Proc. Nati. Acad. Sci. USAVol. 89, pp. 11706-11710, December 1992Immunology

Identification of a functionally important sequence in the Cterminus of the interferon-y receptorMICHAEL A. FARRAR, JACQUELINE D. CAMPBELL, AND ROBERT D. SCHREIBER*Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110

Communicated by Paul E. Lacy, September 21, 1992

ABSTRACT We have previously shown that the intracel-lular domain of the interferon-v (IFN-y) receptor plays anobligate role in receptor-medited al transduction. More-over, we have specificaly identified two regions within thehuman IFN-y receptor's intraceflular domain required forfunctional activity: the membrane-proximal 48 aino acidsrequired for both functional activity and receptor-mediatedligand internalization and the C-terminal 39 amino acidsrequired exclivel for biologic response induct. Herein wereport the identcation of the 3 amino acids within theC-terminal region of the receptor that are obligtorily requiredfor receptor function. By using a set of overlapping t omutants, the inimal conal sequence within the C-termi-nal region was localized to residues 434-444 (APTSFGYD-KPH). By m utating each individual residue within this se-quence to alanine, three residues (Tyr-440, Asp-441, andHis-444) were idetifias being critical forIFN-t(i) upregulation of major ili complex a Iproteins, (i) activation ofthe IFN regulatory factor 1 gene, and(iii) stimulation of cells to produce nitric oxide. The moreconservative Tyr-440 Phe substitution also resulted in anonfunctional receptor. Subsequent mutational analysis of allfive of the IFN-y receptor's intracellular tyrosine residuesrevealed that Tyr-440 was the sole tyrosine required forreceptor activity. These results thus identify a unique sequencein the EFN-y receptor that is required for intitiop o IFN-y-dependent biologic responses and highlight the importance ofthe hydroxyl side chain of Tyr-440 in this process.

Interferon y (IFN-y) is a pleotropic cytokine produced byactivated T cells and natural killer cells that plays an impor-tant role in regulating host defense and immunopathologicprocesses (1, 2). IFN-y mediates its effects on cells byinteracting with a single high-affinity receptor expressed atthe target-cell surface (3-8). Functionally active human ormurine IFN-y receptors are composed ofat least two species-matched polypeptides: the IFN-y receptor itself and anaccessory component encoded on human chromosome 21 ormurine chromosome 16, respectively (9-13). Furthermore,the species-specific interaction between the receptor and theaccessory component is now known to occur within thereceptor's extracellular domain (14-16).We have demonstrated (12) that the intracellular domain of

the IFN-y receptor is also required to form a functionallyactive receptor, although its action is not species-specific.These experiments were performed with murine fibroblastlines that either lacked (L cells) or contained (SCC16-5 cells)a single copy of human chromosome 21. L cells stablytransfected with the full-length human IFN-y receptor ac-quired the ability to bind and internalize human IFN-ybut didnot respond to it. In contrast, SCC16-5 cells expressing thehuman IFN-y receptor not only bound human IFN-y but alsoresponded to it. Expression in SCC16-5 of a mutant IFN-y

receptor that lacked all but 3 amino acids of the intracellulardomain resulted in the formation of a nonfunctional receptorunable to internalize ligand or induce biologic responses.Using a family of intracellular-domain deletion mutants, weidentified two large regions within the receptor's intracellulardomain that contained all the elements required for receptor-mediated ligand internalization and functional activity. Onewas localized to the 48 amino acids closest to the membrane(residues 256-303) and was required for receptor-mediatedligand internalization and functional activity. The other waslocalized to the 39 amino acids at the receptor's C terminus(residues 434-472) and was required exclusively for biologicresponse induction.This observation has led to subsequent studies aimed at

identifying the specific residues required for receptor func-tion. In this way we hope to gain insights into the molecularmechanisms used by the receptor to effect IFN-t-dependentbiologic responses. In the current study, we have focused onthe C-terminal region of the receptor since it contains ele-ments that are only required for biological activity and not forreceptor internalization. We demonstrate herein that threeamino acids in the receptor's C-terminal functional domainare specifically required for receptor activity (Tyr440, Asp-441, and His-444) and document that the hydroxyl group ofTyr,440 plays a particularly important role in this process.

MATERIALS AND METHODSReagents. Purified recombinant human and murine IFN-y

were generously provided by Susan Kramer of Genentech.Purified recombinant human IEN-y and murine IFN-'y wereradioiodinated using the Bolton-Hunter reagent (ICN) asdescribed (12). Recombinant human IFN-a2 was generouslyprovided by Marvin Siegel (Schering-Plough). IFN-a2 is aspecies-specific form of human IFN-a. G418 was fromSigma. Lipopolysaccharide (LPS) derived from Escherichiacoli 0127:B8 by the method of Westphal was from Difco.

Antibodies. Human IFN-y receptor-specific monoclonalantibody (mAb) (GIR-208) was purified and biotinylated asdescribed (17). Goat anti-murine immunoglobulin-Sepharosewas prepared as described (18). mAb 114.1 recognizesmurine H-2Kk and was purified- from hybridoma culturesupernatants by protein A-Sepharose chromatography.Cels and Cell Culture. Normal murine L929 cells were

obtained from American Type Culture Collection. SCC16-5,a murine fibroblast cell line that contains a single copy ofhuman chromosome 21 (19), was generously provided byDavid Cox (University of California, San Francisco). Celllines were cultured as described (12).PlamId ContructIon. cDNAs encoding full-length, wild-

type, or mutant receptors were produced by PCR using

Abbreviations: IFNr'y, interferon y; IRF-1, interferon regulatoryfactor 1; IRU, international reference unit(s); LPS, lipopolysaccha-ride; MHC, major histocompatibility complex; NO, nitric oxide;mAb, monoclonal antibody.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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primers based on the nucleotide sequence of the humanIFN-y receptor (hgR) cDNA (3). The constructs were clonedinto the pSFFV expression vector as described (12). Theaccuracy of all PCR-generated DNA was confirmed bydideoxynucleotide sequencing (Sequenase; United StatesBiochemical).DNA Transfection. SCC16-5 and L929 cells (5 X 105 cells)

were stably transfected with 25 Ag of plasmid by usingthe calcium phosphate precipitation method (20) and se-lected by using G418 (12). Selected cell lines (SCC.hgRA445-472, YF440, and PA443) were subsequently cloned by lim-iting dilution. Results shown for SCC.hgRA445-472, YF440,and PA443 are of representative clones. Similar results wereobtained with bulk transfected populations.

Demonstration of Human and Murine IFN-y Receptors onTransfected Murine Cells. Receptor expression was moni-tored by flow cytometry (using biotinylated GIR-208 andstreptavidin-phycoerythrin) and by radioligand binding anal-ysis as described (12).

Analysis of Transfected Murine Cells for Responsiveness toHuman IFN-y. The ability ofhuman IFN-y to enhance majorhistocompatibility complex (MHC) class I antigen expressionon transfected murine fibroblasts was examined as described(12). For induction of nitric oxide (NO), cells were seeded at1 x 105 cells per well in a Costar 24-well tissue culture plateand allowed to adhere overnight. Cells were then stimulatedwith LPS (10 pug/ml), recombinant murine IFN-y (500 IRU/ml), recombinant human IFN-y (500 IRU/ml), or variouscombinations of the above agents at the same concentration.After 48 h of stimulation, supernatants were harvested andthe level of nitrite was determined by the Greiss reaction (21).To measure interferon regulatory factor 1 (IRF-1) gene in-duction, cells were grown to 90o confluency in Costar T-162tissue culture flasks and treated with medium, recombinanthuman IFN-'y (1000 IRU/ml), or recombinant murine IFN-y(1000 IRU/ml) for various amounts of time. RNA wasprepared using published protocols (22). RNA (10 jig perlane) was subjected to electrophoresis in a 1% agarose/formaldehyde gel and analyzed on a Northern blot as de-scribed (23) using full-length IRF-1 cDNA (24) labeled by therandom-primer method (25). To control for equal loading ofRNA, membranes were stripped and reprobed with a full-length /3-actin probe.

RESULTSIdentification ofan 11-Amino Acid Linear Sequence at the C

Terminus of the IFN-y Receptor Required for Biologic Re-sponse Induction. Our study (12) showed that the C-terminal39 amino acids of the IFN-y receptor contained elements thatwere obligatorily required for IFN-y-mediated enhancementofMHC class I protein expression. To map the functionallyimportant residues within this portion of the receptor using asingle amino acid mutagenesis approach, we had to morenarrowly define the limits of this region. A truncation mutantwas constructed that lacked the C-terminal 28 amino acidsand was stably expressed in SCC16-5 cells. This particulartruncation site was chosen because human, murine, and ratIFN-y receptors show remarkable sequence identity in the 11amino acids upstream from this site and considerably lessidentity in the residues downstream from this position. Asshown in Fig. 1 Left, all three receptor constructs could bestably expressed in SCC16-5. SCC16-5 cells expressing eitherthe full-length human IFN-y receptor (SCC.hgR, 1300 recep-tors per cell) or the receptor truncation mutant lacking theC-terminal 28 amino acids (SCC.hgRA445-472 expressingcomparable numbers of human IFN-y receptors based onflow cytometry analysis) responded to human IFN-y whereasSCC16-5 cells expressing the receptor truncation mutantlacking the C-terminal 39 amino acids (SCC.hgRA434472,

SCC.hgR

SCC.hgRtA445-472'

SCC.hgRA434472

APTSFGYDKPH

: / i

:Isl ::iH::N Reetr :H ls

Expression OdUtifs:Huorecenc Intnsit

FIG. 1. Functionally important residues of the human IFN-yreceptor's C terminus are localized to 11 amino acids. (Left) HumanIFN-y receptor expression using the biotinylated mAb GIR-208.(Right) Five hundred thousand SCC.hgR (sorted bulk line) (Top),SCC.hgRA445-472 (representative clone) (Middle), or SCC.hgRA434-472 (sorted bulk line) (Bottom) cells were cultured in 10 mlof medium for 72 h either alone (dotted lines) or with human IFN-y(1000 IRU/ml; solid lines). Cells were harvested, washed, and thenstained forMHC class I expression by using 114.1 mAb. Expressionwas quantitated by flow cytometry. Hu, human.

2100 receptors per cell) did not (Fig. 1 Right). As measuredusing enhancement ofMHC class I expression, the responseinduced by the full-length receptor (56-channel shift) and thehgRA445-472 truncation mutant (65-channel shift) were in-distinguishable. This data thus localized the functionallyimportant C-terminal region to the 11-amino acid sequence ofresidues 434-444 (APTSFGYDKPH).

Point Mutational Analysis of the IFN-yR 's C Ter-minus. To identify the critical functional residues within this11-amino acid sequence, a panel of receptor point mutantswas generated wherein each residue was changed individu-ally to alanine (Fig. 2) and then stably expressed in SCC16-5cells. All receptor-expressing transfected cell lines wereisolated by bulk sorting and expressed equivalent amounts ofhuman IFN-'y receptor mutants by flow cytometry (data notshown). The cell lines were then tested for their ability torespond to IFN-yby quantitating enhancement ofMHC classI expression. Whereas eight of the cell lines when stimulatedwith human IFN-'y responded normally, three lines contain-ing receptors with alanine replacements at Tyr-440, Asp-441,or His444 were completely devoid of a response. All celllines remained responsive to their homologous murine IFN-yligand, indicating that there was no defect in the general

Amino Acid Sequence

A P T S F G Y D K P HMean Charnel

Shift

A.. . . . . . . . 54**A * * * * * * * * 59

A**A * * * * * ' * 64****A * * * * * * 47*****A * * * * * 56*** * * * A * * * * 0*** * * * A * * * 0********A * * 60*****A * 65

* A 0

FIG. 2. Identification offunctionally important amino acids in theC terminus of the human IFN-y receptor. IFN-y receptor pointmutants were generated by replacing each residue between positions435 and 444 (Left) with alanine, and stably expressed in SCC16-5.Five hundred thousand cells of each cell line were cultured in 10 mlof medium for 72 h with or without human IFN-y at 1000 IRU/ml.Cells were stained for MHC class I expression. (Right) Valuesrepresent the mean channel shift increase over basal levels of classI MHC expression after stimulation with human IFN-y. Valuesshown are the average of two or more experiments.

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hgR-y44ctl,

hgR-A440z/

FluorescencelItensity'uMHC ClassI)

hgR-F440

FIG. 3. Tyr-440 is required for receptor function. Five hundredthousand SCC.hgR (Top) or SCC16-5 cells expressing the Tyr440-Ala mutation (YA440; cell line) (Middle) or the Tyr-440-* Phemutation (YF440; representative clone) (Bottom) were cultured in 10ml of medium for 72 h either untreated (dotted lines) or treated withhuman IFN-'y (1000 IRU/ml). Class I MHC expression was quanti-tated by flow cytometry.

pathway ofMHC class I induction. Furthermore, all cell linescontained human chromosome 21 (as assessed by monitoringexpression ofthe human IFN-a receptor) and thus expressedthe human IFN-'y receptor accessory component (data notshown).Tyr-440 Is Required for Receptor Function. Since tyrosine

residues have been shown to be important for signaling in avariety of other receptor systems (26, 27), we proceeded toexamine the functional significance of Tyr-440 more fully. Amore conservative Tyr-440 receptor mutant was constructedin which tyrosine was replaced by phenylalanine instead ofalanine. This receptor construct was then stably transfectedinto SCC16-5 cells, and receptor expression and responsive-ness to murine IFN-y and human IFN-a2 were confirmed.SCC16-5 expressing either the Tyr -* Ala receptor mutant(YA440) or the Tyr -- Phe mutant (YF440) were unrespon-sive to human IFN-y for MHC class I enhancement (Fig. 3).This result not only confirms that Tyr-440 plays a critical rolein effecting IFN-y-receptor-mediated enhancement of class IMHC expression but also demonstrates that the hydroxylgroup present on the tyrosine side chain is needed for thisprocess.To examine whether Tyr-440 was unique in its functional

importance among the other tyrosine residues in the recep-tor's intracellular domain, each of the other four intracellulardomain tyrosines were altered and the mutant receptors weretested for functional activity in mediating the upregulation ofMHC class I. This was accomplished either by point mutationofTyr-287 or -294 to Ala or by deleting the regions containingTyr-380 or -462. None of these mutations ablated receptorfunction. Thus Tyr-440 is the only tyrosine residue in theIFN-y receptor's intracellular domain that is important formediating IFN-y-dependent MHC class I enhancement.

Tyr-440, Asp-44l, and His-444 Are Required for MultipleIFN-r-Inducible Functions. We next examined whether thefunctional importance of these three intracellular domainresidues was generalizable to other IFN--inducible re-sponses, specifically induction of IRF-1 gene expression andstimulation of NO production in the transfected cell lines.IRF-1 gene induction was monitored because it has beencharacterized as a relatively rapid (1-2 h) cellular response toall forms of IFN (24) and, therefore, may involve a less-complex signal transduction pathway than that needed for the

enhancement ofMHC class I proteins (which requires 48-72h for maximal effects). Northern blot analysis showed thateither murine or human IFN-y could induce IRF-1 geneexpression in SCC.hgR (data not shown). IRF-1 mRNA wasmaximally induced by human IFN-y at 2 h and did notsignificantly diminish over the next 30 h. As shown in Fig. 4A (Northern blot) and B (densitometry), IRF-1 mRNA waspresent only at low levels in resting SCC.hgR cells (lane 1) butwas induced 6-fold after stimulation with human IFN-y for 2h (lane 2). In contrast, human IFN-y did not induce IRF-1mRNA in SCC16-5 expressing the mutant human IFN-yreceptors YA440, YF440, DA441, or HA444. Unresponsive-ness was not due to a generalized inactivation of the IRF-1induction signal transduction pathway since each cell linebearing a mutant human IFN-y receptor responded to its ownmurine IFN-y (lanes 5, 8, 11, and 14; range, 5.1- to 6.3-fold).Comparable RNA loading in each lane was verified byprobing the filters with a 3-actin probe (Fig. 4C). In asubsequent experiment, the functional importance of Lys-442 and Pro-443 within the YDKPH sequence was assessedusing IRF-1 gene induction. Exposure of SCC16-5 cell linesexpressing the KA442 and PA-443 receptor mutants tohuman IFN-y led to expression of IRF-1 mRNA at levelscomparable to cells expressing the wild-type human receptor(data not shown). Thus the same receptor residues requiredfor enhancement of MHC class I expression were alsorequired to induce IRF-1 gene expression.We next examined the ability of human IFN-y to prime

cells for production of NO. This biologic response is inter-mediate in the kinetics of its induction (18-48 h) and isdifferentiated from both MHC class I enhancement and IRF-1gene induction by its absolute requirement for two activationsignals. NO is a product of the enzymatic conversion ofL-arginine to L-citrulline (28). Whereas in certain cells thisconversion occurs at a low level by a constitutively expressed

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FIG. 4. Tyr-440, Asp-441, and His-444 are required for IRF-1mRNA induction. SCC.hgR or SCC16-5 expressing the YA440 (cellline), YF440 (representative clone), DA441 (cell line), or HA444 (cellline) receptor mutants were untreated or stimulated for 2 h withhuman IFN-y (1000 IRU/ml) or murine IFN--y (YA440, YF440,DA441, or HA444 only; 1000 IRU/ml). Cell monolayers were lysed,RNA prepared, and 10 ,ug loaded per lane. Blots were sequentiallyprobed with a full-length IRF-1 cDNA probe (A) and then a full-length P-actin cDNA probe (C). IRF-1 mRNA levels were quanti-tated by densitometry (B). Integrated area is expressed as OD xmm2.

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A

:-B

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.-Em L929.SCC.hgR-h-R--

[3LIk 1luFtlyEHuIFNy

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3.

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o. 4.: YFL44=A4 KA4jA443YA440 YF440 DA441 KA442 PA443

FIG. 5. The same receptor residues required for MHC class Iupregulation and IRF-1 mRNA induction are also required for NOpriming by IFN-y. (A) Establishment ofthe minimal requirements forNO production by murine fibroblasts. SCC.hgR or L929.hgR cellswere cultured in medium for 48 h alone or with murine (Mu) IFN--y(500 IRU), human (Hu) IFN-y (500 IRU), LPS (10 u&g), or thecombinations of murine IFN-y (500 IRU) plus LPS (10 Mg) or humanIFN-y (500 IRU) plus LPS (10 Mg). Supernatants were harvested andnitrite determined by the Greiss reaction. (B) Demonstration thatTyr-440, Asp-441, and His-444 are selectively required for NOinduction. SCC.hgR or SCC16-5 cells expressing the YA440 (cellline), YF440 (representative clone), DA441 (cell line), KA442 (cellline), PA443 (representative clone), or HA444 (cell line) pointmutants were cultured for 48 h with 10 ,g of LPS and either 500 IRUof murine IFN-y or human IFN-y. Supernatants were harvested andnitrite determined by the Greiss reaction.

NO synthase enzyme, most cells can be stimulated to gen-erate high amounts of NO by a distinct NO synthase that isinduced when cells are treated with IFN-y and triggered byeither endogenous or exogenous stimuli such as tumor ne-crosis factor, interleukin 1, or LPS (29, 30). NO is a short-lived reactive nitrogen species. However, its generation canbe monitored by quantitating the accumulation of its stableoxidation product, nitrite (NO2) by using the Greiss reaction(23).SCC.hgR did not produceNO either constitutively or when

treated with recombinant murine IFN-'y, human IFN-'y, orLPS alone (Fig. 5A). In contrast, NO was induced in thesecells after stimulation with LPS plus murine or human IFN-y(3.8 or 2.8 nmol of nitrite per 1 x 105 cells, respectively).Normal murine L cells, expressing wild-type human IFN-yreceptors, produced NO in response to treatment with mu-rine IFN-y and LPS but not to human IFN-y plus LPS (Fig.5A). Thus NO production shows the same requirement forthe IFN-y receptor accessory molecule as does MHC class Ienhancement. In these and all subsequent experiments, theproduction ofNO was shown to be effected by the cytokine-inducible NO synthase because NO generation was inhibitedby aminoguanidine, a specific inhibitor of this enzyme (31).

Fig. SB demonstrates that the same three amino acids in theC terminus of the human IFN-y receptor required for signal-

ing for upregulation of MHC class I and induction of IRF-1gene expression are also required for human IFN-y-dependent priming of cells for NO production. Tyr,440(YA440 and YF440), Asp-441 (DA441), and His-444 (HA444)mutants did not signal forNO induction in stably transfectedSCC16-5 cells treated with human IFN-y and LPS. In con-trast, the same cells were fully responsive when exposed tothe homologous murine IFN-y ligand in combination withLPS. The two receptor point mutants within the YDKPHsequence that were functional in MHC class I enhancementin SCC16-5 cells treated with human IFN-y (KA442 andPA443) were also functional in the NO induction assay (Fig.5B). In a subsequent experiment, the receptor mutants withalanine replacements at positions 435-439 (PTSFG), whichwere functional in the class I MHC induction assay, weretested for the ability to signal for NO induction. All wereactive in this assay as well (data not shown). Thus, the resultsobtained using three distinct biologic response assays docu-ment that the sequence of YDXXH (Tyr-440, Asp441, andHis 444), as it occurs in the membrane distal portion of theIFN-'y receptor, is of broad importance in promoting IFN--mediated cellular responses.

DISCUSSIONIn this communication we identify three distinct amino acidspresent in the C-terminal region ofthe human IFN-y receptorthat are obligatorily required for receptor function. Theseresidues are Tyr440, Asp441, and His 444. Replacement ofany of these amino acids with alanine abrogated the capacityof the IFN-y receptor to induce three temporally distinctcellular responses: IRF-1 gene induction, NO production,and enhancement ofMHC class I protein expression. More-over, replacement of Tyr44O with Phe also led to -thegeneration of an inactive receptor. The latter result demon-strates that the hydroxyl group of Tyr440 is of particularimportance for expression of a functional IFN-y receptor.These observations thereby identify an amino acid sequencethat is important in receptor-dependent biological responses.These studies relied on the use of an experimental system

wherein the function of different human IFN-'y receptormutants could be evaluated by expressing them in murinefibroblasts containing a single copy ofhuman chromosome 21(SCC16-5) (12). The use of SCC16-5 was critical since theformation of a functional human IFN-y receptor requires thepresence of a species-specific accessory molecule encodedby a gene on human chromosome 21. The goal of the currentstudy was to map the functionally important amino acids thatreside within a region at the receptor's C terminus, which wehave shown (12) to be required for receptor activity. Using atruncation analysis approach similar to that used in ourprevious report (12), we restricted the functionally importantregion at the receptor's C terminus to an 11-amino acid linearsequence. Through a subsequent point mutational analysis,we identified the three critical amino acids needed for induc-tion of different biological responses. The functionally crit-ical residues occur within a pentameric sequence, YDKPH(residues 440-444), that is absolutely conserved in human,murine, and rat IFN-y receptors. The preservation of thissequence is remarkable since the intracellular domains ofthehuman and murine IFN-y receptors are only 55% identical(4). This observation is consistent with previous findingsusing chimeric human-murine IFN-y receptors, which indi-cated that the intracellular domains of the human and murinereceptors are functionally interchangeable (14-16). More-over, the results also support the generally held concept thatfunctionally critical residues in proteins tend to be conserved.

In contrast to the high degree of homology shown by thissequence between IFN-y receptors of different species, theYDXXH sequence does not match any known consensus

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signaling motif nor does the 11-amino acid linear sequence inwhich it is found exhibit significant sequence homology tosequences found in other known signaling receptors. Thismay not be surprising as the IFN-y receptor belongs to thetype II cytokine receptor family (32, 33) about which little iscurrently known.The second messenger pathways utilized by the IFN-y

receptor to transduce signals remain obscure. Analysis of theprimary structures of the human and murine IFN-y receptorshas failed to reveal any sequence homology to any knownkinases or phosphatases (3-8, 33). Therefore, the mechanismby which the functionally critical amino acids identifiedherein are involved in signaling is not clear. The resultsobtained with the YF440 mutant indicate that the hydroxylgroup present on the tyrosine residue's side chain is criticalfor function. This suggests that Tyr-440 is involved in astructurally critical hydrogen bond or that it is a site fortyrosine phosphorylation. We have obtained preliminarydata suggesting the importance of tyrosine phosphorylationin the development of IFN-y-mediated biologic responses.Treatment of Colo-205 or U937 with several different tyrosinekinase inhibitors (i.e., herbimycin A, tyrphostin, or genistein)inactivated their ability to respond to human IFN-'y andexpress MHC class II proteins (M.A.F. and R.D.S., unpub-lished observations).

Previous studies in which IFN-y receptor phosphorylationwas examined did not detect the presence ofphosphotyrosine(34, 35). In these studies the kinetics of phosphorylation werefound to be rather slow, with maximal phosphorylationoccurring 30 min after stimulation with a saturating dose ofIFN-y. Subsequent phosphoamino acid analysis detectedphosphoserine and phosphothreonine but not phosphoty-rosine (34). However, the previous observations do notexclude that a rapid and transient phosphorylation of tyrosinemay be involved in receptor signaling. In particular, theoriginal phosphoamino acid studies were carried out on cellsstimulated for 30 min with ligand because that was the timepoint at which total receptor phosphorylation was maximal.Tyrosine phosphorylation, however, is often a rapid andtransient event occurring as early as a few seconds anddisappearing as quickly as a few minutes after receptorstimulation. A recent example of this point is the ligand-dependent tyrosine phosphorylation of the IgE Fc6 receptorI, which becomes maximal 5 sec after receptor crosslinkingand subsequently decays to background levels within 5 secafter receptor disengagement (36). Therefore, if a rapidtyrosine phosphorylation pathway exists for the IFN-y re-ceptor, as for the Fc6 receptor, it would not have beendetected in the earlier studies.

We thank Drs. H. Harada and T. Taniguchi, (Osaka University,Japan) for supplying the IRF-1 cDNA; Dr. J. Matthews (WashingtonUniversity) for supplying the f-actin probe; and Dr. J. Corbett(Washington University), for his invaluable advice on NO. We arealso grateful to M. Florea for secretarial assistance. This work wassupported by National Institutes of Health Grants CA43059 andA124854.

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