The human IFN-α receptor (hIFNAR) is a complex composed of at least two chains, hIFNAR1 and hIFNAR2. We have performed a structure-function analysis of hIFNAR2 extracellular domain regions using anti-hIFNAR2 mAbs (1D3, 1F3, and 3B7) and several type I human IFNs. These mAbs block receptor activation, as determined by IFN-stimulated gene factor 3 formation, and block the antiviral cytopathic effects induced by type I IFNs. We generated alanine substitution mutants of hIFNAR2-IgG and determined that regions of hIFNAR2 are important for the binding of these blocking mAbs and hIFN-α2/α1. We further demonstrated that residues E78, W101, I104, and D105 are crucial for the binding of hIFN-α2/α1 and form a defined protrusion when these residues are mapped upon a structural model of hIFNAR2. To confirm that residues important for ligand binding are indeed important for IFN signal transduction, we determined the ability of mouse L929 cells expressing hIFNAR2 extracellular domain mutants to mediate hIFN signal. hIFN-α8, previously shown to signal a response in L929 cells expressing hIFNAR1, was unable to signal in L929 cells expressing hIFNAR2. Transfected cells expressing hIFNAR2 containing mutations at residues E78, W101, I104, or D105 were unresponsive to hIFN-α2, but remained responsive to hIFN-β. In summary, we have identified specific residues of hIFNAR2 important for the binding to hIFN-α2/1 and demonstrate that specific regions of the IFNAR interact with the subspecies of type I IFN in different manners.

Type I IFNs are a family of cytokines defined by their antiviral activities. Human IFNs include at least 14 subspecies of hIFN-α,2 one hIFN-β, one hIFN-ω, and one hIFN-τ (1, 2). These type I hIFNs share a common receptor (IFNAR) (3, 4), which is composed of two chains, a 135-kDa α subunit (hIFNAR1) (5) and a 115-kDa β subunit (hIFNAR2) (6, 7). Three different forms of hIFNAR2 have been reported: a 40-kDa soluble form designated hIFNAR2a (6), a 55-kDa short form known as hIFNAR2b (6), and a 115-kDa long form known as hIFNAR2c (7). These three forms are derived by alternative splicing of the same gene. Only hIFNAR2c mediates a biological response when associated with hIFNAR1 (5) and is the form of the receptor we have utilized in this study.

When hIFNAR1 is expressed alone in mouse cells, there is no significant IFN-α binding; however, the expression of hIFNAR2 alone produces a low affinity ligand-binding receptor (0.5–1 nM). The coexpression of hIFNAR1 and hIFNAR2 results in a high affinity receptor complex (10–100 pM) (8, 9, 10). These results demonstrate that hIFNAR2 is the ligand-binding subunit, but hIFNAR1 contributes to the formation of a high affinity receptor. It has been shown that the coexpression of hIFNAR1 and hIFNAR2c in a murine background can mediate the antiviral response of human IFNs, but not an antiproliferative response (11). This suggests that there may be additional species-specific components required for the antiproliferative response (12). Recently, Petricoin et al. (13) reported that the antiproliferative action, but not the antiviral action or the activation of the JAK-STAT pathway, of IFN-α requires TCR signaling components.

hIFNAR2 is a 515-aa protein composed of an ECD of 217 residues. The ECD of hIFNAR2 is composed of two domains (∼100 residues/domain), domain 1 and domain 2. IFN-mediated signaling is initiated by ligand-induced receptor dimerization via the ECD, tyrosine phosphorylation of the Tyk2 and Jak1 tyrosine kinases, and subsequent phosphorylation of the Stat1 and Stat2 proteins. Activated STATs translocate to the nucleus as an IFN-stimulated response element 3 (ISGF3) complex and induce the transcription of IFN-stimulated genes (13). There are multiple IFNs in the type I IFN family that initiate receptor dimerization. To understand how the same receptor interacts with these different IFN subtypes, in this study we have investigated the interaction of type I IFNs with hIFNAR2 using soluble hIFNAR2-IgG immunoadhesin and blocking mAbs. Using alanine-scanning mutagenesis, we have determined residues on hIFNAR2 that are important for type I IFN binding. We have extended the binding data by transfecting wild-type and mutant hIFNAR2 cDNAs into murine L929 cells and have studied the effect of several type I IFNs on mediating signal transduction.

hIFNAR2-IgG and various subspecies of hIFN-α were prepared as described (14) with the following modification: A c-DNA encoding the hIFNAR2-IgG molecules was constructed based on the ECD (residues 1–216) of hIFNAR2. hIFNAR2-IgG was expressed in 293 cells and the immunoadhesin was purified using a protein A column. Human IFN-α2/α1 (IFN-α2 residues 1–62/α1 residues 64–166) (15, 16) were a gift from Dr. M. J. Brunda (Hoffman-LaRoche, Nutley, NJ). hIFN-β was obtained from Sigma (St. Louis, MO). The specific activities of the various type I IFNs are as follows: IFN-α2/α1 (2 × 107 IU/mg), IFN-α1 (3 × 107 IU/mg), IFN-α2 (2 × 107 IU/mg), IFN-α5 (8 × 107 IU/mg), IFN-α8 (19 × 107 IU/mg), and IFN-β (1.5 × 105 IU/mg).

BALB/c mice were immunized with 2.5 μg of hIFNAR2-IgG into each hind footpad, and mAbs were generated as described (17). Three days after the final boost, popliteal lymph nodes were fused with myeloma cell line P3 × 63Ag.U.1 (18). Culture supernatants were initially screened for their ability to bind to hIFNAR2-IgG, but not to CD4-IgG in a capture ELISA, as described previously (19). The selected mAbs were further tested for their ability to block ligand-receptor binding in a capture ELISA and for their ability to recognize cell membrane receptors on U266 cells by flow-cytometric analysis, as described (19). After cloning the selected hybridomas twice, their Ag specificity as well as blocking activities were confirmed in a ligand receptor-binding assay. The Western blot and isotype analyses were done as described (20).

Microtiter plates (Maxisorb; Nunc, Kamstrup, Denmark) were coated with 50 μl/well of 2 μg/ml of goat Abs specific for the Fc portion of human IgG (goat anti-hIgG Fc; Cappel, ICN, Costa Mesa, CA) in PBS overnight at 4°C and blocked with 2% BSA for 1 h at RT. A total of 50 μl/well of 50 ng/ml of hIFNAR2-IgG (or hIFNAR2-IgG mutant) was added to each well for 1 h. Plates were then incubated with 50 μl/well of 2 μg/ml of anti-hIFNAR2 mAbs (or 50 ng/ml of hIFNAR2-IgG mutants) for 1 h. Wells were then incubated with 50 μl/well of HRP goat anti-mouse IgG. The bound enzyme was detected by the addition of TMB (3,3′,5,5′-tetramethylbenzidin) substrate, the reaction was stopped by the addition of stop solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and the plates were read at 450 nm with an ELISA plate reader. Between each step, plates were washed three times in wash buffer (PBS containing 0.05% Tween-20).

To determine whether mAbs recognized the same or different epitopes, a competitive binding ELISA was performed using biotinylated mAbs (Bio-mAb). mAbs were biotinylated using N-hydroxyl succinimide, as described (20).

The equilibrium dissociation and association constant rates of anti-hIFNAR2 mAbs were determined using KinExA, an automated immunoassay system (Sapidyne Instruments, Boise, ID), as described, with a modification (21, 22). Briefly, 1 ml of anti-human IgG agarose beads (56 μm; Sigma, St. Louis, MO) was coated with 20 μg of hIFNAR2-IgG in PBS by gentle mixing at RT for 1 h. After washing with PBS, nonspecific binding sites were blocked by incubating with 10% human serum in PBS for 1 h at RT. The blocked beads were diluted into 30 ml of assay buffer (0.01% BSA/PBS). The diluted beads (550 μl) were drawn through the flow cell with 20-μm screen and then washed with 1 ml of running buffer (0.01% BSA + 0.05% Tween-20 in PBS). The beads were then disrupted gently with a brief backflush of running buffer, followed by a 20-s setting period to create a uniform and reproducible bead pack. A bead pack (∼4 mm high) was created in the observation flow cell. For equilibrium measurements, mAbs (5 ng/ml in 0.01% BSA/PBS) were mixed with a serial dilution of hIFNAR2-IgG (starting from 2.5 nM to 5 pM) and were incubated at RT for 2 h. Once equilibrium was reached, 4.5 ml of this mixture was drawn through the beads, followed by 250 μl of running buffer to wash out the unbound mAbs. The anti-IFNAR2 mAbs bound to beads were detected by 1.5 ml of PE-labeled goat anti-mouse IgG. Unbound labeled material was removed by drawing 4.5 ml of 0.5 M NaCl through the bead pack over a 3-min period. The equilibrium constant was calculated using the software provided by the manufacturer (Sapidyne).

HeLa cells (5 × 105 cells) were incubated with each hIFN-α (25 ng/ml) in 200 ml of DMEM for 30 min at 37°C. In experiments using the anti-hIFNAR2 mAbs, cells were incubated with 5–500 μg/ml for 15 min at 4°C before addition of the hIFN-α. Cells were lysed using 0.025% Nonidet P-40, and gel-shift assays were performed using a 32P-labeled double-stranded oligonucleotide containing the ISG15 (19). DNA-protein complexes were resolved in 6% nondenaturing polyacrylamide gels (Novex, San Diego, CA) and analyzed by autoradiography. The specificity of the assay was determined by the addition of 350 ng of unlabeled ISG15 probe in separate reaction mixtures. Formation of an ISGF3-specific complex was confirmed by a supershift assay using anti-murine Stat1 or Stat2 Ab (Santa Cruz Biotechnology, Santa Cruz, CA).

The assay was done as described, in duplicate 96-well microtiter plates using human lung carcinoma A549 cells challenged with encephalomyocarditis virus (23). Serial dilutions of mAbs were incubated with various units of type 1 IFNs for 1 h at 37°C in a total volume of 100 μl. These mixtures were then incubated with 5 × 105 A549 cells in 100 μl of medium for 24 h. Cells were then challenged with 2 × 105 PFU of encephalomyocarditis virus for an additional 24 h. At the end of the incubation, cell viability was determined by visual microscopic examination. The neutralizing Ab titer (EC50) was defined as the concentration of Ab that blocks 50% of the antiviral cytopathic effect by 100 U/ml of type I IFNs. The units of type I IFNs used in this study were determined using National Institute of Health reference human rIFN-α2 as a standard.

The cDNAs encoding 1–216 residues of the extracellular domain of type 1 hIFNAR2 were expressed as immunoadhesins (14). Alanine substitution mutants were generated according to the method of Kunkel (24). Mutant receptor IgGs were expressed transiently in human 293 cells. Transfected 293 cells were grown overnight in F-12:DMEM (50:50) containing 10% FCS, 2 mM l-glutamine, 100 μg/ml of penicillin, and 100 μg/ml of streptomycin, and then were placed in a serum-free media. Three days later, culture supernatants were collected. For the hIFNAR2-IgG mutant analysis, the concentrations of immunoadhesin molecules in 293 transfected culture supernatants were determined by ELISA using CD4-IgG as a standard, and were adjusted to be 50 ng/ml. The degree of mAb binding to these mutants was compared with the degree of mAb binding to the wild-type receptor using a capture ELISA.

A 648-bp fragment, containing the entire coding region of the hIFNAR2 cDNA, was inserted into the mammalian cell expression vector pRSV and designated pRSVHAR2. cDNAs encoding mutant receptors were produced in a repair deficient Escherichia coli strain using two oligonucleotide primers and pRSVHAR2 as a template according to the manufacturer’s instructions (Clontech Laboratories, Palo Alto, CA). The accuracy of all cDNA was confirmed by supercoiled DNA sequencing with an automated DNA sequencing system. The murine fibroblast cell line L929 was cotransfected with 1 μg of expression plasmid and 50 ng of pSVE neo DNA per dish by a liposome-mediated transfection technique (Superfect, Qiagen, Chatsworth, CA). Forty-eight hours after transfection, the cells were split and transfectants were selected in G418. Twenty-four individual G418-resistant clones were analyzed for each construct. Human IFNAR2-expressing clones were initially screened by RT-PCR using hIFNAR2-specific primers because the mutations created in the ECD of hIFNAR2 might affect mAb binding to the receptor. Those clones that expressed mRNA for hIFNAR2 were then analyzed by flow cytometry using mAb 3B2 and PE-conjugated goat anti-mouse IgG (Caltag, San Francisco, CA) to determine the level of membrane receptor expression. At least three clones of each construct were tested for a functional response to several IFNs.

mAbs to the ECD of hIFNAR2 were generated using a soluble immunoadhesin as an Ag, as described in Materials and Methods. Initially, we selected 20 strong positive binding mAbs to hIFNAR2 by an ELISA. After flow-cytometric analysis on 9D cells (a human B cell line), we selected seven mAbs that recognized membrane hIFNAR2. Six mAbs are of the IgG2 isotype and one, mAb 3B2, is an IgG1. mAb 3B2 was used for routine flow cytometry to evaluate receptor expression since our PE goat anti-mouse IgG (Caltag, San Francisco, CA) appears to bind murine IgG1 with higher affinity than murine IgG2. From the results of competitive binding ELISA, we were able to group these seven mAbs into three groups depending upon regions of hIFNAR2-IgG they recognized (data not shown). Three mAbs, 1D3, 1F3, and 3B7, representing each group, were selected for our study, and the general characteristics of these three mAbs are outlined in Table I. mAb 1D3 recognizes the reduced form of hIFNAR2 in a Western blot assay (data not shown), suggesting that it may recognize a linear epitope, while mAbs 1F3 and 3B7 recognize conformational epitopes. Receptor affinities were determined using a KinExA system that allows the measurement of mAb affinities in a solution phase. The affinities (Kd−1) of mAbs 3B7, 1F3, and 1D3 are 1, 5, and 242 pM, respectively, demonstrating that these are relatively high affinity mAbs.

Table I.

General characteristics of mAbs to hIFNAR2

mAbIsotypeaCytometrybEpitopecWestern BlotdIPeAffinityfKd−1 (pM)
1D3 IgG2a ++ ND 242 
1F3 IgG2a ++ − 
3B2 IgG1 ++ − ND 
3B7 IgG2a ++ − 
mAbIsotypeaCytometrybEpitopecWestern BlotdIPeAffinityfKd−1 (pM)
1D3 IgG2a ++ ND 242 
1F3 IgG2a ++ − 
3B2 IgG1 ++ − ND 
3B7 IgG2a ++ − 
a

The mAb isotype was determined using an isotype-specific goat anti-mouse Ig.

b

All mAbs were selected for positive staining of a 9D human B cell line.

c

mAbs were shown to recognize different epitopes by a competitive binding ELISA.

d

The immunoblot was performed using hIFNAR2-IgG reduced with DTT.

e

U266 cells were biotinylated using NHS-LC-biotin and lysed with 1% Nonidet P-40. Biotinylated hIFNAR2 was precipitated by mAbs bound to protein-G-4B Sepharose and separated on a 7.5% SDS-PAGE gel. Biotinylated hIFNAR2 transferred onto nitrocellulose paper was detected by HRP-strepavidin.

f

The affinity of mAbs were determined using the KinExA system.

The blocking ability of each mAb was determined in a competitive binding ELISA (Fig. 1). mAbs 1F3 and 3B7 at 0.6 nM (0.1 μg/ml) were able to block >90% of hIFN-α2/α1 binding to the soluble receptor, hIFNAR2-IgG. mAb 1D3 showed no significant blocking activity even at a concentration 10 times higher, of 6 nM (1 μg/ml). To extend these findings in functional assays, we analyzed the ability of mAbs 1D3, 1F3, and 3B7 to prevent receptor activation via the ISGF3 complex conformation by an EMSA and by an IFN-induced antiviral assay.

FIGURE 1.

mAbs to hIFNAR2 inhibit the binding of hIFN-α2/α1 to hIFNAR2-IgG, as determined by an ELISA. hIFNAR2-IgG was captured onto ELISA wells precoated with goat anti-human IgG Fc. Bio-hIFN-α2/α1 plus various concentrations of mAbs were added into each well. The level of Bio-hIFN-α2/1 bound was detected by the addition of HRP-streptavidin.

FIGURE 1.

mAbs to hIFNAR2 inhibit the binding of hIFN-α2/α1 to hIFNAR2-IgG, as determined by an ELISA. hIFNAR2-IgG was captured onto ELISA wells precoated with goat anti-human IgG Fc. Bio-hIFN-α2/α1 plus various concentrations of mAbs were added into each well. The level of Bio-hIFN-α2/1 bound was detected by the addition of HRP-streptavidin.

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In HeLa cells treated with 25 ng/ml of several type I IFNs, all three mAbs at a concentration of 500 μg/ml prevent receptor activation and subsequent ISGF3 complex formation (Table II). At lower concentrations, only 1F3 and 3B7 are effective. These results show that all three mAbs are blocking Abs, although mAb 1D3 is relatively weak. The blocking activities of these mAbs were also tested in an antiviral assay using A549 cells challenged with ECM virus in the presence of 100 U/ml of hIFN-α1, hIFN-α2, hIFN-α5, hIFN-α8, hIFN-α2/α1, or IFN-β (Table III). Serial dilutions of mAbs in the concentration range of 0.1–30 μg/ml were tested in duplicate. The EC50 was arbitrarily determined as the mAb concentration that neutralized 50% of the antiviral cytopathic effect induced by 100 U/ml of type I IFN. All three mAbs blocked the activity of all type I IFNs tested, and the potency of these mAbs was variable depending upon the IFN subtypes tested. mAb 3B7 demonstrated its blocking activity at an EC50 ≤ 1 μg/ml, while mAb 1F3 required 1–3 μg/ml to block activity. mAb 1D3 was able to block the activity of all IFNs tested, except IFN-β, although a much higher Ab concentration (EC50 = 10–20 μg/ml) was required. From these biological assays, we conclude that all three mAbs are blocking Abs and mAb 3B7 is the most potent.

Table II.

Effects of anti-hIFNAR2 mAbs on ISGF3 formation in HeLa cellsa

HeLa Cells1D3 (μg/ml)1F3 (μg/ml)3B7 (μg/ml)
550500550500550500
hIFN-α2/α1 − − 
hIFN-α1 − − +/− 
hIFN-α2 − +/− 
hIFN-α5 − − 
hIFN-α8 − − 
hIFN-β − − − 
HeLa Cells1D3 (μg/ml)1F3 (μg/ml)3B7 (μg/ml)
550500550500550500
hIFN-α2/α1 − − 
hIFN-α1 − − +/− 
hIFN-α2 − +/− 
hIFN-α5 − − 
hIFN-α8 − − 
hIFN-β − − − 
a

EMSA detecting ISGF3 complex formation during IFN activation was carried out using HeLa cells treated with 25 ng/ml of human type 1 IFNs plus 5–500 mg/ml of mAbs as described in Materials and Methods. Results were expressed as complete blocking (+), partial blocking (+/−), or no blocking (−).

Table III.

Effects of anti-hIFNAR2 mAbs on the antiviral effects of type I IFNsa

mAbEC50 of mAb (μg/ml)
hIFN-α2/α1hIFN-α1hIFN-α2hIFN-α5hIFN-α8hIFN-β
1D3 20 10 20 10 20 NB 
1F3 
3B7 0.6 0.1 0.1 0.3 0.3 
mAbEC50 of mAb (μg/ml)
hIFN-α2/α1hIFN-α1hIFN-α2hIFN-α5hIFN-α8hIFN-β
1D3 20 10 20 10 20 NB 
1F3 
3B7 0.6 0.1 0.1 0.3 0.3 
a

The neutralizing Ab titer (EC50) was defined as the concentration of Ab that neutralizes 50% of the antiviral cytopathic effects induced by 100 U/ml of type I IFNs on A549 cells. The experiment was done using serial dilutions of mAbs in the range of 0.1–30 μg/ml in duplicate. When mAbs at 30 μg/ml showed no blocking effect in this assay, we arbitrarily designated these as a nonblocking mAb (NB).

To determine areas in the ECD of hIFNAR2 that are important for ligand binding, we generated alanine substitution hIFNAR2-IgG mutants. We initially selected 12 charged areas for analysis and substituted clusters of two to seven residues with alanines (Table IV). Eleven of the twelve mutants could be expressed as immunoadhesins, as detected in an anti-human IgG-Fc ELISA. Mutant 8 (residues 145–149: EEQSE/AAQSA) did not express at all, suggesting that this region is important for maintenance of the structural integrity of hIFNAR2. The binding of hIFN-α2/α1 (50 nM) and mAbs (5 nM) to hIFNAR2-IgG mutants (0.5 nM) was determined in a capture ELISA. The degree of binding to the receptor mutants was compared with the degree of binding obtained with the wild-type receptor. Alanine substitution mutants, 1 (7–11), 2 (29–33), 10 (159–163), 11 (172–173), and 12 (187–192), showed significant binding (>70% of the wild-type control) to Bio-hIFN-α2/α1, while 3 (49–55), 4 (68–72), 5 (74–78), 6 (105–109), 7 (133–139), and 9 (153–157) showed an 80% reduction in their ability to bind Bio-hIFN-α2/α1. Residues 49–157 are in the middle portion of hIFNAR2 occupying portions of domains 1 and 2, suggesting that this area interacts with ligand. We also determined the binding of the three anti-hIFNAR2 mAbs, 1D3, 1F3, and 3B7, to these 11 hIFNAR2 mutants. In comparison with the wild-type hIFNAR2-IgG, all of these mutants show 35–100% binding to at least one of these mAbs, suggesting that these multiple mutants retain the general structural integrity of hIFNAR2. When the alanine substitution, in particular residues of hIFNAR2-IgG, resulted in <25% of the wild-type binding, we arbitrarily considered these residues as being important for the mAb binding. The most potent blocking mAb, 3B7, recognizes an epitope of the receptor in a small area containing residues 49–55 and 68–72. The areas recognized by mAb 1F3 span a larger region in residues 49–55, 68–72, 74–78, 105–109, 133–139, and 153–156, which closely overlaps with the ligand-binding region of hIFN-α2/α1. The main binding area of mAb 1D3 is in a small area, including residues 133–139 and 153–157, in domain 2 of hIFNAR2.

Table IV.

Binding of hIFN-α2/α1 and mAbs to multiple alanine hIFNAR2 mutantsa

No.ResiduesAlanine Substitution% Wild-Type hIFNAR2 Binding
IFN-2/11D31F33B7
7–11 DYTDE /AYTAA 73 ± 10 105 ± 13 96 ± 1 101 ± 13 
29–33 ELKNH /ALANA 87 ± 22 80 ± 1 87 ± 6 82 ± 7 
49–55 KPEDLK /APAALA 18 ± 2 39 ± 1 6 ± 0 4 ± 0 
68–72 DLTDE /ALTAA 16 ± 1 38 ± 2 5 ± 0 3 ± 0 
74–78 RSTHE /ASTAA 16 ± 1 95 ± 1 16 ± 1 89 ± 2 
105–109 DMSFE /AMSFA 19 ± 2 65 ± 1 8 ± 1 40 ± 1 
133–139 EEELQFD /AAALQFA 16 ± 1 5 ± 1 9 ± 0 35 ± 1 
145–149 EEQSE /AAQSA b — — — 
153± 157 KKHKP /AAHAP 19 ± 1 14 ± 1 14 ± 0 35 ± 1 
10 159–163 EIKGN /AIAGN 92 ± 5 89 ± 7 96 ± 5 93 ± 3 
11 172–173 DK /AA 73 ± 7 73 ± 5 91 ± 7 80 ± 3 
12 187–192 HEWED /AASAAQ 82 ± 3 82 ± 10 83 ± 9 66 ± 10 
No.ResiduesAlanine Substitution% Wild-Type hIFNAR2 Binding
IFN-2/11D31F33B7
7–11 DYTDE /AYTAA 73 ± 10 105 ± 13 96 ± 1 101 ± 13 
29–33 ELKNH /ALANA 87 ± 22 80 ± 1 87 ± 6 82 ± 7 
49–55 KPEDLK /APAALA 18 ± 2 39 ± 1 6 ± 0 4 ± 0 
68–72 DLTDE /ALTAA 16 ± 1 38 ± 2 5 ± 0 3 ± 0 
74–78 RSTHE /ASTAA 16 ± 1 95 ± 1 16 ± 1 89 ± 2 
105–109 DMSFE /AMSFA 19 ± 2 65 ± 1 8 ± 1 40 ± 1 
133–139 EEELQFD /AAALQFA 16 ± 1 5 ± 1 9 ± 0 35 ± 1 
145–149 EEQSE /AAQSA b — — — 
153± 157 KKHKP /AAHAP 19 ± 1 14 ± 1 14 ± 0 35 ± 1 
10 159–163 EIKGN /AIAGN 92 ± 5 89 ± 7 96 ± 5 93 ± 3 
11 172–173 DK /AA 73 ± 7 73 ± 5 91 ± 7 80 ± 3 
12 187–192 HEWED /AASAAQ 82 ± 3 82 ± 10 83 ± 9 66 ± 10 
a

Wild-type and mutant hIFNAR2-IgGs (0.5 nM) were captured with goat anti-human IgG Fc reagent precoated onto microtiter wells. Biotinylated hIFN-α2/α1 (50 nM) or mAbs (5 nM) were allowed to interact for 1 h. After washing, the amounts of the ligand or mAbs bound were determined by the addition of HRP-streptavidin or HRP-goat anti-mouse IgG, respectively.

b

No expression.

To define specific residues of hIFNAR2 that bind hIFN-α2/α1 and interact with the blocking mAbs, 26 single alanine mutants were generated in the region encompassing residues 49–156 (Table V). Compared with the wild-type binding, hIFN-α2/α1 lost >75% binding to the hIFNAR2 mutants, D68A, E78A, W101A, I104A, and D105A. All of these mutants except D68A retained the high binding to the mAbs, demonstrating their structural integrity. In contrast, the binding of all three mAbs to mutant D68A was significantly diminished. This suggests that D68 may be structurally important, as mutations of it affect the binding of ligand and all three mAbs. The binding ELISA was done by capturing 0.5 nM hIFNAR2-IgG mutants onto an anti-human IgG-Fc-coated well, followed by the incubation with 10-fold molar excess (5 nM) of Bio-hIFN-α2/α1. Under these conditions, we should be able to detect the majority of the receptor-ligand interactions if there is a significant affinity. However, to confirm the result shown in Table III, we compared the binding of these IFNAR2-IgG mutants/wild type to various concentrations (0.5–50 nM) of Bio-hIFN-α2/α1 (Fig. 2). Compared with the binding of wild hIFNAR2-IgG to various doses of Bio-hIFN-α2/α1, mutants W101A, I104A, and D105A, and E78A demonstrate <50% binding of the wild type at various concentrations of IFN, while K49A and K54A show <75% binding. From these results, we concluded that the most crucial residues in IFNAR2 for the interaction with hIFN-α2/α1 are E78, W101, I104, and D105, while K49 and K54 have some influence on this interaction. Residues E78, W101, I104, and D105, which are important for binding to the ligand, comprise a small region of the receptor according to our proposed computer model (Fig. 5 A).

Table V.

Binding of hIFN-α2/α1 and anti-hIFNAR2 mAbs to hIFNAR2-IgG mutantsa

Mutants% Wild hIFNAR2 Binding
hIFN-α 2/11D31F33B7Polyclonal
K49A 68 ± 10 101 ± 1 104 ± 6 99 ± 1 98 ± 13 
E51A 95 ± 9 103 ± 4 101 ± 4 67 ± 0 92 ± 3 
D52A 97 ± 11 101 ± 1 107 ± 6 83 ± 2 94 ± 15 
K54A 53 ± 1 87 ± 1 83 ± 2 87 ± 12 91 ± 13 
K57A 111 ± 2 143 ± 2 260 ± 15 144 ± 1 107 ± 5 
D68A 41 ± 1 8 ± 1 8 ± 0 38 ± 1 
D71A 113 ± 26 100 ± 2 91 ± 3 110 ± 13 103 ± 12 
E72A 129 ± 29 91 ± 7 106 ± 19 90 ± 13 94 ± 2 
R74A 122 ± 28 92 ± 2 103 ± 12 93 ± 16 93 ± 13 
H77A 83 ± 19 150 ± 4 121 ± 137 132 ± 16 150 ± 12 
E78A 1 ± 1 92 ± 3 54 ± 4 94 ± 2 89 ± 14 
W101A 99 ± 4 90 ± 2 98 ± 2 100 ± 13 
I104A 23 ± 2 104 ± 1 94 ± 6 104 ± 1 99 ± 13 
D105A 8 ± 2 89 ± 3 62 ± 8 87 ± 2 97 ± 14 
E109A 99 ± 10 99 ± 4 107 ± 19 99 ± 4 100 ± 17 
E133A 99 ± 9 92 ± 3 96 ± 12 147 ± 5 80 ± 12 
E134A 86 ± 3 87 ± 1 82 ± 3 88 ± 12 92 ± 3 
E135A 72 ± 2 88 ± 1 74 ± 1 80 ± 12 90 ± 1 
Q137A 85 ± 12 63 ± 1 55 ± 4 61 ± 14 82 ± 1 
D139A 109 ± 1 87± 100 ± 4 139 ± 5 89 ± 14 
E145A 78 ± 9 84 ± 2 82 ± 12 93 ± 12 91 ± 11 
E146A 87 ± 4 93±± 92 ± 3 98 ± 12 97 ± 5 
E149A 97 ± 8 95 ± 1 92 ± 3 104 ± 11 99 ± 12 
K153A 71 ± 1 91 ± 1 86 ± 5 96 ± 11 93 ± 13 
K154A 82 ± 18 90 ± 1 78 ± 6 89 ± 19 91 ± 4 
K156A 106 ± 2 83 ± 11 97 ± 10 87 ± 7 96 ± 17 
Mutants% Wild hIFNAR2 Binding
hIFN-α 2/11D31F33B7Polyclonal
K49A 68 ± 10 101 ± 1 104 ± 6 99 ± 1 98 ± 13 
E51A 95 ± 9 103 ± 4 101 ± 4 67 ± 0 92 ± 3 
D52A 97 ± 11 101 ± 1 107 ± 6 83 ± 2 94 ± 15 
K54A 53 ± 1 87 ± 1 83 ± 2 87 ± 12 91 ± 13 
K57A 111 ± 2 143 ± 2 260 ± 15 144 ± 1 107 ± 5 
D68A 41 ± 1 8 ± 1 8 ± 0 38 ± 1 
D71A 113 ± 26 100 ± 2 91 ± 3 110 ± 13 103 ± 12 
E72A 129 ± 29 91 ± 7 106 ± 19 90 ± 13 94 ± 2 
R74A 122 ± 28 92 ± 2 103 ± 12 93 ± 16 93 ± 13 
H77A 83 ± 19 150 ± 4 121 ± 137 132 ± 16 150 ± 12 
E78A 1 ± 1 92 ± 3 54 ± 4 94 ± 2 89 ± 14 
W101A 99 ± 4 90 ± 2 98 ± 2 100 ± 13 
I104A 23 ± 2 104 ± 1 94 ± 6 104 ± 1 99 ± 13 
D105A 8 ± 2 89 ± 3 62 ± 8 87 ± 2 97 ± 14 
E109A 99 ± 10 99 ± 4 107 ± 19 99 ± 4 100 ± 17 
E133A 99 ± 9 92 ± 3 96 ± 12 147 ± 5 80 ± 12 
E134A 86 ± 3 87 ± 1 82 ± 3 88 ± 12 92 ± 3 
E135A 72 ± 2 88 ± 1 74 ± 1 80 ± 12 90 ± 1 
Q137A 85 ± 12 63 ± 1 55 ± 4 61 ± 14 82 ± 1 
D139A 109 ± 1 87± 100 ± 4 139 ± 5 89 ± 14 
E145A 78 ± 9 84 ± 2 82 ± 12 93 ± 12 91 ± 11 
E146A 87 ± 4 93±± 92 ± 3 98 ± 12 97 ± 5 
E149A 97 ± 8 95 ± 1 92 ± 3 104 ± 11 99 ± 12 
K153A 71 ± 1 91 ± 1 86 ± 5 96 ± 11 93 ± 13 
K154A 82 ± 18 90 ± 1 78 ± 6 89 ± 19 91 ± 4 
K156A 106 ± 2 83 ± 11 97 ± 10 87 ± 7 96 ± 17 
a

Experiments were carried out as described in Table IV.

FIGURE 2.

The binding of hIFN-α2/α1 to hIFNAR2-IgG alanine mutants, as determined by an ELISA. Experiments were performed as described in Fig. 1 using various concentrations of Bio-hIFN-α2/α1.

FIGURE 2.

The binding of hIFN-α2/α1 to hIFNAR2-IgG alanine mutants, as determined by an ELISA. Experiments were performed as described in Fig. 1 using various concentrations of Bio-hIFN-α2/α1.

Close modal
FIGURE 5.

A, Location of residues on hIFNAR2 that are important for binding of hIFN-α2/α1. The model of hIFNAR2 was made by displaying its sequence on the structure of tissue factor (28 ). The crucial binding residues for hIFN-α2/α1 are shown in orange. B, Hypothetical model for the hIFNAR1 and hIFNAR2 interaction. C, Regions on hIFNAR2 important for binding of mAbs 1D3, 1F3, and 3B7. The binding regions of 1D3 and 3B7 are shown in dark blue and royal blue, respectively. The binding region of 1F3 includes all blue colors.

FIGURE 5.

A, Location of residues on hIFNAR2 that are important for binding of hIFN-α2/α1. The model of hIFNAR2 was made by displaying its sequence on the structure of tissue factor (28 ). The crucial binding residues for hIFN-α2/α1 are shown in orange. B, Hypothetical model for the hIFNAR1 and hIFNAR2 interaction. C, Regions on hIFNAR2 important for binding of mAbs 1D3, 1F3, and 3B7. The binding regions of 1D3 and 3B7 are shown in dark blue and royal blue, respectively. The binding region of 1F3 includes all blue colors.

Close modal

In our initial analysis of the multiple alanine substitution hIFNAR2-IgG mutants, we demonstrated a significant reduction in the binding of the anti-hIFNAR2 mAbs to some of these mutants. However, in the more detailed analysis of single alanine substitution mutants, we were not able to detect any single residue that had a significant effect on the binding of these mAbs. This suggests that multiple regions of hIFNAR2-IgG are involved in mAb binding and that the regions on hIFNAR2 interacting with mAbs are much larger than the region interacting with hIFN-α2/α1 as shown in other systems (25).

Soluble hIFNAR2 immunoadhesin molecules may assume a tertiary structure in solution that is different from the receptor cellular membrane structure. To extend the findings obtained with the soluble hIFNAR2-IgG, we generated murine L929 cells that express wild-type and mutant human IFN receptors. The species specificity of the IFN system has been widely used as a means to understand the receptor function. Murine cells, such as L929 cells, that express mIFNAR1 and mIFNAR2 will respond to all murine IFNs and some, but not all, human IFNs. For example, hIFN-α8, hIFN-α2, and hIFN-β signal with human IFNAR, while hIFN-α1, hIFN-α5, and hIFN-α10 can signal with human or murine IFNARs. Mouse L929 cells transfected with hIFNAR1 respond to hIFN-α8, but do not respond to hIFN-α2 or hIFN-β, demonstrating that the mIFNAR2 can interact with hIFNAR1 to effect a signaling complex (5). We utilized this system to test the signaling ability of the hIFNAR2 mutants.

We transfected L929 cells with a vector that encodes the cDNA for hIFNAR2 and established a stable cell line that expresses the full-length hIFNAR2 chain. The level of hIFNAR2 expression from a representative clone (L929.R2.19) was determined by flow cytometry using mAb 3B2 (Fig. 3,A). To demonstrate that the expressed human receptor is able to bind hIFN-α2, we determined the binding of biotinylated hIFN-α2 (Bio-hIFN-α2) to the L929.R2.19 cells by flow cytometry (Fig. 3,B). The L929.R2.19 cell line was then tested for its ability to respond to type I IFNs in an EMSA (Fig. 3,C). Mouse fibroblast L929 cells constitutively express murine IFN receptors. Thus, L929.R2.19 cells respond to murine IFN-α11 (mIFN-α11) with formation of an activated ISGF3 complex (Fig. 3,C). Human IFN-α1 is recognized by the murine receptor, so mouse L929 cells will respond to the hybrid human type I IFN (hIFN-α2/α1) we used in the binding studies cited above. Therefore, this hybrid human IFN could not be used in the studies utilizing the transfected L929 cells. To examine the response to other human IFNs, hIFN-α2, hIFN-α8, and hIFN-β were tested. After incubation of L929.R2.19 cells with 25 ng of hIFN-α2/ml or 100 U of hIFN-β/ml, an ISGF3 complex is observed. This complex is the same as that formed in response to mIFN-α11 and can be shifted by Abs to murine Stat1 or murine Stat2 (data not shown). In contrast, there is no response of L929.R2.19 cells to hIFN-α8 (Fig. 3,C). The hIFN-α8 is biologically active, because in a L929.R1 cell line expressing the hIFNAR1 chain, an ISGF3 complex is observed after hIFN-α8 treatment (Fig. 3 C). Thus, from this experiment, we conclude that in the presence of the hIFNAR2 chain, hIFN-α2 and hIFN-β, but not hIFN-α8, will signal a biological response in a mouse cell line.

FIGURE 3.

Receptor expression, binding of hIFN-α2, and signaling ability of L929 cells expressing hIFNAR2. A, The level of expression of a representative clone, L929.R2.19, was determined using FITC-mAb 3B3 (solid line, FITC-mAb 3B3; dotted line, unstained control).B, The ligand-binding ability was determined using Bio-hIFN-α2, followed by PE-streptavidin (solid line, Bio-hIFN-α2 plus PE-streptavidin; dotted line, PE-streptavidin alone). C, The ability of L929.R2.19 cells to mediate the IFN signal was determined by EMSA, which detects ISGF3 complex formation. As a control, L929. R1 cell line expressing hIFNAR1 is included.

FIGURE 3.

Receptor expression, binding of hIFN-α2, and signaling ability of L929 cells expressing hIFNAR2. A, The level of expression of a representative clone, L929.R2.19, was determined using FITC-mAb 3B3 (solid line, FITC-mAb 3B3; dotted line, unstained control).B, The ligand-binding ability was determined using Bio-hIFN-α2, followed by PE-streptavidin (solid line, Bio-hIFN-α2 plus PE-streptavidin; dotted line, PE-streptavidin alone). C, The ability of L929.R2.19 cells to mediate the IFN signal was determined by EMSA, which detects ISGF3 complex formation. As a control, L929. R1 cell line expressing hIFNAR1 is included.

Close modal

Two cDNAs were constructed that encode receptor mutants with alanine substitutions for multiple hydrophobic residues (2 mutant, residues 29–33, and 5 mutant, residues 74–78) in the ECD. Six cDNAs were constructed that encode receptor mutants with single alanine substitutions (R74A, E78A, W101A, I104, D105A, and E109A) in the ECD. Each construct was transfected into the mouse L929 cell line, and stable cell lines were established (Table VI). Fig. 4 illustrates the level of hIFNAR2 expression using mAb 3B2 and the binding of biotinylated hIFN-α2 (Bio-hIFN-α2) of representative clones (L929.R2.74–78, L929.R2.78, and L929.R2.74) from three mutant cell lines, as determined by flow cytometry.

Table VI.

Summary of signal transduction of L929 hIFNAR2 ECD mutantsa

hIFNAR2 MutantsReceptor ExpressionIGSF Complex Formation
mIFN-α11hIFN-α2hIFN-β
WT +++ 
29–33 ++ 
74–78 − +/− 
R74 ++ 
E78 ++ − 
W101 − 
I104 − 
D105 − 
E109 
hIFNAR2 MutantsReceptor ExpressionIGSF Complex Formation
mIFN-α11hIFN-α2hIFN-β
WT +++ 
29–33 ++ 
74–78 − +/− 
R74 ++ 
E78 ++ − 
W101 − 
I104 − 
D105 − 
E109 
a

The degrees of response (−, +/−, +, ++, and +++) were arbitrarily determined by comparing the results shown in Fig. 4.

FIGURE 4.

Receptor expression, binding of hIFN-α2, and signaling ability of L929-transfected cells expressing hIFNAR2 mutants. All of the experiments were conducted as described in Fig. 3. L929.R2.74–78, L929 cells expressing hIFNAR2 with multiple mutation in residues 74–78; L929.R2.78, L929 cells expressing hIFNAR2 with a single alanine mutation in residue E78; L929.R2.74, L929 cells expressing hIFNAR2 with a single alanine mutant in residue R74.

FIGURE 4.

Receptor expression, binding of hIFN-α2, and signaling ability of L929-transfected cells expressing hIFNAR2 mutants. All of the experiments were conducted as described in Fig. 3. L929.R2.74–78, L929 cells expressing hIFNAR2 with multiple mutation in residues 74–78; L929.R2.78, L929 cells expressing hIFNAR2 with a single alanine mutation in residue E78; L929.R2.74, L929 cells expressing hIFNAR2 with a single alanine mutant in residue R74.

Close modal

All of these clones expressed hIFNAR2, although the level of receptor expression was variable among all of the mutant clones examined due to the clonal variation (Fig. 4,A). The L929.R2.74 mutant receptor cell line expressed a low level of BiohIFN-α2 binding, while this binding was undetectable on the L929.R2.74–78 and the L929.R2.78 cell line (Fig. 4,B). All clones, including L929.R2.19 (Fig. 3 B), tested demonstrated a low level of Bio-hIFN-α2 binding that did not correlate well with the level of receptor expression. This difference may be a result of the biotinylation of the IFN.

The cell lines that express a mutant hIFNAR2 were tested for their ability to respond to mIFN-α11, hIFN-α2, and hIFN-β in an EMSA. The L929.R2.74–78 cell line that contains the receptor with alanine substitutions in residues 74–78 does not respond to hIFN-α2 and weakly responds to hIFN-β, yet remains responsive to mIFN-α11, indicating that there is no defect in the general pathway of ISGF3 formation. The single alanine substitution mutants of residue R74 and E78 were examined to determine which of these two residues are important for signal transmission. The L929.R2.74 cell line, which contains a hIFNAR2 with a single alanine substitution at residue R74, is responsive to hIFN-α2 and hIFN-β, even though the binding of the Bio-hIFN-α2 is low. The L929.R2.78 cell line does not respond to hIFN-α2, yet remains responsive to hIFN-β. These data indicate that residue E78 in this small mutated region is involved in mediating a functional response to hIFN-α2.

The results obtained in all L929 transfectants, including multiple mutants (residues 29–33 and residues 74–78) and single mutants (R74, E78, W101, I104, D105, and E109), are summarized in Table VI. All of these mutants expressed the ECD-hIFNAR2 on the cell surface membrane, as determined by flow cytometry, and responded to mIFN-α11 and hIFN-β. While all single mutants responded to hIFN-β, only two single mutants, R74A and E109A, responded to hIFN-α2. These results further support the finding that residues E78, W101, I104, and D105 are crucial for mediating the hIFN-α2 response. These findings in conjunction with the results from the ligand-binding studies performed with the soluble immunoadhesin suggest that the conformation of the soluble hIFNAR2-IgG closely mimics the structure of the membrane-bound receptor. We demonstrate that the regions on the soluble receptor that are recognized by the hybrid hIFN-α2/α1 are the same as those recognized by the hIFN-α2 on the hIFNAR2-expressing cell lines. While all type I IFNs utilize IFNAR2 to signal, we have demonstrated that different receptor regions are utilized by different IFNs in generating a biological response. Thus, in this system, hIFN-α8 is unable to signal without the hIFNAR1 receptor, while hIFN-β most likely uses residues on hIFNAR2 that are distinct from those utilized by hIFN-α2.

On the basis of their structural homology, the cytokine receptor superfamily can be divided into two classes (26, 27). The first class of the cytokine superfamily includes the receptors for human growth hormone, erythropoietin, GM-CSF, IL-3, IL-4, IL-6, and IL-7, while the class 2 cytokine receptor family includes the receptors for IFN-γ, IFN-α, and IL-10. The structures of the human growth hormone receptor (28) and tissue factor, which belong to class 1 and class 2 cytokine receptor superfamily, respectively, have been well characterized. The main difference between these two receptors is the angle between ∼100-aa domains. The angle between domains in the class 1 receptor family is ∼85°, while that of class 2 receptor family is ∼120°. Using the structure of tissue factor as a backbone (29), we have constructed a computer model of the ECD of hIFNAR2 (Fig. 5,A) to have a better understanding of the possible structure of hIFNAR2. The multiple alanine substitutions in hIFNAR2 residues 49–54, 68–72, 74–78, 105–109, 133–139, and 153–156 completely abolished the binding of hIFN-α2/1. Although it appears to be puzzling that so many residues from various portions of the hIFNAR2 sequence contribute to the binding of hIFN-α2/1, our computer model of hIFNAR2 reveals that all of these residues come together to form a protrusion. Furthermore, results obtained with single mutant analysis (Table V and Fig. 2) demonstrate that residues E78, W101, I104, and D105 are crucial for the binding of hIFN-α 2/1, and these residues form a small protrusion in our model (Fig. 5 A).

In our previous work describing the neutralizing epitope on hIFNAR1 recognized by blocking mAbs, we proposed a structural model of hIFNAR1 (19). The 409-residue ECD of hIFNAR1 is almost twice as big as the 217-residue ECD of hIFNAR2. It would be interesting to know how these hIFNAR1/hIFNAR2 come together in the presence of IFN. To visualize the spatial interaction of these two receptor chains with an IFN, we considered the following information. First, our present study demonstrates that both domains of IFNAR2 interact with the ligand, as proposed by Seto et al. (30). Second, the second and third domains of hIFNAR1 may be involved in mediating an IFN signal, and the region around the residue K249 is crucial (19). Finally, hIFNAR2 has ∼15 residues at the C-terminal portion of the ECD that may allow the hIFNAR2 to come into close proximity to the second and third domains of hIFNAR1. Based on this information, we designed a model that shows how hIFNAR1 and hIFNAR2 may come together in the presence of an IFN (Fig. 5 B). This needs to be confirmed using crystal structure analysis.

The results shown in Table IV and Fig. 5,C demonstrate that the binding of mAb 1F3 overlaps most closely with that of IFN-α 2/1. These binding areas are located both on domain 1 and 2 of IFNAR2. The main area recognized by mAb 3B7 includes residues 49–55 and 68–72 in domain 1 of hIFNAR2, while mAb 1D3 recognizes mainly residues 133–139 and 153–157 in domain 2. Residues important for the most potent blocking mAb 3B7 overlap only partially, but are in close proximity to the crucial binding region of IFN-α 2/1 (Fig. 5 C). These results suggest that mAb 3B7 (Kd−1 = 1 pM) exerts its potent blocking activity due to its high affinity to hIFNAR2 and its close proximity to the crucial ligand-binding region on hIFNAR2. In contrast, mAb 1D3, which binds to a region in domain 2 of hIFNAR2, showed no blocking activity in the ligand receptor-binding ELISA, but demonstrated weak blocking activities in the antiviral assay and in EMSA. This suggests the possibility that mAb 1D3 binding may interfere with the interaction of hIFNAR1 and hIFNAR2 since the residues that are important for mAb 1D3 binding are in domain 2, a lower portion of hIFNAR2.

It is well known that all of the type 1 IFNs, hIFN-α, hIFN-β, and hIFN-ω, bind to the same cell surface receptor, but there is some disparity in the biological effects demonstrated by different members of the type 1 IFN family. For example, IFN-β is more effective than other type 1 IFNs in the treatment of multiple sclerosis (31, 32) and induces a very strong cytoplasmic association between hIFNAR1 and hIFNAR2 of the type 1 IFNAR (33, 34). It has also been shown that hIFN-α2 and hIFN-β require distinct intracytoplasmic regions of the hIFNAR2 for signaling (35, 36). The results shown in Fig. 5 demonstrate that there are differences in the hIFNAR2 residues interacting with hIFN-α2 and those with hIFN-β. Our findings further support the notion that there are subtle but important differences between IFN-α and IFN-β in the way they interact with IFNAR.

Results shown in Fig. 3 demonstrate that murine L929-transfected cells with hIFNAR2 respond to hIFN-α2 and hIFN-β, but not hIFN-α8. The fact that hIFN-α8 mediates a signal through hIFNAR1 expressed on murine L929 cells, but not through hIFNAR2 expressed on L919 cells, indicates the important role of hIFNAR1 in mediating the hIFN-α8 signal on the murine cell background. By understanding the subtle differences among different subspecies of the type 1 IFNs, we may have a better understanding of how a single receptor sorts the various type 1 IFN signals. This is an area for future investigation. We believe that the information provided in this study will facilitate the understanding of type 1 IFN receptor structure.

We thank Brian Fendly for careful reading of the manuscript and A. Mironov for secretarial assistance.

2

Abbreviations used in this paper: hIFN, human IFN; ECD, extracellular domain; hIFNAR, hIFN-α receptor; ISGF3, IFN-stimulated gene factor 3; RT, room temperature.

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