JOURNAL OF INTERFERON RESEARCH 9:719-730 (1989)Mary Ann Liebert, Inc., Publishers

Characterization of Human Interferon-y ReceptorPurified from Placenta

SIMON STEFANOS, YONG HO AHN, and SIDNEY PESTKA

ABSTRACT

Membranes prepared from human placenta were used for characterization of the receptorfor human interferon-Y (HuIFN- y) after large-scale purification. HuIFN-y linked covalentlyto Affigel-10 was used for the purification of the receptor from octylglucoside-solubilizedplacental membranes. Radiolabeled IFN-y [32P]HuIFN-y, was used in binding and cross-

linking studies to detect the receptor at different stages of the purification. From bindingassays it was calculated that an average placenta contained 90-120 ;tg of receptor with a

Revalue of 1.3 x 10"9 M. Thus, human placenta is a rich and convenient source of receptorfor IFN- y. When purified receptor was cross-linked to [32P]HuIFN-y, a variety ofcross-linked bands were detected dependent on the preparation conditions. The use ofprotease inhibitors in the course of processing prevented degradation of the 90-kD intactreceptor, showing that the lower-molecular-weight products detected in previous studies are

degradation products of the receptor. Furthermore, a 20-kD fragment of the receptor wasfound to be active in binding HuIFN-y.

INTRODUCTION

Interferons (IFNs) are a group of distinct molecular species comprising three major classes, a, ß, andy.'1"*' They exhibit a variety of antiviral and immunoregulatory activities.'1"3,5,6' For IFNs to induce

physiological activity, they must first bind to a specific cell-surface receptor.'3,7"14' Binding inhibition andcross-linking studies have shown that IFN- a and IFN-ß bind to the same receptor.'3,14,15' The receptor forIFN- y is, however, distinct from the a/ß receptor.'3,14,16"18' The number of receptors for IFN-y varies fromcell to cell, usually ranging from undetectable levels to 20,000 per cell.'3,11,14,1*"22' However, a clearcorrelation between the number of the receptors on cells and the magnitude of the responsiveness of cellsto IFN has not been demonstrated. The gene for the human (Hu) IFN-y receptor has been mapped tochromosome 6q.'23' It has also been demonstrated that human chromosome 21 is required along with thechromosome 6 for HuIFN-y to induce HLA antigen expression on somatic cell hybrids.'24' Cross-linking

University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Department of MolecularGenetics and Microbiology, 675 Hoes Lane, Piscataway, NJ 08854-5635.

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STEFANOS ET AL.

studies show the existence of one or more specific bands (50 kD to 160 kD)!311141920-25-26) by using 125I-or 32P-labeled IFN-y, depending on the cells used or the experimental conditions, indicating that differentstructural subunits may exist. Other reports'26~28'suggested that the lower-molecular-weight species couldbe degradation products. Calculation of binding affinity from several laboratories showed that IFN- y bindsto a single class of high-affinity receptor with a Ká of 10~9 to 10"11 ^f^311141719-20) but low-affinity receptorshave also been reported.'314' 9) Ambiguity in the study of the IFN-y receptor can only be clarified by makingavailable large amounts of purified receptor, allowing studies of its structure and function as well as

distinction between low- and high-affinity receptors. Purification of limited amounts of HuIFN-y-receptorfrom cell cultures has been reported.'25'26' We previously presented preliminary data demonstrating that humanplacenta plasma membranes are a rich source of IFN- y receptors.'27' Here we report the characterizationof the HuIFN-y receptor purified from placenta. During the preparation of this manuscript reports appeareddescribing the purification of the HuIFN-y receptor from placenta'28' and cloning of the receptor.'29'

EXPERIMENTAL PROCEDURES

Chemicals and Reagents: Protease inhibitors (leupeptin, aprotinin, antipain, chymostatin, benzamidine,pepstatin, and PMSF), octylglucoside, and PEG 8000 were purchased from Sigma (St. Louis, MO); EDTAand Triton X-100 from Mallinckrodt (KY); NP-40 from BRL (Bethesda,MD); Mega-10 and Mega-8 fromBoehringer Mannheim Biochemicals (Indianapolis, IN). The bifunctional cross-linker disuccinimidylsuberate (DSS) was from Pierce (Rockford, IL). HuIFN- y was cloned material'31'32' that was purified as

described.'33'34' Purified HuIFN-y was prepared with the help of J. Langer (UMDNJ-Robert Wood JohnsonMedical School, Piscataway, NJ) and F. Khan (Hoffmann-La Roche, Nutley, NJ); several batches were

generously supplied by Y. Furuichi (Nippon Roche).Membrane Preparations: Fresh placenta was collected for each preparation from Robert Wood Johnson

University Hospital or St. Peter's Medical Center in New Brunswick, NJ, and plasma membranes were

prepared as reported.'35' Briefly, placenta was washed thoroughly with cold 0.1 M phosphate-buffered saline(PBS) containing a mixture of protease inhibitors (Leu-peptin 1 ng/ml, aprotinin 10 kallikrein U/ml, antipain2 p-g/ml, benzamidine 10 (ig/ml, chymostatin 1 p.g/ml, pepstatin 1 pg/ml, PMSF 0.5 mM, and EDTA 10 mAf).The connective tissue and umbilical cord were removed and the resulting tissue was then minced into smallpieces and homogenized with a Waring Blendor in the same buffer; debris was removed by low-speedcentrifugation (2,500 rpm for 30 min at 4°C). The supernatant was recovered and membranes were pelletedby high-speed centrifugation at 4°C (30 min at 100,000 x g). Each gram of the pellets was homogenizedin 100 ml of PBS containing protease inhibitors and stored in a liquid nitrogen freezer until use. A yieldof about 1 gram of membrane proteins was obtained per 400 grams of placenta.

Membrane Solubilization: Frozen membranes (100 ml) were thawed and solubilized by addition of 5 mlof octyl-ß-D-glucopyranoside (octylglucoside; 25%) to a final concentration of 1.25%, and solubilizationcontinued for 2 h at 0°C. Insoluble material was removed by centrifugation at 100,000 x g for 1 h at 4°C.The supernatant was diluted to 525 ml with PBS containing protease inhibitors to a final concentration of0.25% octylglucoside. Other detergents examined include Triton X-100, NP-40, Mega-8, and Mega-10.

Binding ofIFN-y to Membranes or Solubilized Receptor: The radiolabeled ligand ([32P]HuIFN-y) was usedfor binding experiments. IFN was labeled as reported previously with minor modifications.'36' Briefly, 1 p.gof purified HuIFN- y was incubated for 1 h at 30°C in the presence of cAMP-dependent protein kinase and0.5 mCi [ y-32P]ATP. The reaction was stopped by cooling to 0°C with subsequent addition of 200 pi of5 mg/ml of bovine serum albumin in 10 mM sodium pyrophosphate. The free ATP was removed by overnightdialysis against 10 mAf sodium pyrophosphate buffer, pH 6.5. Aliquots of [32P]IFN-y were stored in a liquidnitrogen freezer for further use. After labeling, a cytopathic effect inhibition assay with WISH cells andvesicular stomatitis virus'37' showed no loss of IFN- y activity.

For the binding assay, [32P]HuIFN-Y was incubated with placental membranes or with detergent-solubilized membrane supernatants at room temperature for 2 h. Reactions contained the following in a totalvolume of 0.2 ml of PBS: 1.5-2 x 105 cpm of [32P]HuIFN- y; 100-150 [ig of placental membranes, or 50

720

CHARACTERIZATION OF THE RECEPTOR FOR HuIFN-y

u;l of soluble receptor as noted in the legends to each figure. Placenta membranes were directly centrifugedfor 2 min at maximum speed in an Eppendorf microfuge to separate the receptor-ligand complex from freeIFN. Radioactivity in the pellet was measured in a scintillation spectrometer. The radiolabeled complex fromthe detergent-solubilized membranes was precipitated by means of polyethylene glycol 8000 (PEG 8000)as follows. After incubation, the reaction mixture was transferred to 4°C and carrier protein (1 mg of humanIgG in 25 p.1 of PBS or 25 pil of fetal calf serum) was added. To precipitate the complex, 106 u,l of 25%PEG 8000 (wt/vol) was added to a final concentration of 8%. The reaction was held at -20°C for exactly10 min, centrifuged at maximum speed in a tabletop Eppendorf centrifuge in a cold room (4°C) for 10 min,the supernatant discarded, and the radioactivity in the pellet counted. Nonspecifically bound counts weredetermined in the presence of a 500- to 1,000-fold excess of unlabeled HuIFN-y.

Binding of [32P]HuIFN- y to whole cells was performed as reported'20': The ligand bound to cells was

separated from free [32P]HuIFN-y by centrifugation over a 10% sucrose cushion, and the radioactivity in thepellet and supernatant were measured separately.

Purification of the HuIFN-y Receptor: Soluble detergent extract of human placental membranescontaining protease inhibitors and 0.25% octylglucoside were applied to a column containing HuIFN-ybound to Affigel-10. HuIFN-y was coupled covalently to Affigel-10.'38' The packed column was thenthoroughly washed with PBS containing 1 u.g/ml leupeptin, 10 kallikrein units/ml aprotinin, 10 mM EDTA,0.5 mM PMSF, and was eluted with 2 M guanidine hydrochloride in 20 mM sodium phosphate, pH 7.0. Thefractions were monitored by cross-linking with radiolabeled [32P]HuIFN-y for binding activity, and fractionscontaining receptor activity were pooled and concentrated about five-fold under vacuum (Spin-Vac, Savant,Inc.) before loading onto a new Affigel-10-IFN-y column. The second column was washed and elutedsimilarly and fractions that contained receptor activity as monitored by cross-linking were again pooled,concentrated with a Spin-Vac, and used for further studies.

Cross-linking of [32P]HuIFN-y to Placental Membranes: Either 150 [ig to 200 u.g of membrane proteins,or 30-50 p.1 of solubilized membranes or 5-10 |xl of purified receptors (20-30 ¡xg/ml as determined by aminoacid analysis) were incubated for 2 h with [32P]HuIFN-y (total volume 200 u.1) at room temperature. Abifunctional cross-linking reagent, dissuccinimidyl suberate (DSS), was then added to a final concentrationof 1 mM and incubation continued on ice for 20 min. The excess cross-linker was then neutralized with 10[ú of 1 M Tris-HCl, pH 7.0, followed by addition of an equal volume of two-fold concentrated sample buffer(125 mM Tris-HCl, pH 6.8, containing 0.02% bromophenol blue, 1% SDS, and 20% glycerol). Proteins were

separated on SDS-PAGE according to the method of Laemmli.'39' The gel was dried under vacuum and thenexposed overnight for autoradiography on X-Omat R film (Kodak).

SDS-polyacrylamide Gel Electrophoresis: Depending on the material to analyze, 8%, 10%, 12%, or

7-15% gradient gels were used as appropriate.'39' Samples were loaded with (reducing gels) or without(nonreducing gels) 1% 2-mercaptoethanol. Electrophoresis was performed overnight at 50 V or at 150-200V for 4 h for 16-cm gels and 150 V for 1 h for minigels (mini-protean II, Biorad). The gels were then stainedwith either Coomassie blue or silver, or dried directly and exposed for autoradiography.

RESULTS

Membranes from human placenta were prepared as described under Experimental Procedures.[32P]HuIFN-y was bound to placental membranes or to HL-60 cells, covalently cross-linked by treatmentwith DSS and analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 1). The HuIFN-y-receptor fromHL-60 cells can be visualized as one specific broad band at 110-120 kD, whereas the HuIFN-y-receptor fromplacental membranes appears as multiple bands with approximate sizes at 55, 70, 90, and 115 kD. The115-kD band is similar or very close to the 110- to 120-kD band detected with HL-60 cells. The 55, 70, and90-kD bands are not detected in HL-60 or other cell lines (data not shown).

In the initial experiments (Fig. 1), the placenta membranes were prepared by first washing the placentawith 20 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 0.5 mM PMSF, and 10 mM EDTA andthen homogenizing in 10 mMHEPES buffer, pH 7.4, with 150 mM NaCl, 0.5 mM PMSF, and 10 mM EDTA.

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STEFANOS ET AL.

92kd-

67kd-

43kd-

CROSS LINKING OF[32p]Hu-IFN-T

HL60 Cells

II i

r •

PLACENTAMEMBRANES

92kd

67kd

43kd

FIG. 1. Comparative cross-linking of [32P]HuIFN-y to HL-60 cells and placental membranes. HL-60 cells(1 x 107/ml, in 100 pi) or placenta membrane proteins (150-200 [ig) were incubated with 1 x 105 cpm of[ P]HuIFN-y in the presence (+) or absence (-) of 0.15 pg of unlabeled HuIFN- y. Free HuIFN-y was thenwashed away with PBS and cross-linking was performed with DSS as described under ExperimentalProcedures. The cross-linking reaction was quenched with 10 pi 1 M Tris-HCl, pH 7.0, and proteins were

analyzed by SDS-PAGE with a 10% gel. Prestained molecular weight markers (arrows) were used to estimatethe apparent molecular weight of the cross-linked specific bands. Placental membranes were preparedwithout the protease inhibitor cocktail: only 0.5 mM PMSF and 10 mM EDTA were present (see text).

The mixture of protease inhibitors was then added and the membranes were stored in a liquid nitrogen freezer.To assess the possibility of protease degradation during processing of placenta, we used a cocktail ofdifferentprotease inhibitors (see Experimental Procedures). The placenta was washed in PBS containing the inhibitorcocktail, and homogenization, solubilization, and purification steps were carried out in the same buffercontaining the protease inhibitors. Figure 2 shows the cross-linking pattern of such preparations, where one

major specific 115-kD band is labeled. Aprotinin and leupeptin were critical for the protection of the intactreceptor in the course of the processing and purification of the placenta. EDTA and PMSF were maintainedin the protease-inhibitor mixture.

The membranes were solubilized in PBS containing the inhibitor cocktail and used for purification.Different detergents were screened for their ability to solubilize the receptor with maximum recovery ofbinding and cross-linking activity. Triton X-100, Nonidet P40 (NP-40), octylglucoside, Mega-8, andMega-10 were used. Only octylglucoside proved to be capable of maintaining maximum receptor activity

722

CHARACTERIZATION OF THE RECEPTOR FOR HuIFN-y

92kd

67kd

43kd -*

PROTEASE INHIBTORS

NO

^^

YES

FIG. 2. Protection of HuIFN- y-receptor from placental membranes by protease inhibitors. Cross-linkingwas performed with placental membranes prepared in the absence (NO) or in the presence (YES) of a mixtureof protease inhibitors as described under Experimental Procedures. Prestained molecular weight markers(arrows) were used to estimate the apparent molecular weight of the labeled complexes.

as determined by ligand-binding and minimum degradation as assessed by the cross-linking assay. Figure3 shows the banding pattern after cross-linking of [32P]HuIFN-y to octylglucoside or Triton X-100-solubilized membranes. Two bands, a major band at 70 kD and a minor band at 115 kD, were detected withTriton X-100 solubilization, whereas solubilization with octylglucoside yielded a major single band at 115kD.

Binding of [32P]HuIFN- y was performed with membranes solubilized with octylglucoside as describedunder Experimental Procedures. The binding data were linearized by the method of Scatchard (Fig. 4). Fromthis analysis, the affinity constant was estimated to be 0.8 x 109 M"1 with a total of 90-120 p.g of bindingsites (assuming a size of about 90 kD for the receptor) per placenta. Consequently, we decided to use

octylglucoside-solubilized membranes for any further study of the receptor.The octylglucoside extract of placenta, in the continuous presence of protease inhibitors, was applied to

a first HuIFN-y-Affigel-10 column, washed with PBS containing all the protease inhibitors, and eluted with2 M guanidine hydrochloride in 20 mM sodium phosphate, pH 7.0. The eluate was dialyzed against 0.25%octylglucoside in PBS in the presence of aprotinin and leupeptin and assayed by ligand binding or

723

NO

STEFANOS ET AL.

FIG. 3. Solubilization of receptor-HuIFN-y complexes with different detergents. Placenta membranes (nodetergent) or octylglucoside (1.25% octylglycopyranoside) and a nonionic detergent (1% Triton X-100)solubilized membranes were centrifuged at 100,000 x g and supernatant (or wash) and pellets were separatelyincubated with 1 x 105 cpm [32P]HuIFN- y. Binding was followed by cross-linking with DSS and specificbands analyzed by SDS-PAGE in 10% polyacrylamide gels. Molecular weight standards are indicated witharrows.

cross-linking before loading to a second HuIFN-y column and eluted as detailed above. The cross-linkingassay shows one broad band at 90 kD to 115 kD [Fig. 5, autoradiography (+)2-ME] corresponding to a majorband at 70 kD and two minor bands at 85 and 90 kD on silver-stained gels, respectively [Fig. 5, silver staining(+)2-ME]. If the placenta is not processed immediately in the presence of buffer with protease inhibitors,the cross-linking of [32P]HuIFN-y to the purified receptor shows at least five specifically labeled bands at40, 55, 70, 90, and 115 kD, with corresponding bands on silver staining at 20, 30, 55, 70, and 90 kD,respectively (Fig. 6), showing that a fragment of 20 kD still binds [32P]HuIFN-y.

Proteins in the purified samples were quantitated by amino acid analysis. From one placenta, 20-40 p.gof purified receptor was recovered, resulting in a 20-40% yield of the calculated receptor per placenta. Thepresence of sulfhydryl groups was indicated by SDS-PAGE analysis in reducing and nonreducing conditionsas shown in Fig. 5. In the presence of 2-mercaptoethanol, the cross-linked purified receptor appears as a broadband at 90-115 kD [autoradiography (+)2-ME] and as a major band at 70 kD in a silver-stained gel [silverstaining (+)2-ME]. When this same [32P]HuIFN-y cross-linked receptor is revealed by autoradiography afterseparation of proteins on nonreducing gels [autoradiography (-)2-ME], it appears as a very high-molecular-weight complex at around 300 kD. Similarly, the direct visualization of the receptor by silver staining showeda major band that shifted to a complex of very high molecular weight (higher than 250 kD) in a nonreducinggel [silver staining (-)2-ME]. This behavior is not observed if placenta membranes are used instead of thepurified material as source of receptor for the cross-linking assay.

724

CHARACTERIZATION OF THE RECEPTOR FOR HuIFN-y

—i-1-1-1-1-r

0 25 50 75 100 125 150

P-P] HulFN-"} FREE (CPMxIO3)

FIG. 4. Binding of [32P]HuIFN-y to detergent-solubilized human placental membranes. Octylglucoside(1.25% wt/vol) solubilized membranes were centrifuged at 100,000 x g for 1 h at 4°C and 100-jil samplesof the resultant supernatant were incubated with various concentrations of [32P]HuIFN-y (and 0.1 ¡xg ofunlabeled IFN-y for determination of nonspecific binding) at room temperature for 2 h. The reaction mixturewas then cooled to 0°C and 25 u.1 of fetal calf serum was added as a carrier. The radioactive receptor-IFN-ycomplex was then precipitated with 8% PEG 8000 as described under Experimental Procedures. Specificallybound radioactivity was calculated by subtracting the nonspecifically bound counts from the total binding.The data obtained were linearized by the method of Scatchard (insert).

DISCUSSION

An important step toward understanding the mechanism of action of HuIFN-y and its structure-functionrelationship will be the characterization of its receptor. Unfortunately purification of the receptor is hamperedby its low abundance on cells and the difficulty of maintaining the functional integrity of these glycoproteinsonce solubilized.

In this study we show that the HuIFN-y receptor from human placenta is an excellent source for large scalepurification of HuIFN-y-receptors. The receptor from placenta membranes was monitored by cross-linkingand binding of [32P]HuIFN-y to follow extraction, degradation, and purification. Octylglucoside proved tobe the most effective detergent for extraction of receptor from placental membranes. Solubilized receptorsfrom placental membranes exhibited the highest molecular weight as a cross-linked complex whenmembranes were prepared in the presence of protease inhibitors. The most effective protease inhibitors were

aprotinin and Leu-peptin (see Results).The cross-linked receptor complex prepared with placental membranes migrated as a 115 kD cross-linked

725

STEFANOS ET AL.

EFFECTS OF 2-MERCAPTOETHANOLON THE Hu-iFN

-

7-RECEPTOR

200kd"

92.5kd.

68kd-

43kd-

200kd

92.5kd68kd

K-43kd

FIG. 5. Behavior of purified HuIFN-y receptor under reducing and nonreducing conditions. HuIFN-yreceptor purified by affinity chromatography after solubilization with octylglucoside was analyzed bySDS-PAGE either after cross-linking with [32P]IFN-y (autoradiography) in the presence (+) or absence (-)of excess unlabeled IFN-y or directly by silver staining the gel (silver stain). Electrophoresis was performedin the presence [autoradiography (+)2-ME and silver staining (+)2-ME] or absence of 2-mercaptoethanol[autoradiography (-)2-ME and silver staining (-)2-ME]. The first autoradiography (-)2-ME (center twolanes) is a l-to-5 dilution of purified receptor in the second autoradiography (-)2-ME (right two lanes).Arrows indicate molecular weight markers.

band in the presence or absence of 2-mercaptoethanol (results not shown). After solubilization, most of thecross-linked receptor complex is no longer detected as a 115-kD band in the absence of 2-mercaptoethanol:after cross-linking or silver staining, it migrates at a molecular weight higher than 250 kD. The parallel resultsbetween the cross-linked material and the silver-stained gel indicates a high degree of purification. Theseresults also suggest a role for unpaired or free sulfhydryls in the aggregation of the detergent-solubilizedreceptor.

Our studies with different protease inhibitors and several detergents showed that the molecular weight forthe intact receptor for HuIFN-y is 90kD, in agreement with that predicted from the DNA sequence of thecloned receptor.'29' This receptor is highly sensitive to placental proteases and is cleaved to lower-molecular-weight bands, as low as 20 kD, which are still capable ofbinding HuIFN- y as shown in Fig. 6 by cross-linkingof [32P]HuIFN-y bound to the intact receptor and degraded fragments (see Results). In fact, several studieshave shown low- and high-affinity receptors in binding studies with 125I- or 32P-labeled HuIFN-Y (3,n,i4,i9,20)jn cr0SS_ijn]Qng studies with intact cell lines, complexes with molecular weights of 115 to 160kD were identified.'18' In contrast, at least three distinct complexes (70, 92, and 160 kD) were demonstratedwhen cross-linking was performed on membranes prepared from several cultured cell lines in the absence

726

CHARACTERIZATION OF THE RECEPTOR FOR HuIFN-y

RADIOGRAPHY

95.5kd'

55kd'

46kd-H

36kd-

29kd'

SILVERSTAIN

•95.5kd

55kd>36kd 29kd

FIG. 6. Cross-linking pattern of HuIFN-y receptor purified from placental membranes prepared in theabsence of protease inhibitors. The receptor was cross-linked with [32P]HuIFN-y in the absence (-) or

presence (+) of excess unlabeled IFN-y and analyzed by SDS-PAGE on a 10% polyacrylamide gel (16-cmgel), exposed overnight for autoradiography, or directly analyzed by SDS-PAGE followed by silver staining(minigel). Arrows on the side of each gel indicate molecular weight markers.

of protease inhibitors'19'; a two-subunit model (53 kD + 75 kD = 128 kD receptor) was proposed based on

these results. However, cross-linking studies on several of the same lines demonstrated that a 115-kD bandwas produced when intact cells were used, whereas smaller bands were observed when membranes were

prepared by the procedure of Ucer et al. (i.A. Langer, personal communication). More recently, partialpurification of the receptor has been reported. Aguet and Merlin'25' identified two receptor bands at 90 kDand 50 kD. Novick et a/.'26' identified three bands at 95, 80, and 60 kD. It is highly likely that 90-kD bandrepresents the intact receptor and the 50-kD (-60 kD) band observed represents a degraded receptor fragmentas noted below.

Our results clarify some of the previous results. When cells were used for binding and cross-linking, theintact receptor with a molecular weight of 115 kD (corresponding to the 90-kD receptor in silver-stainedgels) was detected. When using membrane preparations, Ucer et a/,'19' Aguet et a/.,'25' and Novik eta/.'26'identified, in addition to the intact receptor, degraded forms of the receptor in the course of processingmembranes and purification. In the later stages of our current experiments, we learned of a similarpurification of HuIFN-y receptor from placental membranes with similar results.'28'

Solubilized receptor was also used for binding assay and Scatchard analysis. These assays were used to

727

STEFANOS ET AL.

quantitate the concentration of receptor in the placenta membranes. The calculated content was estimatedto be about 100 pg per placenta (250 pg/kg of placenta). The recovery after purification from two HuIFN-ycolumns was 20-40 pg per placenta, a yield of 20-40%. From these data the affinity constant of HuIFN-yto its receptor was calculated and found to be approximately 109 AT1. This value is slightly lower than theaffinity constant calculated for the receptor of intact cells, but still indicative of high affinity.

The purified receptor was used for characterization studies. Previously, we demonstrated thatintramolecular disulfide bonds were important for maintenance of a functional binding site.'35' Data fromSDS-PAGE gel elctrophoresis under reducing and nonreducing conditions (Fig. 5) of the purified receptorprotein demonstrated significant changes in the size of the cross-linked receptor on reduction. Together withthe observation that reduction eliminates the activity of the HuIFN-y receptor,'35' it is apparent cysteineresidues play a significant role in the binding domain of the IFN-y receptor. Many other receptors have a

cysteine-rich region, usually in the extracellular domain. Thus, the intact HuIFN-y receptor has an apparentmolecular weight of 90,000, is rich is cysteine residues (and disulfide bonds'35') and is highly glycosylated.'40'The predicted amino acid sequences of both the cloned murine'41' and human'29' IFN-y receptors also exhibita cluster of cysteine residues in the extracellular domain and a high level of asparagine-linked glycosylationsites. In fact, 17% and 20% of the human and murine IFN-y receptor, respectively, consists of serine andthreonine, with serine alone representing about 12% of all residues. Thus, the high level of glycosylationof the IFN-y receptor observed'28'40' is consistent with the sequence and composition predicted from therespective cDNA recombinants. Further analysis and characterization of the receptor from placenta shouldprovide information about the amino terminus and the overall primary structure of the natural IFN-y receptor.

ACKNOWLEDGMENTS

We thank Sean Porbin and Adrienne Simone for helping prepare placenta membranes, Jerome A. Langerfor discussions and advice, and Margaret Icangelo and Lois Zapolsky for assistance in the preparation ofthis manuscript. We thank Fazul Khan and J.A. Langer for assistance in the preparation of HuIFN- y fromE. coll We are grateful to Yasuhiro Furuichi for a generous supply of HuIFN- y as well; to Jimmy Calacay,John Shively, Stanley Stein, Henri Lackland, and Femi Akinyosoye for initial amino acid analyses of severalof the fractions. These studies were supported in part by United States Public Health Services Grant #R01CA46465-01 from the National Cancer Institute awarded to S.P.

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10. FINBLOOM, D.S., HOOVER, D.L., and WAHL, L.M. (1985). The characteristics of binding of human recombinantIFN-y to its receptor on human monocyte-like cell line. J. Immunol. 135, 300-305.

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CHARACTERIZATION OF THE RECEPTOR FOR HuIFN-y

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chromosome 21 coded cell surface components block binding of human ct-interferon but not y-interferon to humancells. J. Virol. 137, 422-427.

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interferon a2 to its receptor on human cells. J. Biol. Chem. 257, 13884-13887.16. BRANCA, A.A., and BAGLIONI, C. (1981). Evidence that type I and II interferons have different receptors. Nature

294, 768-770.17. ANDERSON, P., YIP, Y.P., and VILCEK, J. (1982). Specific binding of 125I-human interferon- y to high affinity

receptors on human fibroblasts. J. Biol. Chem. 287, 11301-11304.18. SARKAR, F.H., and GUPTA, S.L. (1984). Receptors for human v-interferon: Binding and crosslinking of 125I-labeled

recombinant human y-interferon to receptors on WISH cells. Proc. Nati. Acad. Sei. USA 81, 5160-5164.19. UCER, U., BARTSCH, H., SCHEURICH, P., BERKOVIC, D., ERTEL, C, and PFIZENMAIER, K. (1986).

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Address reprint requests to:Dr. Sidney Pestka

Department of Molecular Genetics MicrobiologyL'MDNJ-Robert Wood Johnson Medical School

675 Hoes LanePiscataway, NJ 08854-5635

Received 27 February 1989/Accepted 18 May 1989

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