The human interferon αβ receptor: Characterization and molecular cloning

Cell, Vol. 77, 391-400,

May 6, 1994, Copyright

0 1994 by Cell Press

The Human Interferon a/p Receptor: Characterization and Molecular Cloning Daniela Novick, Batya Cohen, and Menachem Rubinstein Department of Molecular Genetics Weizmann Institute of Science Rehovot 76100 Israel

and Virology

Summary We describe a universal ligand-binding receptor for human interferons a and interferon 3 (type I IFNs). A soluble 40 kDa IFN-a/3 receptor (~40) that blocks the activity of type I IFNs was purified from urine and sequenced. Antibodies raised against p40 completely block the activity of several type I IFNs and lmmunoprecipitate both a cellular 102 kDa IFN-a/f? receptor and its cross-linked complexes with IFN-a2 The receptor is a disulfide-linked dlmer, consisting of 51 kDa subunits. We isolated and expressed a 1.5 kb cDNA, coding for the IFN-a&l receptor. Its 331 amino acid sequence includes a leader and a transmembrane region, while Its ectodomain corresponds to ~40. IFN-aIf3 receptor is physically associated with the cytoplasmic Tyr klnase JAKl , hence, in addition to ligand binding, it is directly involved in signal transduction. introduction lnterferons (IFN) a and 8 are a group of structurally and functionally related proteins, induced by viruses or doublestranded RNA and defined by their ability to establish an antiviral state in cells. Since their original discovery (Isaacs and Lindenmann, 1957), the proteins and their genes have been identified (Knight, 1976; Rubinstein et al., 1978; Nagata et al., 1980; Taniguchi et al., 1980), and their mechanism of action has been established. In addition to antiviral activity, many other biological activities of IFNs, including inhibition of cell proliferation and immunomodulation, were described (reviewed by Pestka et al., 1987). Human IFN-a is a family of at least 23 polypeptides, coded by related genes. A high level of sequence homology is displayed among the various IFN-a subtypes, and about 25% identity exists between these species and the single human IFN-8 subtype. The structural differences affect the potency (specific activity) of the various IFNs, which correlates with the affinity of a given subtype to the IFN receptor (Aguet et al., 1984). The various IFNs exhibit a high level of species specificity, with only few exceptions, notably the high activity of all human IFN-a subtypes on bovine cells (Stewart, 1979). Human IFN receptors were characterized in several cell types. According to an earlier nomenclature, IFN-a and IFN-8 are grouped together as type I interferons, whereas the mitogen-induced IFNr is a type II interferon. A type

I receptor, common to IFNs-a and IFN-8, is present on human (Branca and Baglioni, 1981) and mouse (Aguet, 1980) cells, whereas a different (type II) cell surface receptor binds IFNr (Orchanskyet al, 1984; Novicket al., 1987). The cross-linking of radioiodinated IFN-a to whole cells or to isolated cell membranes revealed 11 O-l 50 kDa protein bands, indicating that the type I receptor has a molecular mass of 100-l 30 kDa (Joshi et al., 1982; Colamonici and Domanski, 1993). The type I IFN receptor is present in almost every cell type, albeit at a rather low abundance (100-5000 molecules per cell), therefore, attempts to isolate it have so far been unsuccessful. A human IFN-a receptor, responding mainly to IFN-aB, was cloned by transferring human DNA into mouse cells and selecting for cells responding to IFN-aB. However, the response of these cells to other human IFN-a subtypes and to human IFN-8 was insignificant. Since various human cells respond equally well to IFN-8 and to almost all IFN-a subtypes, it was proposed that additional components of the receptor must be present (Uze et al., 1990). The signal transduction pathway of the IFN-al8 receptor is well characterized. After the binding of a type I IFN to its receptor, the cytoplasmic STAT (signal transducers and activators of transcription) proteins p84/p91 and ~113 undergo Tyr phosphorylation and combine with another cytoplasmic 48 kDa protein (~48) to form the IFN-a-stimulated gene factor 3 (ISGF3) complex (Schindler et al., 1992). ISGF3 rapidly translocates to the nucleus and binds to cis-acting IFN-stimulated response elements (ISRE), present in IFN-induced genes, to initiate their transcription. Two cytoplasmic Tyr kinases, TykP and JAKl , were identified as the enzymes that phosphorylate the subunits of ISGF3 (Velasquez et al., 1992; Miiller et al., 1993). However, a physical linkage between an IFN receptor and these kinases, as demonstrated with some other receptors (Witthuhn et al., 1993), have not been reported so far. Soluble proteins, corresponding to ligand-binding regions of many receptors, were previously identified in cell culturesupernatants and in body fluids, including the soluble receptors to interleukin-2 (IL-2) IFN-y, 11-6, the two tumor necrosis factor receptors, IL-l, IL-4, and low density lipoprotein (Marcon et al., 1988; Novick et al., 1989 Engelmann et al., 1989; Engelmann et al., 1990; Eastgate et al., 1990; Maliszewski et al., 1990; Fischer et al., 1993). Recently, we demonstrated the presence of an IFN-abinding protein in serum and urine (Novick et al., 1992). Here, we present the isolation of this binding protein and its identification as a soluble receptor, corresponding to the ligand-binding region of a novel type I IFN receptor. We have cloned and expressed the receptor cDNA. The deduced amino acid sequence of its ectodomain corresponds to that of the urinary binding protein. Unlike the previously described receptor, the one presented here binds and responds effectively to IFN-8 and to several IFN-a subtypes.

Cdl 392

A. Agarose-IFN-a2

3

I A m=7 ‘0 2

f

aC aB a2

p

-

B. Agarose-IFN-P y

m

-

a2

p

y

C

z-200

p

z Iw lF2 CL 0

I 0

I IO

I

I 20

I

I 30

1

I 40

-

97

-

69 4-57

5(

Figure 2. Competition Cross-Linking to p40

between

Various

IFNs

and

[‘251]lFN-a2

for

Homogenous p40 was cross-linked to [‘9]IFN-a2 in the absence (minus) or presence of excess (30- lo 200-fold) of various IFNs, precipitated by agarose anti-IFN-a monoclonal antibody (MAb) and analyzed by SDS-PAGE ano’ autoradiography. The type of competing IFN is indicated at the top of each lane. Lane m, molecular mass markers (in kilodaltons); lane C, control cross-linking without ~40. The crosslinked 57 kDa product is indicated by an arrow. (A) Autoradiogram of p40 from the agarose-IFN-a2 column. (B) Autoradiogram of p40 from the agarose-IFN-b column. Figure

1. Chromatography

and Analysis

of p40

(A) Size exclusion chromatography of ~40, affinity-purified by the agarose-IFN-a2 column. The protein peak in fractions 26-26 is ~40. (B) SDS-PAGE. Aliquots of various fractions were electrophoresed in 10% acrylamide gel (nonreducing), and the gel was stained with silver. Lane 1, crude urinary proteins; lane 2, affinity purified p40 from the agarose-IFN-a2 column; lanes 16-26, aliquots of corresponding fractions from the size exclusion chromatography; lane 27’, aliquot of fraction 27 from a size exclusion chromatography of ~40, purified by agarose-IFN-fl. Molecular mass markers (in kilodaltons) indicated on the lefl side. (C) Autoradiogram of p40 cross-linked lo [1zI]IFN-u2. Aliquots of various fractions were cross-linked lo [‘“IjlFN-a2 in the absence or presence (denoted by plus) of excess unlabeled IFN-a2 and immunoprecipitated by the anti IFN-a antibody. Lanes 1, affinity-purified p40 from IFN-P-agarose; lanes 2, affinity-purified p40 from IFN-aP-agarose; lanes 26-26, p40 from corresponding fractions of the size exclusion chromatography. Molecular mass markers (in kilodaltons) are denoted on the right side.

Results Isolation and Sequencing of a Soluble IFN-a@ Receptor (~40) The IFN-a-binding protein, previously identified in body fluids (Novick et al., 1992), was purified to homogeneity from human urine by ligand-affinity chromatography on an agarose-IFN-a2 column, followed by size exclusion chromatography (Figure 1A). The purification steps were monitored by silver staining by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and by cross-linking with (‘251]IFN-a2. One of the peaks in the size exclusion step (fractions 28-28, Figure 1A) contained a homogenous 40 kDti protein (~40, Figure 16). About 20 hg of pure p40 was recovered from 250 liters of urine (11 g of crude protein). p40 had the same apparent molecular mass when analyzed by SDS-PAGE under nonreducing (Figure 16) and reducing (data not shown) conditions, indicating that p40 represents a single polypeptide chain. The cross-

linking of p40 to [1Z51]IFN-a2 gave a single 57 kDa radiolabeled band, corresponding to a 1: 1 complex of p40 and the (apparent) 17 kDa IFN-a2. In the presence of excess unlabeled IFN-a2, the 57 kDa radiolabeled band was not obtained, indicating the specificity of the interaction between p40 and IFN-a2 (Figure 1C). We also purified p40 on an agarose-IFN-P column (Figure lB, lane 271, hence p40 binds both IFN-a and IFN-0. The IFN-binding properties of p40 were further studied. Several recombinant type I human IFNs (including IFN-a2, IFN-aB, IFN-aC, and IFN-P) inhibited the binding of [?]IFN-a2 to p40 when added to the above mentioned cross-linking reaction, while (type II) IFNr did not inhibit this binding (Figure 2). Moreover, in a biological assay, p40 inhibited the antiviral activity of natural human leukocyte IFN (a mixture consisting of many IFN-a species), as well as the activity of the individual type I recombinant IFNs, including IFN-0, but not IFN-r. In these inhibition experiments, the specific anti-IFN activity of p40 was >1.5 x 10s Ulmg for the various IFNs. Based on the binding properties of p40 and by analogy with other receptor systems, we assumed and further proved that p40 is a soluble receptor corresponding to the ligand-binding ectodomain of a novel type I IFN receptor. As such, p40 is an IFN inhibitor exhibiting a broad spectrum of binding activity to various type I interferons. The N-terminal sequence, as well as internal sequences of three cyanogen bromide (CNBr)-cleaved peptides of p40 were determined by protein microsequencing. Altogether, 82 amino acid residues were identified. In addition to the major N-terminal sequence, a minor one corresponding to the same protein with three extra N-terminal amino acid residues was obtained. These sequences were compared against GenBank and specifically against the

The Human 393

Interferon

!m

25

u/g Receptor

12

6.2

3.1

1.6

.8

.4

.2

123

4

a9 +ab: aB: leuko+ab: leuko: a2 +ab:

-

69

Cd?:

fl+ab: B: *(+ab: +Y:

Figure 3. Blocking of IFN Activity by p40 Antiserum Human WISH cells were pretreated (plus ab) or not with p40 antiserum (1:300), serial Z-fold dilution of the indicated IFN was added, followed by challenge with VSV. Leuko is a natural mixture of human leukocyte IFN consisting of various IFN-a subtypes.

Dreviously cloned human IFN-a receptor and its mouse and bovine analogs. No significant homology was found between these partial sequences and any one of these receptors or other known proteins.

p40 Corresponds to the Ectodomain of a Novel IFN-alp Receptor, Exhibiting a Broad Specificity for Type I IFNs To characterize the cell surface counterpart of ~40, antibodies were raised in a rabbit against homogeneous ~40. Later, a panel of monoclonal anti-p40 antibodies (suitable for immunoaffinity purification, immunoprecipitation, immunoblotting, and enzyme-linked immunosorbent assay [ELISA]) were developed as well. Treatment of human WISH cells with the rabbit p40 antiserum completely blocked the antiviral activity of natural and recombinant IFN-a subtypes, including IFN-aB, as well as the activity of IFN-8 (Figure 3). The neutralizing titer of the p40 antiserum in these experiments was 50,000 U/ml. In contrast, this antiserum did not block the antiviral activity of either human IFNr in human WISH cells, human IFN-a2 in bovine MDBK cells, or murine IFN-a/8 in murine L cells. These experiments indicated that the p40 antiserum was specifically reactive against a human type I IFN receptor, and further proved that this receptor exhibits a broad spectrum of response to type I IFNs and is essential for IFN activity. The nature of the cell surface protein that interacts with this antiserum was then studied. A detergent extract of

Figure 4. lmmunoblot Analysis of Cellular IFN-o/p Receptor The following samples were subjected to SDS-PAGE (7.5% acrylamide) under nonreducing conditions. Lane 1, clarified detergent extract of Daudi cells (108), affinity purified on agarosemonoclonal antip40 antibody (MAb 5.73); lane 2, crude detergent cell extract of 5 x I@ Daudi cells; lane 3, crude detergent cell extract of control NIH 3T3 cells (5 x lv). The following sample was subjected to SDS-PAGE (7.5% acrylamide) under reducing (100 mM Dll) conditions. Lane 4 shows clarified detergent extract of Daudi cells (5 x lo’), affinity purified on agarose-monoclonal anti p40 antibody (MAb 5.73). Immunoblotting was done with the p40 antiserum. The 102 kDa and the 51 kDa bands are indicated by arrows. Molecular mass markers (in kilodaltons) are indicated.

human Daudi cells was subjected to SDS-PAGE under nonreducing conditions, followed by immunoblotting with the rabbit p40 antiserum. A major 102 kDa protein band was obtained (Figure 4, lane 2); however, additional bands were seen. When the immunoblotting was done with an extract of control murine NIH 3T3 cells, the 102 kDa band was not seen, while some of the other bands were apparent (Figure 4, lane 3). The 102 kDa protein was further studied with the monoclonal anti-p40 antibodies. Agarosebound monoclonal antibody 5.73 was reacted with the detergent cell extract. Bound proteins were eluted at a low pH in the presence of a detergent and were subjected to SDS-PAGE under reducing and nonreducing conditions and to immunoblotting with the polyclonal p40 antiserum. The major 102 kDa band as well as a minor 51 kDa band were obtained under nonreducing conditions (Figure 4, lane 1). Initial experiments employing 8-mercaptoethanol as a reducing agent yielded both the 102 and 51 kDa bands at a similar intensity. However, when stronger reducing conditions (100 mM dithiothreitol [DlTj) were used, a complete reduction of the 102 kDa band occurred, yielding the 51 kDa protein as a single cross-reactive band (Figure 4, lane 4). Hence, the soluble p40 and the cellular 51 and 102 kDa proteins are all immunologically cross-reactive. Furthermore, the 102 kDa protein is made of subunits held together by disulfide bonds. It should be noted that, in a shorter exposure, the 51 kDa band appears as a doublet, possibly owing to microheterogeneity at the polysaccharide side chains.

Cdl 394

A. (WISH) -m&B

B. (Daudi) Y

-mcQPy

-69 ,132, -115-

Figure 5. Detection to [‘261]lFN-a2

of the Cellular

IFN-a/6

Receptor

by Cross-Linking

[9]lFN-a2 was cross-linked to cells in the absence (minus) or presence of excess (200-fold) of various IFNs. The clarified detergent extract of these cells was immunoprecipitated by p40 antiserum and analyzed by SDS-PAGE and autoradiography. Lane m, molecular mass markers (in kilodaltons) are indicated. The major cross-linked products of 115 kDa and 132 kDa are indicated by arrows. (A) Autoradiogram of [‘251]IFN-a2-IFNa/6 receptor complexes from WISH cells. (8) Autoradiogram of [‘251]lFN-a2-IFNa/6 receptor complexes from Daudi cells.

On the basis of these neutralization and immunoprecipitation experiments, we concluded that p40 is indeed a soluble receptor capable of binding various type I IFNs. Furthermore, p40 is cross-reactive with the ligand-binding ectodomain of a novel 102 kDa cell surface IFN-al3 recep tor, consisting of two disulfide-linked 51 kDa subunits. This ligand-binding receptor is essential for the antiviral action of various type I IFNs, including IFN-aB and IFN-6. The Cell Surface IFN-a/fl Receptor Binds Type I IFNs The nature of the complex between IFN-a and its cell surface receptor was studied. [‘251]IFN-a2 was cross-linked to either human WISH or Daudi cells alone or in the presence of excess IFN-a2, IFN-6, or IFN-y. The cells were detergent solubilized and immunoprecipitated with the p40 antiserum. Analysis of the immunoprecipitate by SDSPAGE under reducing conditions revealed two major radiolabeled bands of 132 and 115 kDa. Slower migrating bands of >200 kDa were seen as well, but they were less abundant. All these radiolabeled bands were specific complexes of IFN and its receptor, since they were not formed when the cross-linking was done in the presence of excess unlabeled IFN-a or IFN-3, whereas IFNq did not prevent their formation (Figure 5). The Cell Surface IFN-a/f! Receptor Is Physically Associated with the Cytoplasmlc Tyr Klnase JAKl We studied the possible physical association between IFN-al6 receptor and cytoplasmic proteins involved in signal transduction by coimmunoprecipitation experiments. Detergent extracts of Daudi cells were immunoprecipitated with either the p40 antiserum or control rabbit antiserum to IL-6 receptor (Novick et al, 1991). The immunoprecipitates were analyzed by immunoblotting with antisera

Figure

6. Association

between

the IFN-a/5

Receptor

and JAKl

A clarified extract of Daudi cells (5 x 103 was detergent solubilized, immunoprecipitated, either by p40 antiserum (lane 1) or by control anti IL-6 receptor antiserum (lane 2). Daudi cells (5 x 1Or) were treated for 7 min with either medium alone (lane 3), IFN-a2 (200 U/ml, lane 4) or IFN-6 (200 U/ml, lane 5) washed, and detergent solubilized. The clarified detergent extracts were immunoprecipitated by p40 antiserum (lanes 3-5). All samples were subjected to SDS-PAGE (7.5% acrylamide) under reducing (5% b-mercaptoethanol) conditions, followed by immunoblotting with either p40 antiserum (lanes 1-2) or with JAKl antiserum (lanes 3-5). The 102 kDa band of IFN-a/f3 receptor is indicated by an arrow on the left side. The specific 130 kDa JAKl bands are indicated by an arrow on the right side. The bands near the 45 kDa markers are the heavy chain of rabbit immunoglobulin.

to either ~40, JAKl, JAK2, or Tyk2. SDS-PAGE under reducing (j3-mercaptoethanol) conditions, followed by immunoblotting with the p40 antiserum, revealed the 102 kDa band of IFN-al3 receptor, whereas the accompanying 51 kDa band was probably masked under that of the immunoglobulin heavy chain (Figure 6, lane 1). lmmunoblotting with JAKl antiserum gave a clear - 130 kDa band (corresponding to JAKl), indicating that this kinase is physically associated with the receptor. The intensity of the JAKl band was increased upon pretreatment (7 min) of the cells with IFN-6 but not with IFN-o.2 (200 U/ml each, Figure 6). However, a comprehensive kinetic study must be done to evaluate the possible effect of various IFNs on the extent of association between the receptor and JAKl. Immunoblotting with JAK2 antiserum failed to show a JAKP band, whereas immunoblotting with TykP antiserum gave a very faint - 135 kDa band (data not shown). Isolation of cDNAs Encoding the IFN-a/j3 Receptor and Its Expression In Murlne 3T3 Dells. A 27 amino acid sequence of an internal CNBrcleaved peptide of p40 was used as a starting point for the cloning of the cDNA of the receptor. Fully degenerate oligonucleotides, corresponding to amino acids 151-l 56(sense direction, see Figure 7A for the positions of the amino acids) and amino acids 177-170 (antisense direction), were synthesized. Total RNA from Daudi and WISH cells was reverse transcribed, using the antisense oligonucleotide mixture as primers. The resulting cDNA fragments were then amplified by a polymerase chain reaction (PCR), using the combined sense and antisense degenerate primers. Analysis of the PCR products showed an expected 101 bp band obtained with the cDNA of both Daudi and WISH cells. The 101 bp fragment was cloned into pBluescript II KS(+), and five clones were sequenced. The se-

The Human 395

Interferon

al8 Receptor

B

Figure 7. Nucleotide and Amino Acid Sequences of the Human Pre-IFN-al8 Receptor and Northern

and Hydropathy Plot Blotting of its mRNA

(A) Nucleotide and amino acid sequences, Amino acid residues in single letter codes are numbered in bold, starting at the translationinitiation codon. Hydrophobic leader and transmembrane regions are underlined. N-terminal protein sequences of ~40 (from codon 27) and the internal CNBr peptides are dot underlined (Cys and N-glycosylated Asn residues are not detectable). N-glycosylation signals are indicated by asterisks and the polyadenylation signal is double underlined. (9) Hydropathy plot of the amino acid sequence of the human IIFN-al8 receptor. Positive values indicate hydrophobic regions (Kyte and Doolittle, 1982). (C) Northern blot analysis of RNA from Daudi cells. Poly(A)’ RNA (2 ug) was electrophoresed through a formaldehyde-containing agarose gel and transferred to a nylon membrane. The membrane was hybridized to the 397 bp probe defined in the experimental procedure. The 28s and 18s ribosomal RNA is indicated.

quence of the region flanked by the sense and antisense degenerate primers was invariant and encoded the expected sequence of amino acid residues 159-169. A 35 bp probe, corresponding to this nondegenerate internal sequence, was used for screening of a ligtl 1 library, generated from random and oligo(dT) primed HeLa cDNA. Screening of lo6 independent clones gave five positive clones sharing a common DNA sequence. The sequence had an open reading frame, corresponding to the N-terminal and internal peptide sequences of the urinary ~40. In addition, DNA regions coding for a leader and a putative transmembrane domain were identified; however, none of these clones corresponded to a complete mRNA. A 397 bp probe, based on these partial sequences and coding for a region that included the three internal CNBr peptides, was generated by PCR with specific primers. Northern blot analysis of poly(A)C RNA from Daudi cells with this probe revealed two transcripts of 1.55 kb and 4.5 kb. The 1.55 kb transcript was about 1.5 times more abundant than the 4.5 kb transcript (Figure 7C). The same bands were obtained with RNA from WISH cells (data not shown). A human monocyte cDNA library, constructed in phage XpCEV9 (Gutkind et al., 1991) was then screened with the 397 bp probe. We isolated 22 clones with a 1.5 kb insert and two clones with a 4.5 kb insert from lo6 independent phages. DNA sequence analysis of two 1.5 kb clones (XpCEVS-m6 and XpCEV9-m24), as well as the entire open reading frame of the two 4.5 kb clones (hpCEVS-ml9 and hpCEV9-m27), was performed. The 1.5 kb clones coded for a complete precursor of a cell surface receptor, with an open reading frame of 331 codons (Figure 7A). The protein and CNBr peptide sequences, obtained from p40 (dot underlined, Figure 7A), were all identified within the translated DNA sequence. Partial sequencing of the two 4.5 kb clones revealed the same 5’ sequence of 237 codons as present in the 1.5 kb clones, followed by a different sequence that included a termination signal after codon 239. No open reading frame was seen beyond the stop codon in any of the three reading frames in both of the 4.5 kb clones. Hence the 4.5 kb cDNA codes for a truncated soluble receptor. Phage hpCEVS-m6 was digested with Notl and selfligated. The resulting plasmid pCEV9-m6, containing the Moloney murine leukemia virus long terminal repeat promoter, was used for transient transfection of murine NIH 3T3 cells by the DEAE-dextran method. Transfected and mock-transfected cells were incubated with [1251]IFN-a2, cross-linked with disuccinimidyl suberate (DSS), and extensively washed. The cells were solubilized, immunoprecipitated with the p40 antiserum, and counted. It was found that these cells bound [‘251]IFN-a2, whereas very little binding was obtained with the same number of mocktransfected cells. Binding of [‘251]IFN-a2 to human WISH cells under these conditions gave the same order of magnitude of binding as that of the transfected cells. We therefore concluded that cells, transiently expressing the IFN-a/f3 receptor cDNA, can bind human IFN-a2 (Table 1). The ability of the cloned receptor to transduce IFN signals independently upon treatment with type I human IFN

Cell 396

Table

1. Cross-Linking

of [9]lFN-a2

to Cells

Cell Type

Number

of Cells

NIH 3T3, transfected NIH 3T3, mock transfected Human WISH

3.5 x 10’ 3.5 x IO’ 2 x IO’

Sound [‘251]IFN-a2

(cpm)

1140 119 4501

was tested as well. The transfected NIH 3T3 cells were treated with human IFN-8 (500 U/ml, 12 hr) and RNA was isolated and analyzed by Northern blotting with a probe specific for murine 2’-5’oligo(A) synthetase mRNA. In another experiment, a similarly prepared RNA was reverse transcribed with oligo(dT), and the resulting cDNA was subjected to PCR with synthetic oligonucleotide primers based on the sequence of murine 2’-5’oligo(A) synthetase cDNA. In both experiments, no induction of 2’-5’ oligo(A) synthetase mRNA could be seen, whereas specific 2’-5’ oligo(A) synthetase mRNA and DNA bands were obtained upon treatment with murine IFN-al8 (data not shown). Therefore, it is possible that an accessory protein such as the previously cloned IFN-a receptor is required for signaling. Structural Features of the Cloned IFN-a/p Receptor and Its cDNA Hydropathy plot of the protein sequence revealed its main features, including a leader sequence of 26 amino acids, an extracellular region of 217 amino acids, a transmembrane region of 21 amino acids, and an intracellular region of 77 amino acids (Figure 78). The cDNA clone contained a short 3’untranslated sequence, terminated by a polyadenylation signal and a poly(A) tail. The two 4.5 kb clones had a stop codon just prior to the sequences coding for transmembrane and intracellular regions, and therefore they code for a soluble form of the receptor (~40). The N-terminal and internal peptide sequences of p40 corresponded entirely to sequences within the predicted extracellular region. 116-27 is the putative N-terminal amino acid residue, as predicted from the specificity of the signal peptidase (Von Heijne, 1963). Indeed, protein sequencing revealed that lie-27 was an N-terminal amino acid; however, the majority of the protein started at Asp-30, probably owing to further proteolytic processing. Five N-glycosylation signals were identified at Asn residues 58, 87, 116, 188, and 192. Of these, Asn-87 and Asn-192 were confirmed as N-glycosylation points, as judged from the lack of PTHAsn in the corresponding CNBr peptide sequence. Among other motifs, a phosphorylation site for CAMP-dependent protein kinase is found within the cytoplasmic region of the receptor (Lys-Arg-Ala-Ser, residues 323-326). The sequence of the cloned IFN-a/6 receptor was compared with that of the previously cloned IFN-a receptor. The extracellular region of the present IFN-a/8 receptor exhibited a 23.4% identity with the ligand-binding region of the IFN-a receptor. All the Cys residues of the IFN-a receptor within the homology region were conserved in IFN-a/5 receptor. These Cys residues are also conserved

in the murine and bovine homologues of IFN-a receptor. However the novel IFN-a/6 receptor had two additional Cys residues in positions 39 and 123. The intracellular domains of IFN-a/j3 receptor was not significantly homologous to that of the IFN-a receptor. However, comparison of the sequence of IFN-al8 receptor with that of a recently cloned putative cytokine receptor from the same locus as that of the IFN-a receptor (CRF2-4; Lutfalla et al., 1993) revealed a 23.8% identity at the intracellular region. This is an additional indication that IFN-al8 receptor belongs to the IFN receptor gene family.

The present study describes a novel type I IFN receptor having the ability to bind and respond to several subtypes of IFN-a as well as to IFN-8. This 102 kDa-51 kDa receptor is essential for the activity of many type I IFNs, as demonstrated with anti-receptor antibodies. We show that the same receptor is present in various human cells, including monocytes, HeLa, and WISH cells, while itssolubleform is found in body fluids. The receptor is physically associated with the cytoplasmic Tyr kinase JAKl , and therefore it can directly transduce signals across the cell membrane. The novel type I IFN receptor is a member of the IFN receptor family. The soluble form of this receptor, isolated by ligand affinity chromatography from urine (Figure l), is an additional example of this growing family of proteins, whose physiological function is not clear. This soluble receptor is a potent blocker of all type I IFNs, and therefore it may function as a modulator of IFN activity in vivo. It may also be a useful therapeutic agent, since aberrant production of IFN-a was implicated in diseases such as type I diabetes (Stewart et al., 1993). The existence of significant quantities of a 4.5 kb mRNA (Figure 7C), which is translated into the soluble 40 kDa receptor, suggests that this soluble receptor is generated by a mechanism prone to regulation, rather than by proteolysis of the cell surface receptor. The 1.5 and 4.5 kb transcripts are probably derived from the same gene by alternative splicing. The ability of the soluble IFN-al8 receptor (~40) to compete effectively for binding of type I IFNs, as well as the ability of the p40 antiserum to block the activity of various type I IFNs completely, suggest that IFN-a/8 receptor is the major binding receptor for type I IFNs on cell surface. The previously cloned IFN-a receptor was identified by its response to human IFN-aB in transfected murine cells (Uze et al., 1990). However, IFN-aB effectively competes with IFN-a2 for binding to the soluble IFN-al8 receptor, and therefore it can theoretically bind to two different cell surface receptors. Therefore, the finding that IFN-aB activity in human cells is completely blocked by the p40 antiserum (Figure 3) indicates that even if IFN-aB does bind to the IFN-a receptor, such binding is not sufficient for activity. Apparently, both receptors are essential for IFN activity, as can be concluded from our transfection data. This conclusion is supported by a previous study, showing that monoclonal antibodies raised against the IFN-a receptor

The Human 397

Interferon

a/!3 Receptor

blocked the activity of various type I IFNs (Benoit et al., 1993). In terms of receptor structure, IFN-a/b receptor is a ligand-binding or an a chain (Stahl and Yancopoulos, 1993). It is possible that the previously cloned IFN-a receptor funo tions as a signal transducing B chain. Nevertheless, IFN-a/fi receptor appears to also have a role in signal transduction, because it interacts directly with the cytoplasmic Tyr kinase JAKl (Figure 6). This is the first demonstration of a physical association between an IFN receptor and a cytoplasmic Tyr kinase. It fills one of the few remaining gaps in the type I IFN signaling pathway. Our studies do not provide conclusive evidence for a physical interaction between IFN-a/B receptor and Tyk2. However, Tyk2 is essential for type I IFN signaling, and therefore it either interacts with any one of these two receptors, or alternatively, it may be localized further downstream in the signaling cascade. The structure of the IFN-a/P receptor is that of a typical cytokine receptor, with a single transmembrane region (Figures 7A and 78). The minimal homology between the extracellular region of IFN-alo receptor and the ligandbinding region of the IFN-a receptor is mainly seen at the spatial localization of the Cys residues. This homology and the one observed between the intracellular regions of IFN-a@ receptor and CRF2-4 indicate that IFN-a/P receptor belongs to the interferon receptor family, which also includes the two IFNr receptor subunits and the IL-10 receptor (Bazan, 1990; Aguet et al, 1988; Soh, et al., 1994; Hemmi et al., 1994; Ho et al., 1993). The cDNA species isolated in the present study code for a receptor precursor of 331 amino acid residues. The affinity-purified cell surface receptor migrates as a 102 kDa protein under nonreducing conditions; however, a 51 kDa band is seen as well under these conditions (Figure 4., lanes 1 and 2). Under strong reducing conditions (100 mM DTT), the 102 kDa band is >90% reduced to 51 kDa subunits (Figure 4, lane 4). Hence, the 1.5 kb cDNA and mRNA corresponds to the 51 kDa subunit, while the cell surface receptor exists in an equilibrium between a monomeric and dimeric form. The dimeric form is held by one or more disulfide bonds and is the more prevalent one. A dimeric structure is consistent with the appearance of two (115 kDa and 132 kDa) immunologically cross-reactive IFN-receptor complexes upon cross-linking (Figure 5). Such complexes are probably generated by cross-linking of either one or two IFN-a molecules to the dimeric receptor. A 55 kDa “defective” IFN-a receptor was recently described in a human cell line unresponsive to IFN-a (Colamonici and Domanski, 1993). It is possible that the lack of response stems from the inability of the 55 (or 51) kDa protein to form active dimers. Many receptors undergo a ligand-induced dimerization, which is probably the key step in transmembrane signaling (Ullrich and Schlessinger, 1990). However, there is one known case, that of the insulin receptor, in which the receptor is a priori composed of two ligand binding subunits and two signal ,transducing subunits (Ullrich et al., 1985). Hence, IFN-a@ receptor may have some of the gross structural features of the insulin receptor. Additional studies at the posttrans-

lational level are necessary to understand ture of this IFN-a@ receptor.

fully the struc-

Experimental Procedures Cells and Reagents Human WISH (CCL 25), HeLa (CCL 2.1), and Daudi (CCL 213); bovine MDBK (CCL 22), and murine L (CCL 1) cells were from the American Type Culture Collection (ATCC). Vesicular stomatitis virus (VSV, Indiana strain, ATCC VR-158) was grown and plaque assayed on WISH cells. All cells were free of mycoplasma. Natural human leukocyte interferon was produced and purified as previously described (Novick et al., 1982). The following recombinant IFNs were used: IFN-a2 (E. wli) was from Reprogen; IFN-aB (E. coli) was from CIBA-GEIGY Corporation; IFN-aC (E. coli), IFN-p (CHO), and IFNv (CHO) were from InterLab Incorporated. JAKl, JAK2, and TykP antisera were from Upstate Biotechnology Incorporated. [‘“llprotein A was from Amersham International. [‘251]IFN-a2 and [‘=l]p40 (lo5 cpmlng and 2 x IO5 cpml ng, respectively) were labeled by a modification of the chloramine T method (Novick et al., 1992). Crude urinary proteins were provided by C. Serafini, lstituto Di Ricerca Cesare Serono.

Purification and Characterization Receptor (p40)

of the Soluble

IFN-a/B

IFN-a2 (5 mg) or IFN-p (5 mg) were coupled to Affigel-l0 (1 ml, BioRad). Crude urinary proteins were concentrated IOOO-fold from 250 liters of human urine by ultrafiltration (10 kDa cutoff membrane) and loaded on one of the IFN columns. After extensive washing with 0.5 M NaCl in phosphate buffered saline (PBS), bound proteins were eluted with 0.25 mM citric acid (pH 2.2). 1 mM benzamidine and immediately neutralized. Eluted fractions were concentrated by ultrafiltration (Ultrafree MC, cutoff 10 kDa, Millipore) and further resolved by size exclusion chromatography (Superose 12,l x 30 cm, Pharmacia). Elution was in PBS at 0.5 mllmin, and 0.5 ml fractions were collected. Protein purity was checked by SDS-PAGE (10% acrylamide) and silver staining (Oakley et al., 1960). Protein content was determined with fluorescamine, and crystalline bovine serum albumin was used as a primary standard. IFN binding activity was tested in the various fractions by addition of [‘“I]IFN-a2 alone or together with excess unlabeled IFN, cross-linking with disuccinimidyl suberate (DSS), immunoprecipitation with anti-IFN-a antibody, SDS-PAGE under reducing conditions, and autoradiography as described (Novick et al., 1992). This assay was also used for testing the ability of various IFNs to compete for binding to ~40. The N-terminal sequence of homogenous ~40, adsorbed on a PVDF membrane (Pro-Spin, Applied Biosystems) was determined with a protein microsequencer (Model 475, Applied Biosystems). Another sample of p40 was digested overnight with excess CNBr in 70% formic acid, the resulting peptides were resolved by SDS-PAGE and blotted onto a PVDF membrane, and individual peptide bands were similarly sequenced (Matsudaira, 1987). Sequence library searches and alignments were performed against a combined GenBank and European Molecular Biology Laboratory and SwissProt databases. Computer programs of the Genetic Computer Group Incorporated were used.

Generation

of Polyclonal

and Monoclonal

Antlbodles

A rabbit was injected six times subcutaneously with pure p40 (fraction 26 of the Superose 12 column, 5 r(g). Antibody level was followed by an inverted solid-phase radioimmunoassay (see below), and a binding titer of 1:60,000 was obtained. For development of monoclonal antibodies, BALB/c mice were injected five times with p40 (1 pg per mouse per injection). Mouse lymphocytes were fused with an NSO-1 myeloma variant (NSO) cells, provided by C. Milstein of the Medical Research Council (England). Hybridoma supernatants were tested for the presence of anti-p40 antibodies by an inverted sRIA. In brief, 96-well plates coated with affinity purified goat anti-mouse antibodies were reacted with hybridoma supernatants and with [‘+Jl)p40. Positive hybridomas were cloned and recloned by limiting dilution. Monoclonal antibodies (46.10), suitable

fordoubieantibodyELiSA(Novicketal1991), for immunobiotting, and for affinity

purification

forimmunoprecipitation, (5.73), were obtained.

Cross-Linking, immunoprecipitation, Coimmunopreclpitation, lmmunoaffinity Purification, and immunobiotting of Cellular Proteins For cross-linking and immunoprecipitation, ceils (5 x lo’-10’) were incubated with [‘“i]iFN-a2 (5 x 10’ cpmlml; Novick et al., 1992) for 1 hr, alone or in the presence of various IFNs (80 rg/mi). DSS (1 mM) was added for 20 min. Cross-linking was stopped with excess Tris buffer. The ceils were washed and soiubiiized for 1 hr by a iysis buffer (500 pi, 10 mM CHAPS, 20 mM Tris-HCI (pH 7.5), 150 mM NaCi, 10% glycerol, 2 mM EDTA, 1 mM PMSF, 50 pglmi leupeptin, 4 pg/ ml aprotinin, 2 pglmi chymostatin, 1.5 kg/ml pepstatin, and 2 pglmi antipain). The clarified iysate was immunoprecipitated with rabbit ~40 antiserum(l:l OO)overnight.Ail theabovestepsweredoneat4°C. and ail concentrations were final. Protein A-Sepharose beads (Pharmacia) were added at a 1:20 ratio, and after45 min the beads were washed and boiled with SDS-PAGE sample buffer containing 8-mercaptoethanoi. For immunoprecipitation and coimmunoprecipitation experiments, ceils (5 x 10’) were soiubiiized by the lysis buffer, immunoprecipitated with rabbit ~40 antiserum (1:iOO) overnight, and subjected to SDSPAGE under reducing or nonreducing conditions. immunobiotting was done either with anti-p40, antiJAK1, antiJAK2, or anti-Tyk2 rabbit antiserum (1:400, 1500, 1:lOOO and 1:500, respectively), and detection was by (‘25i]protein A. For direct immunobiotting, ceils (5 x 109 were soiubiiized for 1 hr at 4OC by the iysis buffer (50 ~1). The clarified iysate was subjected to SDS-PAGE under nonreducing conditions, followed by immunobiotting with the rabbit p40 antiserum as above. immunoaffinity purification was done by incubating clarified detergent ceil (5 x 10’) extracts with Affigei-lo-bound monocionai antibody5.73 (overnight at 4OC), washing the beads and eiuting bound proteins with a buffer consisting of citric acid (50 mM), Triton X-100 (0.1%) and benzamidine (1 mM). Eluted proteins were subjected to immunobiotting as above.

IFN Assay

and Its Inhibition

The antiviral activity of various human IFNs was measured in human WISH and bovine MDBK cells by a cytopathic effect (CPE) inhibition assay with VSV (Rubinstein et al., 1981). Murine IFN-a/8 was similarly tested on murine L ceils. interferon titers were calibrated against the National institutes of Health standards. Blocking of IFN by ~40 was evaluated by mixing P-fold dilutions of ~40 with an IFN subtype (10 U/ml final). The mixture was then added to preformed monolayers of the above ceils in 98-well plates, and after 2 hr (18 hr in case of IFN-1) at 37OC the cultures were challenged with VSV. The neutralizing titer of p40 in wells showing 50% CPE was taken as 9 U/ml, hence, one neutralizing unit per milliliter is the ~40 concentration required for neutralizing 1 U/ml of IFN under these assay conditions. The blocking titer of the anti-receptor antibodies was evaluated by incubating for 1 hr at 37OC preformed monolayers of the above ceils in 98-well plates with 2-fold dilutions of the antibody. IFN (10 U/ml final) was then added to all wells, followed by VSV challenge as above. The neutralizing titer of the antibodies in wells showing 50% CPE was taken as 9 U/ml. Hence, one blocking unit per milliliter is the antibody concentration needed for blocking the activity of 1 U/ml of IFN under these assay conditions.

amplified by a PCR, using the combined sense and antisense degenerate primers. The amplification was done in 40 cycles of annealing (48OC, 2 min)and extension (72OC, 1 min). The resulting PCRproducts were resolved by agarose (3%) gel electrophoresis, eiuted and cloned into pBiuescript ii KS(+) at the BamHi and Sail sites. DNA from individual clones was sequenced with T3 and T7 primers. An automated DNA sequencer (Model 373A, Applied Biosystems) and DyeDeoxy terminator kits were used for ail DNA sequencing. An oligonucieotide corresponding to the nondegenerate sequence of the above PCR product was synthesized and end labeled with =P by terminal transferase (Boehringer, Mannheim). This probe was used for screening lo6 recombinant clones from a 5’ stretch human HeLa S3 cDNA library in Igtll (Ciontech). Duplicate nitroceiiulose filters were lifted and hybridized with the probe at 50°C in a buffer consisting of 8x SSC, 10x Denhardt’s solution, 0.1% SDS, and 100 &ml Salmon sperm DNA. The filters were washed and exposed overnight at -80°C to KodakXAR film. Doublbpositive clones were plaque purified, and the DNA inserts were cloned into pBiuescript ii KS(+). Sequence analysis of the pBiuescript DNA was performed using sense and antisense internal primers, as well as T3 and T7 primers. Standard protocols were used for these cloning procedures (Sambrook et al., 1989). Based on the overlapping sequence of ail clones, a 397 bp oiigonucieotide probe was prepared by PCR, using a specific sense primer (5’-GAGTAAACCAGAAGATITGAAG), a specific antisense primer (5’-CGTGTTGGAATTAACTGTC), and template DNA from one of the positive clones (clone q10). PCR was done by 35 cycles of annealing (58OC, 2 min) and extension (78OC, 1 min). The resulting PCR product was labeled with “P by random priming. An original (unamplified) human monocyte cDNA library, constructed in IlpCEV9 cloning vector (Gutkind et al., 1991), provided by T. Miki, was plated and screened as above with the 397 bp probe. The size of the insert in positive clones was determined by PCR with sense and antisense primers, derived from the flanking sequences of the cloning vector. Selected clones were digested with Noti, and the resulting piasmids were recovered by self-ligation (Miki et al., 1989). DNA from these plasmids was subjected to further DNAsequencing, with both internal and external primers. Both strands were sequenced. For ceil transfection, batches of 5 x lad ceils in 0.5 pi TD buffer, containing piasmid DNA (5 pg) and DEAE-dextran (300 pg) were incubated for 30 min at room temperature, essentially as described (Sompayrac and Danna, 1981).

RNA Blotting Total RNA was isolated with the TRI reagent (Molecular Research Center incorporated). Poly(A)+ RNA was isolated from the total RNA by oligo(dT)-polystyrene beads (Dynai Incorporated). RNA (2 pg) was resolved by electrophoresis through 1% agarose gel in MOPS-formaldehyde, transferred to a nylon membrane (Hybond N, Amersham) in 20x SSC and UV cross-linked. The membrane was prehybridized (8 hr, 42OC) with denatured salmon-sperm DNA (100 pglml in 50% formamide, 5x SSC, 4x Denhardt’s solution, and 0.5% SDS). A [“P]dCTP DNA probe, prepared by random priming, was then added, and the hybridization continued for 18 hr at 42OC. The membrane was then washed at room temperature (1 x SSC, 0.1% SDS, twice; and 0.2x SSC, 0.1% SDS, twice; 30 min each wash) and autoradiographed.

Acknowledgments Construction of Probes, Libraries and Expresslon

Screening

of cDNA

The longest peptide sequence, obtained from an internal CNBrcleaved peptide of p40, was used for synthesis of a fully degenerate sense oiigonucieotide (5’-TACTGGATCCATGGTNAARTTYCCNWSNATHGT, corresponding to amino acids 151-158; N = A,T,G,C; R = A,G; Y = C,T; W = A,T; S = G,C; H = A,C,T; D = A,G,T) and an antisense oligonucleotide (5’-TCAAGTCGACATNCCWCNSWYTGWCWCDAT, corresponding to amino acids 177-170). Sequences containing the BamHi and Sali endonuciease restriction sequences were included in the C’ends of the sense and antisense oiigonucieotides, respectively. Total RNA from Daudi and WISH ceils was reverse transcribed with Superscript RNAase H reverse transcriptase (GIBCOBRL), using the antisense oligonucieotide mixture as primers and Taq DNA poiymerase (Promega). The resulting cDNA fragments were then

The excellent technical assistance of S. Barak, N. Tai, and S. H. Kim is acknowledged. Monocionai antibodies were developed with the help of 0. Leitner. We are deeply grateful to Y. Groner and M. Walker for critically reviewing the manuscript and to C. Kahana, D. Levanon, Y. Mory, and H. Engelmann for numerous fruitful discussions. This work was supported by a grant from InterLab Incorporated, Israel. M. R. is the Edna and Maurice Weiss Professor of interferon Research. Received

February

22, 1994; revised

March

17, 1994.

References Aguet, M. (1980). High affinity binding of ‘261-labeled mouse to a specific ceil surface receptor. Nature 284, 459-481.

interferon

The Human 399

Interferon

al8 Receptor

Aguet, M., Grobke, M., and Dreiding, P. (1984). Various human interferon alpha subclasses cross-react with common receptors: their binding affinities correlate with their specific biological activities. Virology 732, 211-216. Aguet, M.. Dembic, Z., and Merlin, expression of the human interferonr

G. (1988). Molecular cloning and receptor. Cell 55, 273-280.

Bazan, J. F. (1990). Shared architecture of hormone binding in type I and II interferon receptors. Cell 67. 753-754.

domains

Benoit, P., Maguire. D., Plavec, I., Kocher, H.,Tovey, hf., and Meyer, F. (1993). A monoclonal antibody to recombinant human IFN-alpha receptor inhibits biologic activity of several species of human IFNalpha, IFN-beta. and IFN-omega. Detection of heterogeneity of the cellular type I IFN receptor. J. Immunol. 150, 707-716. Branca, A. A., and Baglioni, C. (1981). Evidence that type interferons have different receptors. Nature 294, 768-770.

I and II

taining

full-length

inserts

at high frequency.

Gene

83, 137-146.

Mfiller, M., Briscoe, J., Laxton, C., Guschin, D., Ziemiecki, A., Silvennoinen, O., Harpur, A. G., Barbieri, J., Witthuhn, B. A., Schindler, C., Pellegrini, S., Wilks, A. F., Ihle, J. N., Stark, G. R., and Kerr, I, M. (1993). The protein tyrosine kinase JAKI complements defects in the interferon-a/8 and -y signal transduction. Nature 366, 129-135. Nagata, S., Mantei. N., and Weissmann, C. (1980). The structure of one of the eight or more distinct chromosomal genes for human interferon-a. Nature 287, 401-408. Novick, D., Eshhar, Z., and Rubinstein, M. (1982). Monoclonal antibodies to human interferon-a and their use for affinity chromatography. J. Immunol. 729, 2244-2247. Novick, D., Orchansky, P., Revel, M., and Rubinstein, M. (1987). The human interferon--r receptor, purification, characterization and preparation of antibodies. J. Biol. Chem. 262, 8483-8487.

Colamonici, 0. Ft., and Domanski, P. (1993). Identification of a novel subunit of the Type I interferon receptor localized to human chromosome 21. J. Biol. Chem. 268, 10895-10899.

Novick, D., Engelmann, H., Wallach, Soluble cytokine receptors are present Med. 770, 1409-1414.

Eastgate, J. A., Symons, J. A., and Duff, G. W. (1990). Identification oi an interleukin-1 beta binding protein in human plasma. FEES Lett. 266213-216.

Novick, D., Engelmann, H., Revel, M., Leitner, O., and Rubinstein, M. (1991). Monoclonal antibodies to the soluble IL-6 receptor: affinity purification, ELISA, and inhibition of ligand binding. Hybridoma 70, 137-146.

Engelmann, H., Aderka, D.. Rubinstein, M., Rotman, D., and Wallach, D. (1989). A tumor necrosis factor-binding protein purified to homoge neity from human urine protects cells from tumor necrosis factor toxicity. J. Biol. Chem. 264, 11974-11980. Engelmann, H., Novick, D., and Wallach, D. (1990). Two tumor necrosis factor-binding proteins purified from human urine. Evidence for immunological cross-reactivity with cell surface tumor necrosis factor receptors. J. Biol. Chem. 265, 1531-1536. Fischer, D. G.,Tal, N., Novick, D., Barak, S., and Rubinstein, M. (1993). An antiviral soluble form of the LDL receptor induced by interferon. Science 262,250-253. Gutkind, J. S., Link, D. C., Katamine, S., Lacal, P., Miki, T., Ley, T. J., and Robbins, K. C. (1991). A novel c-fgr exon utilized in Epstein-Barr vlrus-infected B lymphcytes but not in normal monocytes. Mol. Cell. Biol. 77, 1500-1507. Hemmi, A novel tionality

S., Bijhni, R.. Stark, G., Di Marco, F., and Aguet, M. (1994). member of the interferon receptor family complements funcof the murine interferon y in human cells. Cell 76, 803-810.

Ho, A. S. Y., Liu, Y., Khan, T. A., Bazan, J. F.. and Moore, K. W. (1993) A receptor for interleukin-IO is related to interferon receptors. Proc. Natl. Acad. Sci. USA 90, 11267-I 1271. Isaacs, A., and Lindenmann, J. (1957). Virus interference. feron Proc. Ft. Sot. Lond. (8) 747, 258-267.

I. The inter-

Joshi, A. R., Sarkar, F. H., and Gupta, S. L. (1982). Interferon receptors: cross-linking of human leukocyte interferon a2 to its receptor on human cells. J. Biol. Chem. 257, 13884-13887. Knight, E. (1976). from diploid ceils.

Interferon: purification and initial characterization Proc. Natl. Acad. Sci. USA 73, 520-523.

Kyte. J. and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 757, 105-132. l.utfalla, G., Gardiner, K., and Uze, G. (1993). cytokine receptor family maps on chromosome flrom IFNAR. Genomics 76, 366-373.

A new member of the 21 at less than 35 kb

Maliszewski, C. R., Sato, T. A., Van den Bos. T., Waugh, S., Dower, S. K., Slack, J., Beckmann, M. P., and Grabstein, K. H. (1990). Cytokine receptors and B cell functions. I. Recombinant soluble receptors specifically inhibit IL-l- and IL4induced B cell activities in vitro. J. I’mmunol. 744, 3026-3033. Marcon, L., Fritz, M. E., Kurman, C. C., Jensen, J. C., and Nelson, D. L. (1988). Soluble Tat peptide is present in the urine of normal individuals and at elevated levels in patients with adult T cell leukemia (ATL). Clin. Exp. Immunol. 73. 29-33. Matsudaira, P. (1987). Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262, 10035-10038. Miki, T., Matsui, T., Heidaran. M. A.. and Aaronson, S. A. (1989). An efficient directional cloning system to construct cDNA libraries con-

Novick, D., Cohen, B., and Rubinstein, alpha receptor molecules are present 445-446.

D., and Rubinstein, M. (1989). in normal human urine. J. Exp.

M. (1992). Soluble interferonin body fluids. FEBS Lett. 374,

Oakley, B. R., Kirsh. 0. R., and Moriss, N. R. (1980). A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 705, 361-363. Orchansky, P., Novick. D., Fischer, D. G., and Rubinstein, M. (1984). Type I and type II interferon receptors. J. Interferon Res. 4, 275-282. Pestka, S., Langer, J. A., Zoon, K. C., and Samuel, C. E. (1987). lnterferons and their actions. Annu. Rev. Biochem. 56, 727-777. Rubinstein, M., Rubinstein, S., Familletti, P. C., Gross, M. S., Miller, R. S., Waldmann, A. A., and Pestka, S. (1978). Human leukocyte interferon purified to homogeneity. Science 202, 1289-I 290. Rubinstein, S., Familletti, P. C., and Pestka, assay for interferons. J. Virol. 37, 755-758.

S. (1981).

Convenient

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press). Schindler, C.. Shuai, K., Prezioso, V. R., and Darnell, J. E., Jr. (1992). Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor. Science 257, 809-813. Soh, J., Donnelly, R. J., Kotenko, S., Mariano, T. M., Cook, J. R., Wang, N., Emanuel, S.. Schwartz, B.. Miki, T., and Pestka, S. (1994). Identification and sequence of an accessory factor required for activation of the human interferon y receptor. Cell 76, 793-802. Sompayrac, L. H., and Danna. K. L. (1981). Efficient infection of monkey cells with DNA of simian virus 40. Proc. Natl. Acad. Sci. USA 78, 7575-7578. Stahl, N., and Yancopoulos, kinases of cytokine receptor

G. D. (1993). The alphas, betas, complexes. Cell 74, 587-590.

and

Stewart, T. A., Hultgren, B., Huang, X., Pit&Meek, S., Hully, N. J., and MacLachlan, N. J. (1993). Induction of Type I diabetes by interferon-a in transgenic mice. Science 260, 1942-1946. Stewart, Verlag).

W. E., II (1979).

The Interferon

System.

(Vienna:

Springer

Taniguchi, T., Ohno, S., Fujii-Kuriyama, Y., and Muramatsu, M. (1980). The nucleotide sequence of human fibroblast interferon cDNA. Gene 70, 1 l-15. Ullrich, A., Bell, J. R., Chen, E. Y. Herrara, T. J., Gray, A. Coussens, L., Liao, Y.-C., Seeburg, P. H.. Grunfeld, C.. Rosen, 0. (1985). Human insulin receptor and its kinase family of oncogenes. Nature 378.

R., Petruuelli, L. M., Dull, Tsubokawa, M.. Mason, A., M., and Ramachandran, J. relationship to the tyrosine 756-761.

Ullrich, A., and Schlessinger, J. (1990). Signal transduction tors with tyrosine kinase activity. Cell 67, 203-212. Uze,

G., Lutfalla,

G., and Gresser,

I. (1990).

Genetic

by recep transfer

of a

Cdl 400

functional expression

human interferon a receptor into mouse of its cDNA. Cell 60, 226-234.

Velasquez, L., Fellous, protein tyrosine kinase 70, 313-322.

cells: cloning

and

M., Stark, G. Ft., and Pellegrini, S. (1992). A in the interferon al6 signaling pathway. Cell

Von Heijne, G. (1963). Patterns of amino acids near signal-sequence cleavage sites. Eur. J. Biochem. 133, 17-21. Witthuhn, B.A.,Quelle, F. W., Silvennoinen, O.,Yi, T.,Tang, B., Miura, O., and Ihle, J. N. (1993). JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell 74, 227-236.

GenEank

Acceeelon

The accession x77722.

number

Number for the sequence

reported

in this paper

is