in Grants CA-46465 and AI-25914 (to S. P.). The costs of publication the absence of competing ligand (total binding) minus that bound in of this article were defrayed in part by the payment of page charges. the presence of a 100-fold excess of unlabeled ligand (nonspecific This article must therefore be hereby marked "advertisement" in binding). accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. Covalent Cross-linkingof pzP]ZFN to Cells-Cells were centrifuged 4 Supported by a NationalScience Foundation graduatefellowship. and resuspended a t 5 X lo6 cells/ml. About 1 X lo6 cpm of [ 3 2 P ] H ~ 'The abbreviations used are: IFN, interferon; PBS, phosphate- IFN-aA-P1 or [32P]Hu-IFN-y were added to 0.3 ml of the cell susbuffered saline; Hu,human; DSS, disuccinimidyl suberate;SDS, pension with or without an excess of unlabeled ligand as competitor sodium dodecyl sulfate. (Hu-IFN-aA, -fi, -7, or - w ) and were incubated for 60 min at room
19875
Human Interferono Binds to the (YIPReceptor
19876
FIG. 1. Binding of [82P]Hu-IFNaA-P1 to Daudi cells: competition by unlabeled interferons. [32P]HuIFN-aA-P1 (28 or 34 pCi/pg) was added to Daudi cells in the absence (total binding) or presence (nonspecific binding) of exceas unlabeled Hu-IFN-cuA (2.9 pg/ml; panel A ) , -w (2.9 pg/ml;panel B ) , -0 (4.0 pg/ml; panel C),or -y (2.9 pg/ml; panel D ) , as described under "Experimental Procedures." Specific binding, seen with all interferons but IFN-y, is the difference between the total and the nonspecific binding. Insets show the Scatchard analyses of the specific binding data. B, radioactivity bound to cells; F, radioactivity of unbound ligand.
E
B
Y
3
E D
c
a
m
Total [32P] Hu-IFN-d-P1 (cprn x 109) 20
20
Hu-IFN-UA
A
Hu-IFN-OI
B
FIG. 2. Binding of [3aP]Hu-IFN-y to WISH cells: competition by unlabeled interferons. [32P]Hu-IFN-y (73.3 pCi/pg) was added to WISH cells in the absence (-) or presence (+) of excess unlabeled Hu-IFN-aA ( A ) , -w ( B ) ,-0 ( 0 ,or -Y (Dl (0.88 pglml). Specific binding, only seen with IFN-r, is the difference between the total (-) and the nonspecific binding (+). Inset shows the Scatchard analysis of the specific binding data. B , radioactivity bound to cells; F, radioactivity of unbound ligand.
a
5
10
15
Total [32P] Hu-IFN-y (cprn x 10-4) temperature on a rocking device. Bound 32P-labeledinterferons were cross-linked to thereceptors by the addition of 50 mM disuccinimidyl auberate (DSS Pierce Chemical Co.) freshly prepared in dimethyl sulfoxide to a final concentration of 0.5 mM. After a 20-min incubation at 4 'C, excess unreacted DSS was eliminated by the addition of Tris.
HC1, pH 7.5, to a final concentration of20mM, and incubation for an additional 10 min. Cells were then pelleted by centrifugation at 15,000 X g for 20 s and extracted with 120 pl of 0.5% (v/v) Triton X100 in PBS for 20 min at 4 "C. After centrifugation for 10 min at 4 "C, extractable proteins were analyzed by SDS-polyacrylamide gel
Human Interferon w Binds to the a l p Receptor
19877
IFN-aA-P1 and [32P]Hu-IFN-y to cells in the presence or absence of an excess of unlabeled ligands is shown in Fig. 3. Gel electrophoresis of products from the cross-linking of [“PI Hu-IFN-aAP1 to cells showed that there was competition between Hu-IFN-aA, -0, and -o for the same receptor, whereas cross-linking of [32P]Hu-IFN-y to cellswasonly inhibited by unlabeled Hu-IFN-y. These results provide further evidence to support our hypothesis that Hu-IFN-w binds to the a l p receptor. All the results show definitively that Hu-IFN-o inhibits binding of Hu-IFN-aA, and not of Hu-IFN-y, to cells. Accordingly, as hypothesized, Hu-IFN-a, -P, and -obind to the a/@ receptor but not to they receptor. FIG. 3. Covalent cross-linking of [32P]Hu-IFN-aA-P1 and [32P]Hu-IFN-yto receptors on cells: competition by unlabeled interferons.[32P]Hu-IFN-aA-P1and[“PIHu-IFN-y were crosslinked to Daudi and WISH cells, respectively, and the TritonX-100 extracts were analyzed by SDS-polyacrylamide gel electrophoresis as described under “ExperimentalProcedures.” Binding to thecells was carried out in the absence (-) or presence of an excess of unlabeled Hu-IFN-aA, -u,-8, and -7. Only the portion of the gel that contains the cross-linked bands is shown. Arrows point to the IFN-receptor complexes. electrophoresis on an 8% polyacrylamide gel (Laemmli, 1970). The gel was dried under vacuum and exposed to x-ray film (Kodak XOmat) with an intensifying screen. RESULTS AND DISCUSSION
Acknowledgment-We thank Dr. Jerome Langer for many suggestions throughout this study. REFERENCES Adolf, G. R. (1987) J. Gen. Virol. 68, 1669-1676 Capon, D. J., Shepard, N. M., and Goeddel, D. V. (1985) Mol. Cell. Biol. 5, 768-779 Familetti. P. C.. Rubinstein.. S.. . and Pestka. S. (1981) Methods Enzyrnol. 78,387-394 Feinstein, S. I., Mory, Y., Chernajovsky, Y., Maroteaux, L., Ni, U., Lavie, U.,and Revel, M. (1985) Mol. Cell. Biol. 5, 510-517 Hauptmann, R., and Swetly, P. (1985) Nucleic Acids Res. 13, 47394749
Kumar, C. S., Mariano, T. M., Noe, M., Deshpande, A. K., Rose, P. M., and Pestka, S. (1988)J. Biol Chem. 263, 13493-13496 Kung, H.-F., and Bekesi, E. (1986) Methods Enzymol. 119, 296-301 Kung, H.-F., Pan, Y.-C., Moschera, J., Tsai, K., Bekesi, E., Chang, M., Sugino, H., and Honda, S. (1986)Methods Enzymol. 119,204-
CompetitionbetweenUnlabeled Interferons and f2P]Hu210 IFN-d-PI-The ability of Hu-IFN-a, -P, -7, and -oto block binding of radiolabeled Hu-IFN-aA-P1 was studied by satu- Laemmli, U.K. (1970) Nature 227,680-685 B.-L., Langer, J. A., Schwartz, B., and Pestka, S. (1989) Proc. ration binding experiments in which cells were incubated with Li,Natl. Acad. Sci. U.S. A. 86,558-562 varying amounts of the labeled ligand in the presence or Mariano, T. M., andPestka, S. (1991) in Cytokines: A Practical absence of an excess of non-radioactive interferon. When HuApproach (Balkwill, F. R., ed) pp. 95-108, Oxford University Press, IFN-aA-P1 was usedas thelabeled ligand, IFN-a, -@, and -o, Oxford but not -7, were able to block the binding of the labeled ligand Mariano, T. M., Kozak, C. A., Langer, J. L., and Pestka,S. (1987) J. Biol. Chem. 262,5812-5814 to the cell receptors. These data clearly indicate that IFN-a, Moschera, J. A., Woehle, D., Tsai, K. P., Chen, C., and Tarnowski, IFN-P, and IFN-o bind to the same receptor (Fig. 1). ScatS. J. (1986) Methods Enzymol. 119, 177-182 chard plots (Scatchard, 1949) yielded a Kd of 6.6-8.8 X 10”’ Pestka, S. (1986) Methods Enzymol. 119, 3-14 Pestka, S., and Baron, S. (1981) Methods Enzymol. 78,3-14 M and 12,000-20,000 a/B receptors/Daudi cell. Competition between Unlabeled Interferons and p2P]-Hu- Pestka. S.. Lamer. J. A.. Zoon. K. C., and Samuel, C. E. (1987)Annu. Rev. Biochem. 56,727-777. IFN-y-Binding assays were also performed in which Hu- Pitha-Rowe, P. M., Finter, N., Pestka, S., and Testa, J. (1990) J. IFN-y was used as the radiolabeled ligand. Hu-IFN-aA, -P, Interferon Res. 10, 353 and -owere unable to compete with Hu-IFN-y for binding to Rashidbaigi, A., Kung, H.-F., and Pestka, S. (1985) J. Biol. Chem. 260,8514-8519 the cells. Only an excess of unlabeled Hu-IFN--y wasable to block [32P]Hu-IFN-ybinding to the cells (Fig. 2). This indi- Scatchard. G. (1949)Ann. N. Y. Acad. Sci. 51.660-672 Shirono, H., Kono, K., Koga, J., Hayashi, S., Matsuo, A., and Hiracates that IFN-o, like IFN-a andIFN-@,does not bind to the tani, H. (1990) Biochem. Biophys. Res. Commun. 168, 16-21 IFN-y receptor. Tarnowski, S. J., Roy, S. K., Liptak, R. A., Lee, D. K., and Ning, R. Cross-linking-Analysis of the cross-linking of [ 3 2 P ] H ~ - Y. (1986) Methods Enzymol. 119,153-165 ,
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