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Receptor (hIFNAR1) Using Blocking Monoclonal Antibodies: The Role of Domains 1 and 21
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Department of Antibody and Bioassay Technology, Process Science, Molecular Biology and Protein Engineering, Genentech Inc., South San Francisco, CA 94080; and
San Francisco Veterans Administration Medical Center, San Francisco, CA 94121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor (hIFNAR1) using mAbs
generated by immunizing mice with a soluble hIFNAR1-IgG. Five mAbs
described in this study recognize different epitopes as determined by a
competitive binding ELISA and by alanine substitution mutant analyses
of the hIFNAR1-IgG. Two mAbs, 2E1 and 4A7, are able to block
IFN-stimulated gene factor 3 (ISGF3) formation and inhibit the
antiviral cytopathic effect induced by several IFN- (IFN-2/1,
-1, -2, -5, and -8). None of these anti-IFNAR1 mAbs
were able to block activity of IFN-ß. mAb 4A7 binds to a domain
1-hIFNAR1-IgG but not to a domain 2-hIFNAR1-IgG, which suggests that
its binding region is located in domain 1. The binding of the most
potent blocking mAb, 2E1, requires the presence of domain 1 and domain
2. The most critical residue for 2E1 binding is a lysine residue at
position 249, which is in domain 2. These findings suggest that both
domain 1 and domain 2 are necessary to form a functional receptor and
that a region in domain 2 is important. IFN-ß recognizes regions of
the hIFNAR complex that are distinct from those important for the
IFN-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN-ß,
and IFN-
(4). Human IFN- is a heterogeneous family with at least
23 polypeptides, but with only one IFN-ß polypeptide (4). The IFN-
subtypes show >70% amino acid sequence homology with each other and
25% amino acid identity with IFN-ß (4). Despite these
differences, IFN- and IFN-ß bind and signal through a shared
common species-specific receptor (5, 6).
Two functional components of the IFN- receptor complex have recently
been identified. The cDNA for the first one, human IFN- receptor
(hIFNAR1),3 encodes a
63-kDa receptor protein (7). This receptor undergoes extensive
glycosylation, which causes it to migrate in gel electrophoresis as a
much larger 135-kDa protein. The second IFN receptor, hIFNAR2
(IFN-ßR long), is a 115-kDa protein that binds type I IFNs with
high affinity and mediates a functional signaling complex when
associated with hIFNAR1 (8). Another IFN- receptor, IFN-ßR
short, is a 55-kDa protein that can bind to type 1 IFNs, but cannot
form a functional complex when associated with hIFNAR1, and is an
alternatively spliced variant of IFNAR2 (9).
hIFNAR1 has been shown to be essential for the response to all type 1
IFNs in IFNAR1 gene knockout experiments (10, 11) and for mediation of
all type 1 IFN- signals (12). Although Uze et al. (7) reported
binding of IFN-8 to hIFNAR1, others (11, 13) have been unable to
demonstrate similar binding. When hIFNAR2 is expressed on hamster cells
in the absence of hIFNAR1, these cells display a substantial level of
IFN-2 and IFN-8 binding (11), indicating that hIFNAR2 is
responsible for ligand binding activity. When hIFNAR1 and hIFNAR2 are
expressed on murine cells, the affinity of ligand binding is enhanced
10-fold compared with that on cells expressing hIFNAR2 alone (13).
These findings indicate that hIFNAR1 is one chain of a multicomponent
receptor complex that contributes to ligand binding and is essential
for signal transduction of type 1 IFNs; they also indicate that
hIFNAR2, not hIFNAR1, plays a crucial role in ligand
binding.
hIFNAR1 is a 557-amino acid protein composed of an extracellular domain (ECD) of 409 residues, a transmembrane domain of 21 residues, and an intracellular domain of 100 residues (7). The ECD of hIFNAR1 is composed of two domains, domain 1 and 2, which are separated by a three-proline motif. There is 19% sequence identity and 50% sequence homology between domains 1 and 2 (7). Each domain (D200) is composed of approximately 200 residues and can be further subdivided into two homologous subdomains (SD100) of approximately 100 amino acids.
Benoit et al. (14) developed an anti-hIFNAR1 mAb (64G12) by immunizing mice with a soluble ECD of hIFNAR1 produced in Escherichia coli. This mAb was shown to neutralize the activity of several type 1 IFNs and to recognize an epitope on domain 1 of IFNAR1, which suggests that domain 1 is important in mediating type 1 IFN functions. We have performed a structure-function analysis of the ECD of hIFNAR1 by evaluating the binding of mAbs to wild-type and various mutants of hIFNAR1-IgG. We have chosen this approach to identify regions that may be important for hIFNAR1 function, since direct ligand binding studies are difficult to perform. We found that both domain 1 and domain 2 of hIFNAR1 contain sites that bind to our blocking mAbs and that a lysine residue in domain 2 may be an important site for mediating a functional response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A cDNA encoding the human Ig fusion protein (immunoadhesin) based on the ECD of hIFNAR1 (pRK5 hIFNAR1-IgG clone 53.65) was generated as described (15). hIFNAR1-IgG was expressed in human embryonic kidney 293 cells by transient transfection using a calcium phosphate precipitation technique. The immunoadhesin was purified from serum-free cell culture supernatants in a single step by affinity chromatography on a protein A-Sepharose column (15). Bound hIFNAR1-IgG was eluted with 0.1 M citrate buffer, pH 3.0, containing 20% (w/v) glycerol. The hIFNAR1-IgG purified was >95% pure, as judged by SDS-PAGE.
Production of hIFN- subtypes
Standard cloning procedures were used to construct plasmids that
direct the translocation of the various species of IFN- into the
periplasmic space of E. coli (16, 17, 18, 19, 20). Human IFN-
(-IFN1, -2, -5, and -8) were purified from E.
coli paste containing each of the IFN- subtypes by affinity
chromatography using a mouse anti-IFN-8 Ab (LI-1) (21). LI-1 was
immobilized on controlled pore glass by a modification of the method of
Roy et al. (22). The purified IFN was analyzed by SDS-PAGE and
immunoblotting, and then assayed for bioactivity by the IFN-induced
antiviral assay as described below. IFN-ß was obtained from Sigma
(St. Louis, MO), and IFN- 2/1 was obtained from Hoffmann-La Roche
Inc. (Nutley, NJ).
Generation of mAbs to hIFNAR1
BALB/c mice were immunized into each hind footpad 11 times, at 2-wk intervals, with 2.5 µg of hIFNAR1-IgG fusion protein resuspended in monophosphoryl lipid A/ trehalose dicorynomycolate (Ribi Immunochemical Research Inc., Hamilton, MT). Three days after the final boost, popliteal lymph node cells were fused with murine myeloma cells P3X63AgU.1 (ATCC CRL1597; American Type Culture Collection, Rockville, MD), using 35% polyethylene glycol. Hybridomas were selected in hypoxanthine-aminopterin-thymidine (HAT) medium. Ten days after the fusion, hybridoma culture supernatants were first screened for mAb binding to the hIFNAR1-IgG fusion protein in a capture ELISA. The selected culture supernatants were then tested by flow cytometric analysis for their ability to recognize the hIFNAR1 on human U266 cells as described (23).
Affinity determination using radioimmunoprecipitation
The affinities of anti-hIFNAR1 mAbs were determined in a competitive binding radioimmunoprecipitation assay (24). Briefly, 125I-labeled hIFNAR1-IgG (sp. act. 11.6 µCi/µg) was prepared using a lactoperoxidase labeling method. mAbs were allowed to bind to 125I-labeled hIFNAR1-IgG in the presence of various concentrations of unlabeled hIFNAR1-IgG for 1 h at RT. These mixtures were then incubated with goat anti-mouse IgG for 1 h at RT in the presence of 5% human serum. The immune complexes were then precipitated by the addition of cold 6% polyethylene glycol (m.w. 8000) followed by centrifugation at 200 x g for 20 min at 4°C. Supernatants were removed, and the radioacitivity remaining in the pellet was determined using a gamma counter. The affinity of each mAb was determined by Scatchard analysis (25).
Capture ELISA
Microtiter plates (Maxisorb; Nunc, Flow Lab, McLean, VA) were coated with 50 µl/well of 2 µg/ml of goat Abs specific to the Fc portion of human IgG (Goat anti-hIgG-Fc; Cappel, ICN Pharmaceuticals, Inc., Costa Mesa, CA) in PBS overnight at 4°C and blocked with 2% BSA for 1 h at RT. After washing the plate, 50 µl/well of 2 µg/ml of hIFNAR1-IgG (or hIFNAR1-IgG mutant) were added to each well for 1 h. After washing the plate, the remaining anti-Fc binding sites were blocked with PBS containing 3% human serum and 10 µg/ml of CD4-IgG for 1 h. After washing, plates were then incubated with 50 µl/well of 2 µg/ml of anti-hIFNAR1 mAbs (or hybridoma culture supernatants) for 1 h. After washing, plates were then incubated with 50 µl/well of HRP-goat anti-mouse IgG. The bound enzyme was detected by the addition of the substrate, and the absorbance at 450 nM was read with a plate reader. Between each step, plates were washed in wash buffer (PBS containing 0.05% Tween-20).
Western blot
Reduced hIFNAR1 was prepared by treating the hIFNAR1-IgG fusion protein with 5 mM 2-ME at 95°C for 5 min. The ability of the mAbs to bind to the native and reduced hIFNAR1-IgG was determined by immunoblotting using 12% SDS-PAGE as described (24).
Epitope mapping using a competitive binding ELISA
To determine whether the mAbs recognized the same or different epitopes, a competitive binding ELISA was performed as described (24) using biotinylated mAbs (bio-mAb). Microtiter wells were coated with 50 µl of goat anti-hIgG-Fc and kept overnight at 4°C, blocked with assay buffer for 1 h, and incubated with 25 µl/well of hIFNAR1-IgG (1 µg/ml) for 1 h at RT. After washing microtiter wells, a mixture of a predetermined optimal concentration of bio-mAb and 1000-excess of unlabeled mAb was added into each well. Following 1 h incubation at RT, plates were washed and the amount of bio-mAb was detected by the addition of HRP-streptavidin. After washing microtiter wells, the bound enzyme was detected by the addition of the substrate, and the plates were read at 450 nm with an ELISA plate reader.
Electrophoretic mobility shift assay (EMSA)
Briefly, -IFNs (25 ng/ml) plus various concentrations (5–500
µg/ml) of anti-hIFNAR1 mAbs were incubated with 5 x
105 HeLa cells in 200 µl of DMEM for 30 min at
37°C. Cells were washed in PBS and resuspended in 125 µl buffer A
(10 mM HEPES, pH 7.9, 10 mM KCL, 0.1 mM ETDA, I mM DTT, 1 mM PMSF, 10
µg/ml leupeptin, 10 µg/ml aprotinin) (26). After a 15-min
incubation on ice, cells were lysed by the addition of 0.025% Nonidet
P-40. The nuclear pellet was obtained by centrifugation and was
resuspended in 50 µl of buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl,
0.1 mM EDTA, I mM DTT, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml
aprotinin) and kept on ice for 30 min. The nuclear fraction was cleared
by centrifugation and the supernatant stored at -70°C until use.
Double-stranded probes were prepared from single-stranded
oligonucleotides (ISG15 top, 5'-GATCGGGAAAGGGAAACCGAAACTGAAGCC-3';
ISG15 bottom, 5'-GATCGGCTTCAGTTTCGGTTTCCCTTTCCC-3') utilizing a DNA
polymerase I Klenow fill-in reaction with[32P]dATP (3000
Ci/mM, Amersham, Arlington Heights, IL). Labeled oligonucleotides were
purified from unincorporated radioactive nucleotides using BIO-Spin 30
columns (Bio-Rad, Hercules, CA). Binding reactions, containing 5 µl
of nuclear extract, 25,000 cpm labeled probe, and 2 µg of nonspecific
competitor poly(dI-dC):poly(dI-dC) in 15 µl of binding buffer (10 mM
Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 15%
glycerol) were incubated at RT for 30 min. 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 with anti-STAT1 Ab.
Antiviral assay
The assay was done as described in duplicate in 96-well
microtiter plates (27) using human lung carcinoma A549 cells challenged
with encephalomyocarditis virus. Serial dilutions of mAbs in 50 µl of
DMEM were incubated with various units of type 1 IFNs in 50 µl DMEM
for 1 h at 37°C. These mixtures were then incubated with A549
cells (5 x 105 cells/100 µl DMEM containing
4% FCS) for another 24 h. Culture supernatants were then removed,
and cells were challenged with 2 x 105 plaque-forming
units of encephalomyocarditis virus in 100 µl of DMEM plus 2% FCS
for additional 24 h. At the end of the incubation period, cell
viability was determined by visual microscopic examination. The
neutralizing Ab titer (EC50) was defined as the
concentration of Ab which neutralizes 50% of the antiviral cytopathic
effect by 10 U/ml of type 1 IFNs. The units of type 1 IFNs used in this
study were determined using National Institutes of Health reference
recombinant human IFN-2 as a standard. The specific activities of
the various type 1 IFNs are as follows: IFN-2/1, 2 x
107 IU/mg; IFN-1, 3 x 107 IU/mg;
IFN-2, 2 x 107 IU/mg; IFN-5, 8 x
107 IU/mg; IFN-8, 19 x 107 IU/mg; and
IFN-ß, 1.5 x 105 IU/mg.
Generation of domain 1-IgG, domain 2-IgG, and mutants to hIFNAR1
The cDNAs encoding domain 1 (1–200 residues) and domain 2 (204–404 residues) of hIFNAR1 were separately constructed and expressed as immunoadhesins as described above. Single alanine substitution mutants were generated according to the method of Kunkel (28). The plasmid DNA was isolated using an RPM Kit (BIO 101 Inc., La Jolla, CA) and was sequenced by the Sanger method using an ABI 373A DNA sequencer (Perkin Elmer, Foster City, CA) to verify the mutation. Mutant receptor-IgGs were expressed transiently in human 293 cells as described above. Transfected 293 cells were grown overnight in F-12 DMEM (50:50) containing 10% FCS, 2 mM glutamine, 100 µg/ml of penicillin, 100 µg/ml of streptomycin, 10 µg/ml of glycine, 15 µg/ml of hypoxanthine, and 5 µg/ml of thymidine and were then placed in serum-free media. Three days later, culture supernatants were collected and used in a capture ELISA. For the hIFNAR1-IgG mutant analysis, the concentrations of immunoadhesin molecules in 293-transfected culture supernatants were determined using CD4-IgG as a standard and were adjusted to be equal to the lowest concentration of immunoadhesin molecules. The degree of mAb binding to these mutants was then compared with the wild type of the same concentration.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For this study, we selected five anti-hIFNAR1 mAbs (2E1, 2E8,
2H6, 4A7, and 5A8) that showed different binding epitope and blocking
activities. All of these mAbs are of the IgG2a isotype and recognize
the hIFNAR1 expressed on U266 human myeloma cells as determined by FACS
analysis (Table I
). Western blot analysis
shows that mAbs 2H6, 4A7, and 5A8 bind to reduced hIFNAR1 (data not
shown). This suggests that mAbs 2E1 and 2E8 recognize conformational
epitopes, whereas mAbs 2H6, 4A7, and 5A8 may recognize linear epitopes.
The dissociation constants of these mAbs for hIFNAR1-IgG are in the
range of 52 to 3120 pM, as determined by competitive
radioimmunoprecipitation followed by Scatchard analysis.
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) show that these five mAbs could detect
four different epitopes on hIFNAR1. mAbs 2E1 and 2E8 can compete with
each other, which suggests that they may recognize the same or an
overlapping epitope.
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The blocking activities of mAbs to hIFNAR1 were determined using
an ISGF3 EMSA as well as an antiviral assay. Type 1 IFNs induce the
transcription of IFN-stimulated genes through the formation and
activation of IFN-stimulating response element (ISRE) binding proteins.
One of these binding proteins is ISGF3, which is a multisubunit protein
complex formed in the cytoplasm within minutes of type 1 IFN treatment
(29, 30). By investigating ISGF3 formation in HeLa cells induced by the
addition of 25 ng/ml of several human type 1 IFNs (IFN-2/1, -1,
-2, -5, -8, and IFN-ß), we determined the blocking
activities of mAbs in the range of 5 to 500 µg/ml. Figure 2 shows a typical autoradiograph of ISGF3
formation induced by hIFN-8. mAbs 2E1 and 4A7 at 5 µg/ml inhibited
ISGF3 formation induced by IFN-8; mAb 5A8 inhibited completely the
activity of IFN-8 at 500 µg/ml and inhibited it partially at 50
µg/ml; mAbs 2E8 and 2H6 were unable to block the activity of
IFN-8. Results obtained with all type 1 IFNs tested are summarized
in Table II. Although there is some
variation in the potency of blocking activities of mAbs 2E1 and 4A7
depending upon the subspecies of IFN-, mAbs 2E1 and 4A7 inhibited
the activities of all IFN-s tested; mAb 2E1 was the more potent
inhibitor. mAb 5A8 at 500 µg/ml showed blocking activity on IFN-8
and partial blocking activities on IFN-2/1 and -2. mAbs 2E8 and
2H6 showed no blocking activity on any of these hIFN-. None of these
mAbs to hIFNAR1 were able to block ISGF3 formation induced by
IFN-ß.
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). Assays were
done using serial dilutions of mAbs in the range of 0.1 to 30 µg/ml
and 10 U/ml of type 1 IFNs. The units of these IFNs were determined
using NIH IFN-2 as a standard. mAb 2E1 and mAb 4A7 blocked the
activity of all IFN-s. mAbs 2E8, 2H6, and 5A8 showed no neutralizing
activities in the antiviral assay. None of these mAbs were able to
neutralize the effect of IFN-ß. Similar results were obtained using
100 U/ml of type 1 IFNs (data not shown). The results obtained in the
antiviral assay correlated well with those of the EMSA, although there
were some discrepancies. For example, mAb 4A7 showed partial inhibitory
activity in ISGF3 formation by hIFN-5 at 500 µg/ml, but
neutralized the antiviral effect of hIFN-5 at 6 µg/ml. mAb 5A8
showed complete blocking activity on hIFN-8 and partial blocking
activities on hIFN-2/1 and hIFN-2 at 500 µg/ml in ISGF3 EMSA,
but showed no blocking activity in the antiviral assay. The discrepancy
between the two assays may be due to different cell lines (A549 cells
in antiviral assay and HeLa cells in EMSA) interacting with the
different type I IFNs and the different sensitivities of these assays.
This hypothesis is supported by the previous report of Benoit et al.
(14) that their mAb 6G4 demonstrated different affinities for IFNAR1 on
different cell lines.
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) and the
antiviral assay (Table III) we conclude that mAbs 2E1 and 4A7 are
blocking mAbs against all of the IFN-s tested, mAb 5A8 is a very
weak blocking mAb, and mAbs 2E8 and 2H6 are nonblocking mAbs. None of
these mAbs were able to block the activity of hIFN-ß. The affinity of
mAb 2H6 is very low (Kd-1 =
3120 pM) compared with that of the other mAbs; thus, the nonblocking
activity of mAb 2H6 may be due to its low affinity. Determination of mAb binding to domain 1 and domain 2 of hIFNAR1-IgG
We expressed domain 1 (residues 1–200) and domain 2 (residues
204–404) of hIFNAR1 separately as immunoadhesins (Fig. 3) and determined the binding capacity of
our mAbs to these adhesin molecules in a capture ELISA. The
concentrations of domain 1-IgG and domain 2-IgG in the culture
supernatant were determined by comparison to the known concentrations
of CD4-IgG in an ELISA. mAbs 2H6 and 4A7 bound only to the domain
1-IgG. mAb 5A8 bound to both the domain 1-IgG and the domain 2-IgG,
while mAbs 2E1 and 2E8 were unable to bind to either (Fig. 4). From these results, we conclude that
mAbs 2E1 and 2E8 recognize conformational epitopes composed of regions
in both domains 1 and 2. Blocking mAbs (2E1, 4A7, and 5A8) recognize
epitopes in domain 1, suggesting the importance of domain 1 in
signaling. mAb 5A8 binds to both of the single-domain IgGs, suggesting
the presence of repeating or structurally very similar epitopes in
domains 1 and 2. It is interesting to note that the extent to which mAb
5A8 binds to wild-type hIFNAR1-IgG appears to be the sum of its binding
to each domain-IgG.
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To define areas of hIFNAR1 that play an important role in mAb
binding, we generated multiple alanine substitution mutants in the
hydrophilic regions of hIFNAR1. Residues 19–25, 69–74, 76–80,
103–111, 148–152, 157–162, 244–249, 291–298, 352–359, and
383–388 were selected for mutagenesis (Fig. 3). After adjusting the
hIFNAR1-IgG mutants in the culture supernatants of 293 transfectants to
equivalent concentrations (50 ng/ml), we determined the binding ability
of our mAbs to these mutants in a capture ELISA. Results shown in Table IV were obtained using mAbs at 10 µg/ml
in a capture ELISA; however, similar results were obtained with various
concentrations (0.1–10 µg/ml) of mAbs (data not shown). The binding
capacity of the most potent blocking mAb, 2E1, was significantly
reduced or almost undetectable when the hydrophilic amino acids in
residues 69–74 (domain 1), 244–249 (domain 2), or 291–298 (domain 2)
were substituted with alanines (Table IV). The binding to the alanine
mutant of residues 69–74 was significantly reduced with all mAbs
except mAb 5A8. The binding of mAb 5A8 to this mutant was 67% of that
to the wild type. Since mAb 5A8 was shown to bind to the domain 1-IgG
and to the domain 2-IgG separately (Fig. 4), some of the 67% binding
to this 69–74 mutant by mAb 5A8 was thought to be due to the binding
to domain 2. Thus, the alanine substitution of residues 69–74
drastically affected the binding of all mAbs except mAb 5A8, suggesting
that mutations in this region causes some structural change to occur in
this portion of the receptor.
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). In domain 1,
an arginine at position 74 (R74) was the crucial residue for the
binding of mAb 2H6. In domain 2, charged acidic residues, glutamic acid
at position 291 (E291), and aspartic acid at position 296 (D296) played
important roles in the binding of mAbs 2E1 and 2E8. Interestingly, a
charged basic residue, lysine at position 249 (K249), was important for
the binding of the blocking mAb 2E1 but not for the binding of the
nonblocking mAb 2E8. This suggests that the structure around K249 may
be important in the interaction of ligand with hIFNAR1.
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These epitope mapping studies have been performed using soluble
receptor proteins. It would be useful to demonstrate that the binding
of these mAbs to a soluble hIFNAR1-IgG reflects what is happening on
the ECD of a membrane-associated hIFNAR1. To address this issue, we
determined mAb binding to membrane hIFNAR in the presence of
soluble hIFNAR-IgGs. Fluoresceinated (F-) mAbs were incubated with
wild-type or mutant soluble hIFNAR1-IgGs at RT for 30 min.
These mixtures were then added to U266 human myeloma cells. After
incubation at 4°C for 30 min, cells were washed and analyzed by FACS.
When the wild-type hIFNAR1-IgG was added, the binding of F-2E1 to U266
cells was completely inhibited (Table VI). The same results were obtained with
mAbs 2E8 and 4A7. These results demonstrate that wild-type soluble
hIFNAR1 can effectively inhibit the mAb binding to membrane hIFNAR1 on
U266 cells. This finding suggests that the structure of the soluble
hIFNAR1-IgG indeed mimics the structure of the ECD of membrane hIFNAR1.
Additionally, we performed inhibition experiments using soluble
hIFNAR1-IgG mutants (Nos. 7 and 8 in Table IV). Soluble mutant 7
(alanine substitutions in residues 244–249) inhibited only the binding
of mAbs 2E8 and 4A7, but not 2E1, while soluble mutant 8 (alanine
substitutions in residues 291–298) inhibited the binding of mAbs 4A7,
but not 2E1 and 2E8 as expected. From these results, we conclude that
the epitopes recognized by mAb 2E1 include residues 244–249 and
291–298 and those recognized by mAb 2E8 include residues 291–298 on
hIFNAR1, whether these are expressed as a soluble form or on the cell
membrane.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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8, Seto et al.
(31) ppoposed that type 1 IFNs have two receptor binding sites. Site 1
of type 1 IFNs was proposed to be the region of high affinity binding
to IFNAR2, while site 2 was the region of low affinity interaction with
hIFNAR1. They further proposed from their model that domain 2 of
hIFNAR1 plays an important role in IFN signal transduction. Benoit et
al. (14) reported that their blocking mAb 64G12, which blocks
activities of all type 1 IFNs, binds to domain 1 of hIFNAR1. Their
findings suggest that domain 1 is important in mediating type 1 IFN
activity. The results presented here show that both domain 1 and domain
2 of hIFNAR1 are necessary for the mediation of an IFN- signal.
First, the blocking mAb 4A7 binds to the domain 1-IgG, which implies
the participation of domain 1 in IFN signaling. Second, the binding of
the most potent blocking mAb, 2E1, requires the presence of domains 1
and 2 of hIFNAR1; K249 in domain 2 is crucial in the binding of
this mAb. The mAb binding studies reported here used soluble immunoadhesin molecules, which may have a tertiary structure that is somewhat different from the membrane hIFNAR1 expressed on cells. We have shown that wild-type and mutant soluble receptors can effectively inhibit mAb binding to membrane hIFNAR1 in a specific manner. This suggests that soluble hIFNAR1 retains the structure of the ECD of membrane hIFNAR1, at least in the Ab-binding region. Additional experiments using transfected cells that express hIFNAR1 mutants in regions described herein would be necessary to confirm our findings.
Cytokine receptors have been categorized into two classes based on
structural similarity and the distribution of cysteine residues in
their ECD (32, 33). The class 1 cytokine receptor family includes
receptors for human growth hormone, erythropoietin, IL-3, and IL-4,
while the class 2 cytokine receptor family includes the IFN receptors,
tissue factor, CRF2–4, and IL-10 receptor. Crystal structures recently
have been resolved for members of each class. The angle between the two
subdomains (100 residues/subdomain) of the ECD is significantly
different between members of the two families (34). In the class 1
receptor family, the structures of the human growth hormome (35) and
the prolactin receptor (36) show an angle between subdomains of about
85°, whereas this angle is about 120° in the class 2 members,
tissue factor (37), and the IFN-
R (38). To construct a model of the
ECD of hIFNAR1, we displayed its sequence on the backbone of tissue
factor. As noted previously, the ECD of hIFNAR1 contains four
subdomains each containing 100 amino acids (SD100A and SD100B =
domain 1; SD100A' and SD100B' = domain 2) (Fig. 3). The ECD of tissue
factor contains two subdomains. First, the two subdomains of domain 1
of hIFNAR1 were modeled against the two subdomains of tissue factor.
Then, the orientation between domains 1 and 2 of hIFNAR1 was modeled on
that observed between subdomains. Figure 5 shows a space-filling model of hIFNAR1,
with residues potentially involved in the binding of the mAbs described
above. Residues 69–74 and 103–111 are located in domain 1, in
subdomains SD100A and SD100B, respectively, and residues 244–249 and
291–298 in SD100A' of domain 2. Residues 69–74 are situated away from
the other three, at the top of our model. Substitutions in this region
significantly affect binding of all mAbs except mAb 5A8, which was
shown to bind to both domain 1 and domain 2 of hIFNAR1-IgG. We propose
that residues 69–74 may be structurally important. The remaining three
regions (residues 103–111, 244–249, and 291–298) are clustered near
each other and may constitute a part of the binding sites of the
blocking mAbs 2E1 and 4A7.
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160
kDa) is much greater than that of ligands, resulting in steric
hindrance of the ligand-receptor interaction. Therefore, residues on
hIFNAR1, which are important for the binding of our blocking mAbs, may
not be the residues that interact with the IFN-; however, the
ligand-receptor interaction is likely to be close to the region of
blocking Ab binding. Thus, our findings provide insight into a possible
site of ligand interaction.
None of our anti-hIFNAR1 mAbs blocked IFN-ß activity, which
suggests that IFN- and IFN-ß interact with hIFNAR1 and IFNAR2
differently, although they share a common receptor. Our findings
further support the observation by others (40, 41) that IFN- and
IFN-ß utilize different assembly pathways of hIFNAR1 and IFNAR2. Our
blocking mAbs may be useful tools for understanding these
differences.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. K. J. Kim, Department of Antibody and Bioassay Technology, Genentech, Inc., South San Francisco, CA 94080. E-mail address:
3 Abbreviations used in this paper: hIFNAR1 and -2, human IFN- receptor 1 and 2; D, aspartic acid; E, glutamic acid; ECD, extracellular domain; IFN-ßR long, IFNAR2; hIFNAR1-IgG, hIFNAR1 immunoadhesin molecule; K, lysine; R, arginine; RT, room temperature; HRP, horseradish peroxidase; EMSA, electrophoretic mobility shift assay; bio-mAb, biotinylated mAbs; SD, subdomain (e.g., SD100A); ISG, IFN-stimulated gene; ISGF, IFN-stimulated gene factor.
Received for publication April 3, 1997. Accepted for publication November 3, 1997.
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A. Chuntharapai, K. Dodge, K. Grimmer, K. Schroeder, S. A. Marsters, H. Koeppen, A. Ashkenazi, and K. J. Kim Isotype-Dependent Inhibition of Tumor Growth In Vivo by Monoclonal Antibodies to Death Receptor 4 J. Immunol., April 15, 2001; 166(8): 4891 - 4898. [Abstract] [Full Text] [PDF]
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A. Chuntharapai, V. Gibbs, J. Lu, A. Ow, S. Marsters, A. Ashkenazi, A. De Vos, and K. J. Kim Determination of Residues Involved in Ligand Binding and Signal Transmission in the Human IFN-{alpha} Receptor 2 J. Immunol., July 15, 1999; 163(2): 766 - 773. [Abstract] [Full Text] [PDF]
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