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Receptor 2
*
Departments of Antibody Technology, Molecular Oncology, and Protein Engineering, Genentech Inc., South San Francisco, CA 94080; and
Department of Surgery, San Francisco Veterans Affairs 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 (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-2/1. We further demonstrated that residues E78, W101, I104,
and D105 are crucial for the binding of hIFN-2/1 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-8, 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-2, but remained responsive to hIFN-ß. In
summary, we have identified specific residues of hIFNAR2 important for
the binding to hIFN-2/1 and demonstrate that specific regions of the
IFNAR interact with the subspecies of type I IFN in different
manners. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,2 one
hIFN-ß, one hIFN-
, and one hIFN-
(1, 2). These
type I hIFNs share a common receptor (IFNAR) (3, 4), which
is composed of two chains, a 135-kDa subunit (hIFNAR1)
(5) and a 115-kDa ß 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- 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- 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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hIFNAR2-IgG and various subspecies of hIFN- 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-2/1 (IFN-2 residues 1–62/1 residues 64–166)
(15, 16) were a gift from Dr. M. J. Brunda
(Hoffman-LaRoche, Nutley, NJ). hIFN-ß was obtained from Sigma (St.
Louis, MO). The specific activities of the various type I 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 mAbs to hIFNAR2
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 x 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).
Capture ELISA
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).
Epitope mapping using a competitive binding ELISA
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).
Determination of the affinities of mAbs
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).
Electrophoretic mobility shift assay (EMSA)
HeLa cells (5 x 105 cells) were
incubated with each hIFN- (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-. 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).
Antiviral assay
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 x 105 A549 cells in 100 µl of medium
for 24 h. Cells were then challenged with 2 x
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-2 as a
standard.
Generation of various hIFNAR2-IgG mutants
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.
Plasmid construction and DNA transfection into mouse L929 cells
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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). mAbs 1F3 and 3B7
at 0.6 nM (0.1 µg/ml) were able to block >90% of hIFN-2/1
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.
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). 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-1, hIFN-2, hIFN-5, hIFN-8, hIFN-2/1, or IFN-ß
(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-ß, 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.
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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-2/1 (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-2/1, 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-2/1. 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-2/1. The main binding area of mAb 1D3 is in a
small area, including residues 133–139 and 153–157, in domain 2 of
hIFNAR2.
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2/1 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-2/1 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-2/1.
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-2/1 (Fig. 2). Compared with the binding of wild
hIFNAR2-IgG to various doses of Bio-hIFN-2/1, 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-2/1 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. 5A).
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2/1 as shown in other systems
(25). Residues on hIFNAR2 that are important for ligand binding are functionally important
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-8, hIFN-2, and hIFN-ß signal
with human IFNAR, while hIFN-1, hIFN-5, and hIFN-10 can signal
with human or murine IFNARs. Mouse L929 cells transfected with hIFNAR1
respond to hIFN-8, but do not respond to hIFN-2 or hIFN-ß,
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. 3A). To
demonstrate that the expressed human receptor is able to bind
hIFN-2, we determined the binding of biotinylated hIFN-2
(Bio-hIFN-2) to the L929.R2.19 cells by flow cytometry (Fig. 3B). The L929.R2.19 cell line was then tested for its
ability to respond to type I IFNs in an EMSA (Fig. 3C).
Mouse fibroblast L929 cells constitutively express murine IFN
receptors. Thus, L929.R2.19 cells respond to murine IFN-11
(mIFN-11) with formation of an activated ISGF3 complex (Fig. 3C). Human IFN-1 is recognized by the murine receptor, so
mouse L929 cells will respond to the hybrid human type I IFN
(hIFN-2/1) 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-2, hIFN-8, and hIFN-ß were tested. After incubation of
L929.R2.19 cells with 25 ng of hIFN-2/ml or 100 U of hIFN-ß/ml, an
ISGF3 complex is observed. This complex is the same as that formed in
response to mIFN-11 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-8 (Fig. 3C). The hIFN-8 is
biologically active, because in a L929.R1 cell line expressing the
hIFNAR1 chain, an ISGF3 complex is observed after hIFN-8
treatment (Fig. 3C). Thus, from this experiment, we
conclude that in the presence of the hIFNAR2 chain, hIFN-2 and
hIFN-ß, but not hIFN-8, will signal a biological response in a
mouse cell line.
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). Fig. 4
illustrates the level of hIFNAR2 expression using mAb 3B2 and the
binding of biotinylated hIFN-2 (Bio-hIFN-2) of representative
clones (L929.R2.74–78, L929.R2.78, and L929.R2.74) from three mutant
cell lines, as determined by flow cytometry.
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A). The L929.R2.74 mutant
receptor cell line expressed a low level of BiohIFN-2 binding,
while this binding was undetectable on the L929.R2.74–78 and the
L929.R2.78 cell line (Fig. 4B). All clones, including
L929.R2.19 (Fig. 3B), tested demonstrated a low level of
Bio-hIFN-2 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-11, hIFN-2, and hIFN-ß 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-2 and
weakly responds to hIFN-ß, yet remains responsive to mIFN-11,
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-2 and hIFN-ß, even though the binding of the
Bio-hIFN-2 is low. The L929.R2.78 cell line does not respond to
hIFN-2, yet remains responsive to hIFN-ß. These data indicate that
residue E78 in this small mutated region is involved in mediating a
functional response to hIFN-2.
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-11 and
hIFN-ß. While all single mutants responded to hIFN-ß, only two
single mutants, R74A and E109A, responded to hIFN-2. These results
further support the finding that residues E78, W101, I104, and D105 are
crucial for mediating the hIFN-2 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-2/1 are the same as those
recognized by the hIFN-2 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-8 is unable to
signal without the hIFNAR1 receptor, while hIFN-ß most likely uses
residues on hIFNAR2 that are distinct from those utilized by
hIFN-2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN-, 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. 5A)
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-2/1. Although it appears to be puzzling that so many
residues from various portions of the hIFNAR2 sequence contribute to
the binding of hIFN-2/1, 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- 2/1, and these residues form a small protrusion in
our model (Fig. 5A).
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. 5B). This needs to be confirmed using crystal structure
analysis.
The results shown in Table IV and Fig. 5C demonstrate that
the binding of mAb 1F3 overlaps most closely with that of IFN- 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- 2/1 (Fig. 5C). 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-, hIFN-ß, and
hIFN-, 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-ß 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-2 and hIFN-ß 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-2 and
those with hIFN-ß. Our findings further support the notion that there
are subtle but important differences between IFN- and IFN-ß in the
way they interact with IFNAR.
Results shown in Fig. 3 demonstrate that murine L929-transfected cells
with hIFNAR2 respond to hIFN-2 and hIFN-ß, but not hIFN-8. The
fact that hIFN-8 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-8
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.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: hIFN, human IFN; ECD, extracellular domain; hIFNAR, hIFN- receptor; ISGF3, IFN-stimulated gene factor 3; RT, room temperature.
Received for publication January 29, 1999. Accepted for publication May 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor into mouse cells: cloning and expression of its cDNA. Cell 60:225.[Medline]
/ß receptor: characterization and molecular cloning. Cell 77:391.[Medline]
ß receptor that is required for signaling. J. Biol. Chem. 270:21606. requires components of T-cell-receptor signalling. Nature 390:629.[Medline]
TNF receptor immunoadhesin: comparison to an anti-TNF monoclonal antibody. J. Immunol. 152:1347.[Abstract]
hybrids. Pharmacol. Ther. 66:507.[Medline]
receptor (hIFNAR1) using blocking monoclonal antibodies: the role of domains 1 and 2. J. Immunol. 160:1782.,
ß, and . 1992. In Current Protocols in Immunology, Vol.
1. J. E. Coligan, A. M. Kruisbeek,
D. H. Margulies, E. M. Shevach, and
W. Strober, eds. Wiley-Interscience, New York. Unit 6.9.1.
8 and its receptor complex. Protein Sci. 4:655.[Medline]
and transfer factor in the treatment of multiple sclerosis: a double-blind, placebo-controlled trial. J. Neurol. Neurosurg. Psychiatry 52:566. interferon in patients with chronic progressive multiple sclerosis did not cause clinical deterioration. Neurology 40:259.
and ß signaling: interferon ß selectively induces the interaction of the and ßL subunits of the type I interferon receptor. J. Biol. Chem. 271:23630.. J. Biol. Chem. 271:22878. and IFN-ß. EMBO J. 13:5871.[Medline]
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