From the Department of Molecular Genetics & Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854
Received for publication, October 23, 2000, and in revised form, February 6, 2001
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ABSTRACT |
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Interferons have antiviral, antigrowth and
immunomodulatory effects. The human type I interferons, IFN- The type I interferon receptor,
IFNAR,1 mediates the binding
of all type I interferons (IFNs), which, in the human, derive from
genes for 13 IFN- IFNAR-1 and IFNAR-2 are members of the cytokine receptor superfamily.
All share conserved structural fibronectin type III (FNIII) "building
blocks" forming the extracellular ligand-binding domain (27-29).
Therefore, much can be learned about IFNAR-1 and IFNAR-2 from the
structurally well-defined related receptors, including the
interferon- The low intrinsic affinity of HuIFNAR-1 for IFNs has hampered studies
seeking to identify residues involved in ligand binding and
specificity. Previously, the identity of elements of the ligand binding
site of HuIFNAR-1 could only be deduced indirectly from studies
involving antibody epitope mapping (40-42) or homology modeling based
on other cytokine receptors (40, 43). Without large-scale mutagenesis
and ligand binding analysis, key residues could not be identified in
HuIFNAR-1.
The bovine IFNAR-1 homologue is an attractive target for mutagenesis
and analysis of the IFN binding site. Although the type I interferons
are predominantly species-specific, human interferons display uniformly
high binding and biological activity on bovine cells (3, 44-46). This
appears to reflect the ability of bovine IFNAR-1 (BoIFNAR-1) to bind
human type I IFNs with moderately high affinity. Thus, human or murine
cells expressing BoIFNAR-1 greatly increase their responsiveness to a
variety of human type I IFNs (18, 47, 48). The nanomolar binding
affinity of BoIFNAR-1 for HuIFN- BoIFNAR-1 cDNAs were cloned by independent laboratories (47, 48)
and found to encode a transmembrane protein of 560 amino acids. The
protein consists of a 24-amino acid signal sequence, a 414-amino acid
extracellular domain, a 24-amino acid transmembrane sequence, and a
98-amino acid cytoplasmic domain. The human and murine IFNAR-1 proteins
share 68 and 46% amino acid sequence identity, respectively, with
BoIFNAR-1 (6, 48, 51). This high sequence identity, combined with an
overall structural conservation, has allowed us to use BoIFNAR-1 in
place of HuIFNAR-1 in studies directed at identifying structural
features contributing to HuIFN ligand binding.
The ability of BoIFNAR-1 to bind HuIFNs in the nanomolar range has been
well characterized using a variety of systems (18, 23). COS-1 cells
transfected with BoIFNAR-1 cDNA express very high levels
(0.5-1.0 × 106/cell) of nanomolar affinity binding
sites for HuIFN- Previously, we used BoIFNAR-1 to regionally localize the determinants
that confer strong binding of IFN (49). A series of 14 HuIFNAR-1/BoIFNAR-1 chimeric receptors representing various human/bovine subdomain substitutions were assayed for their ligand binding properties. Only when the two central domains of BoIFNAR-1 (SD2
and SD3) were simultaneously substituted into the HuIFNAR-1 was a
significant increase in the binding of HuIFN- The current study of BoIFNAR-1 identifies amino acids that are critical
for IFN binding, using a series of alanine substitutions throughout its
extracellular domain. By presenting these results in the context of a
new three-dimensional homology model of IFNAR-1, we shed light on the
interaction of type I IFNs with IFNAR-1.
Interferons, Receptor cDNAs, and Antibodies--
IFN- Cells and Media--
Dulbecco's modified Eagle's medium (DMEM,
Life Technologies, Inc.) supplemented with 10% cosmic calf serum
(HyClone), 2 mM glutamine (Sigma), and 10 units/ml
penicillin with 10 µg/ml streptomycin (Life Technologies, Inc.), was
used to maintain all cell lines.
Phosphorylation of IFN--
The HuIFN- Parental Plasmids and Expression--
Bovine and Human IFNAR-1
were transiently expressed from the EF-1 Mutagenesis--
Mutations were engineered into the receptor
using either a two-step splice overlap extension (SOE) polymerase chain
reaction method (56, 57) or the QuikChange site-directed mutagenesis kit (Stratagene). All clones were screened by restriction digests and
confirmed by sequencing. In most cases altered receptors were analyzed
using two independent clones, to eliminate false decreases in binding
due to reasons other than the engineered mutation. Mutants were made
from the BoIFNAR-1 cDNA template in three different versions of
pcDEF-3 at various stages in this project. The BoIFNAR-1 gene was the
same for all three; however, the first had an HA tag at the N terminus
(which was not effective for flow cytometric detection); in the second,
the HA tag was replaced with a FLAG epitope to allow for cell surface
confirmation by flow cytometry; the third version contained an internal
ribosome entry site coupled to enhanced green fluorescent protein
(IRES-EGFP) (CLONTECH) after the FLAG-bovine
IFNAR-1, and a zeocin marker in place of the original neomycin marker.
These plasmids, when transfected into COS cells, were indistinguishable
in their transient BoIFNAR-1 expression and IFN binding.
The two-step Splice Overlap Extension (SOE) polymerase chain reaction
method (56, 57) was carried out using ELongase (Life Technologies,
Inc.). SOE fragments were restriction-digested and then ligated into
the proper vector. All reactions were carried out in a 50-µl volume
in 0.5-ml thin-wall tubes in a PerkinElmer Life Sciences DNA thermal
cycler. The QuikChange site-directed mutagenesis kit was used according
to the manufacturer's directions (Stratagene), except that template
and primer concentrations were doubled; extension time was increased to
3.5 min per kilobase; and DH10B Electromax competent cells (Life
Technologies, Inc.) were used for transformations.
DNA Transfections--
COS-1 cells (58), derived from a simian
kidney CV-1 line, were transfected with 5-10 µg of plasmid using the
DEAE-dextran/Me2SO shock protocol (48, 59, 60). Briefly,
tissue culture dishes (10-cm dish, Falcon) containing 10 ml of 10%
cosmic calf (Hyclone) serum-supplemented DMEM were seeded with
1.75-2.0 × 106 cells. Cells were incubated overnight
at 37 °C before transfecting. Cells were harvested for assays after
48-72 h. Under our transfection conditions, we generally see about
0.25-1 × 106 receptors per cell, averaged over the
entire cell population, as measured by ligand binding to BoIFNAR-1.
This arises from high level expression on 20-50% of the cell
population, as demonstrated by flow cytometry detection of the common
FLAG epitope at the N terminus. Much of this expression variability is
between experiments; within any experiment, the variation of expression
between constructs has a much smaller range. Each clone was transfected
at least two times, and for most mutations binding was measured with
transfections of two independent DNA clones.
Saturation Binding--
Saturation binding assays were done as
previously described (48). Briefly, COS cells were trypsinized and
resuspended to 1 × 106 cells/ml. Aliquots of cells,
with and without excess cold IFN (1-3 µg/ml), were combined with
[32P]IFN- Flow Cytometric Analysis of Receptor Surface
Expression--
Transfected cells were resuspended in DMEM
supplemented with 5% cosmic calf serum (Life Technologies, Inc.).
Cells (7.5 × 105 to 1.25 × 106 in
25 µl) were incubated for 1 h at 4 °C with 25 µl
(0.12-0.25 µg) of primary antibody (M2 anti-FLAG antibody, Sigma) or
medium. The cells were washed with PBS and resuspended in 50 µl
(0.125 µg) of secondary antibody
(R-phycoerythrin-conjugated F(ab')2 goat
anti-mouse IgG, Jackson ImmunoResearch) and incubated for 1 h.
Cells were again washed with PBS and then incubated in 100 µl of 3%
paraformaldehyde at 4 °C for 1 h. The cells were washed once
with 1 ml of PBS containing 50 mM Tris and then resuspended in 500 µl of PBS with 50 mM Tris and stored at 4 °C
until analysis with a Coulter Epics Profile II cell sorter.
Model Building--
Homology models were generated for the
extracellular domains of bovine and human IFNAR-1, based on the
coordinates of the extracellular domain of IFNGR-1 (30, 70) graciously
provided by Drs. Mark Walter (University of Alabama, Birmingham) and
Steve Ealick (Cornell University). The homology model was created using GeneMine (LOOK v.3, Molecular Applications Group) and manipulated with
GeneMine and Sybyl (Tripos, Inc.).
The IFNAR-1 (4-domain receptor) was modeled against IFNGR-1 (2-domain
receptor) in two steps. Because of the size disparity, it was necessary
to separately model the N-terminal half (SD1 and SD2) and the
C-terminal half (SD3 and SD4) of the receptor. This procedure is
consistent with the finding that the four domains of IFNAR-1 likely
evolved from a tandem duplication of a two-domain ancestor, which
diverged to generate the cytokine receptor superfamily (27, 38,
39).
The sequence alignment of the N- and C-terminal halves of bovine and
human IFNAR-1 with HuIFNGR-1 is shown in Fig.
1, with General Considerations--
With 414 amino acids predicted for its
extracellular domain, BoIFNAR-1 presented a large target for
mutagenesis. We therefore created clusters of alanine mutations first,
followed by individual alanine substitutions. Initially we investigated
loops whose amino acid sequences differ between BoIFNAR-1 and
HuIFNAR-1, because their affinities for IFNs differ by >100-fold and
because residues implicated in ligand binding are most often located in
the loops of the cytokine receptors, rather than in the
Transfected COS cells transiently expressing the receptors on the cell
surface at very high levels serve as a reliable, efficient, and
convenient platform for ligand binding experiments. With such high
expression, the binding of IFN to BoIFNAR-1-transfected cells increases
15- to 40-fold over mock-transfected cells (48), providing a very
strong signal. In the same system, surface expression of HuIFNAR-1, at
levels comparable to that of BoIFNAR-1, increases binding of
[32P]IFN- Mutagenesis--
Previous work had emphasized the importance of
subdomains 2 and 3 in generating the nanomolar affinity of BoIFNAR-1
(49), so our mutagenesis focused on these subdomains. In addition, a region corresponding to a well-characterized epitope of SD1 was examined in detail, as were several areas of SD4 whose sequences differ
significantly between the bovine and human homologues. Finally,
differences between human and bovine IFNAR-1 were investigated with several substitutions incorporating the amino acids found in
HuIFNAR-1 into the BoIFNAR-1 structure ("homologue mutants"). The
list of mutants (Tables I and II) is organized by subdomain, and
denoted by the secondary structure location (e.g. 2L4-5 is a mutation in SD2 in the loop connecting
Mutagenesis resulted in altered receptors whose abilities to bind
HuIFN-
For clarity of interpretation, and consistent with mutagenesis studies
of other cytokine receptors (33, 34, 65), we focused on those mutants
that showed decreases in ligand binding of
In SD2 we generated eight alanine clusters, six single alanine
substitutions, and three homologous human substitutions (Tables I and
II). All mutants were expressed efficiently, and their levels of IFN
binding fell into three groups: 1) mutants showing small or no
decreases in binding (0-29% decrease; Table I); 2) mutants with
binding decreases in the range of 67-75% (2L4-5, 2L6-7); 3) a third
and most dramatic series of mutants with decreases of 82-96%. The
clusters in this group were 2L1-2, 2L2-3:2, and 2L4-5:2. Most
striking were the single substitution mutants, 2L2-3W, 2B3F, 2B3Y,
2L4-5Y, corresponding, respectively, to alanine substitutions at
Trp132, Phe139, Tyr141, and
Tyr160, which individually produced decreases in
binding of 82-96% (Fig. 3) while
retaining cell surface expression levels of 126%, 81%, 70%, and 95%
of parental receptor, respectively.
SD3 was examined with a series of 12 cluster mutants, 3 single alanine
mutants, and 3 human substitutions. The majority of mutations showed
minimal changes in ligand binding (0-25% decrease; see Table I).
Three alanine clusters, 3B6, 3L3-4:3, and 3B7, resulted in drastic
decreases of 76-91%, as did the single point mutation 3L3-4W
(Trp253) (Table I; Figs. 2, 3). Although it lost 84% of
it's binding, the Trp253 mutant had a cell surface
expression level of 105% of parental receptor.
Because our previous studies implicated SD1 as having a lesser effect
on ligand binding than SD2 and SD3 (49), a full study of this domain
was not undertaken. A more focused series of mutants came from the
finding that the epitope for the anti-human IFNAR-1-neutralizing monoclonal antibody 64G12 (67) maps to the human IFNAR-1
sequence 62FSSLKLNVY70 in SD1 (sequence
numbering of the mature protein; Fig. 1) (41, 42). We therefore
explored the effect of mutations in the equivalent BoIFNAR-1 sequence
62FSSVELENVF71, focusing on the large aromatic
and charged residues (Table I). Alanine substitutions for
Phe62 or Val65 actually produced an increase in
ligand binding (Table I). A single alanine substitution at
Phe71 (1L5-6F2) caused a moderate decrease of ligand
binding. A slightly larger decrease (57%) was seen for the 64G12
epitope cluster mutant 1L5-6, where the ELEN sequence is substituted
by four alanines. Because of our interest in this region, we also
explored the charged and large aromatic residues in loop 3-4, which in
our three-dimensional homology model is proximal to the 64G12 epitope.
Substitution of alanine for Asp44 or for Trp46
(mutants 1L3-4D and 1L3-4W) did not dramatically decrease IFN binding. Thus, mutants in and near the bovine homologue of the epitope
for mAb 64G12 in SD1 showed moderate but not strong effects on IFN-
In SD4, seven mutants were generated, based on differences between the
bovine and human sequences. This series carried the N-terminal HA
epitope tag, which was not accessible by flow cytometry, compromising
the ability to quantitate surface expression. However, mutants 4L2-3,
4L2-3V, and 4L6-7:2 produced no decrease in IFN binding, and 4L4-5
showed some increase in ligand binding. Therefore, these mutants do not
require independent estimates of surface expression. Mutants 4L1-2,
4B5:1, and 4B5:2 showed moderate decreases of binding (about 50%).
Because of the lack of reliable expression data, the precise
interpretation of these results is uncertain. Nevertheless, it can be
said that none of these mutants in SD4 strongly (4-fold) decreases
ligand binding.
Thus, the alanine-scanning mutagenesis identified several large
hydrophobic residues in SD2 and SD3 of BoIFNAR-1 whose substitution to
alanine dramatically decreased the binding of IFN- BoIFNAR-1, whether as a purified BoIFNAR-1/Fc fusion
protein or when expressed at high levels on COS cells, binds HuIFN- The mutagenesis results are best interpreted in the context of our
three-dimensional homology model of IFNAR-1 derived from the atomic
coordinates of the closely related, two-subdomain
IFNGR-13 (70) (Fig.
5). As there is no related crystal
structure on which to base the interaction of the two separate domains
of IFNAR-1 (specifically, the SD2-SD3 junction), we have chosen to
illustrate the four subdomains in an extended array (rather than, for
instance, a semi-circular or strongly bent arrangement). To orient the
two halves of IFNAR-1 (SD1+SD2 and SD3+SD4) to each other, we logically assume that residues of SD2 and SD3, which strongly affect ligand binding, are on the same face of the molecule.
,
IFN-
, and IFN-
, induce somewhat different cellular effects but
act through a common receptor complex, IFNAR, composed of subunits
IFNAR-1 and IFNAR-2. Human IFNAR-2 binds all type I IFNs but with lower
affinity and different specificity than the IFNAR complex. Human
IFNAR-1 has low intrinsic binding of human IFNs but strongly affects
the affinity and differential ligand specificity of the IFNAR complex.
Understanding IFNAR-1 interactions with the interferons is critical to
elucidating the differential ligand specificity and activation by type
I IFNs. However, studies of ligand interactions with human IFNAR-1 are compromised by its low affinity. The homologous bovine IFNAR-1 serendipitously binds human IFN-
s with nanomolar affinity.
Exploiting its strong binding of human IFN-
2, we have identified
residues important for ligand binding. Mutagenesis of any of five
aromatic residues of bovine IFNAR-1 caused strong decreases in ligand
binding, whereas mutagenesis of proximal neutral or charged residues
had smaller effects. These residues were mapped onto a homology model of IFNAR-1 to identify the ligand-binding face of IFNAR-1, which is
consistent with previous structure/function studies of human IFNAR-1.
The topology of IFNAR-1/IFN interactions appears novel when compared
with previously studied cytokine receptors.
INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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s, one IFN-
, and one IFN-
(1-6). The type I
IFNs produce differential activation of genes and cellular activities
(7-10). This may arise from differential binding to the receptor or
differences in receptor subunit recruitment by the various type I IFNs.
IFNAR consists of two cloned subunits denoted IFNAR-1 and IFNAR-2. On
its own, human IFNAR-2 (HuIFNAR-2) has moderate intrinsic affinity for
the range of human IFNs (HuIFNs) (11-17), whereas its partner, human
IFNAR-1 (HuIFNAR-1), alone binds IFNs weakly (Kd
10
7 M) (6, 11, 13-22). However, IFNAR-1
plays an essential role by contributing to both the final high affinity
and the differential specificity of the IFNAR complex (11, 23) in
addition to its role in signaling (24-26).
receptor (IFNGR-1) (30, 70), human growth hormone
receptor (31-34), tissue factor (35, 36), and erythropoietin receptor
(37). In the cases of IFNGR-1, IFNGR-2, growth hormone
receptor, tissue factor, and IFNAR-2 there are two FNIII domains, each
containing ~100 amino acids with seven
-strands and connecting
loops. The extracellular domain of IFNAR-1 is atypical, consisting of a
tandem array of four FNIII domains, here denoted subdomains 1 through 4 (SD1-4; beginning from the N terminus). The four-domain
structure of IFNAR-1 appears to represent a tandem duplication of the
more common two-domain structure (27, 38, 39).
s provides an elegant way to
circumvent difficulties in the studies of the human IFN type I receptor
complex (47-50).
2a, -
8b, -
1b, -
, and -
(11, 48, 49).
Consistent with these results, a soluble BoIFNAR-1/Fc fusion protein
bound HuIFN-
2a with an affinity of at most 10 nM (the
true affinity is expected to be closer to a Ki of
0.14 nM (23)). All human type I IFNs tested were able to
compete with HuIFN-
2a for binding to BoIFNAR-1/Fc, although they
varied in affinity. Thus, the extracellular domain of BoIFNAR-1 by
itself displays moderate affinity and differential binding of a broad
range of human type I IFNs.
2a measured over the
low affinity binding characteristic of HuIFNAR-1, indicating that
ligand binding was focused in this region. The BoIFNAR-1 N-terminal
(SD1) and membrane-proximal (SD4) domains each further enhanced binding
when transferred with SD2 and SD3 into HuIFNAR-1 (49). Thus, the IFN
binding site is not localized on one FNIII subdomain or even on one
tandem pair of FNIII subdomains, as previously predicted from other
cytokine family members (43); instead the ligand-binding determinants
of IFNAR-1 appear to be distributed in a more complex array centered on
SD2 and SD3. Kumaran et al. (52) used murine/human IFNAR-1
hybrids to confirm that SD1 plays at most a minor role in
species-specific ligand binding.
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2a
(1.56 × 108 IU/mg) was provided by Dr. Sidney Pestka.
The M2 anti-FLAG antibody was purchased from Sigma Chemical Co. R-Phycoerythrin-conjugated F(ab')2 goat
anti-mouse IgG was purchased from Jackson ImmunoResearch.
2a analogue
IFN-
2a-P1 (53) was phosphorylated to a radiospecific activity of
~1 × 103 Ci/mmol with [
-32P]ATP
(6000 Ci/mmol, PerkinElmer Life Sciences) and bovine heart cAMP-dependent protein kinase (Sigma). This corresponds to
a fractional labeling of ~0.15 mol of 32P/mol of protein.
For simplicity, [32P]IFN-
2a-P1 is referred to as
[32P]IFN-
2.
promoter in the vector
pcDEF3 (54). Unique restriction sites were engineered into the bovine
IFNAR-1 cDNA by using oligonucleotides containing silent mutations
RsrII, BspEI, AgeI, and
NheI. These restriction sites were placed between
each respective subdomain and at the beginning of the transmembrane
domain, resulting in a modified version of BoIFNAR-1. This version of
BoIFNAR-1 was also tagged with the FLAG epitope (55) between the signal
peptide and the beginning of the first subdomain of the protein,
allowing for its recognition on the cell surface.
2 (maximum concentration of 1.5-4 × 10
9 M), serially diluted, and then incubated
while rocking for 1 h at room temperature. Cell-bound
[32P]IFN-
2 was separated from unbound by brief
centrifugation of 100 µl of sample through a cushion of 10% (w/v)
sucrose in PBS. Tubes were then frozen and cut, and the tips and tops
were counted separately. Suppliers' determinations of IFN
concentration (in mg/ml) were used for all calculations. Data were
analyzed by non-linear regression to one-site binding using the program
Prism v.2.01 (GraphPad Software, Inc., San Diego, CA). A mutant
receptor that lost 75% of the binding seen for unaltered BoIFNAR-1 was
defined as having a significant loss of binding activity. Among the
mutants, there is a clear demarcation between those mutants that lost
75-100% and a secondary group of mutants, which lost 0-56% of
binding. This criterion is consistent with thresholds used in studies
of other receptors (61-63) and with the accuracy and precision of our
data, thereby guarding against over-interpretation of marginal effects.
-strands from the crystal
structure of IFNGR-1 indicated (underlined in the sequence)
and sequentially labeled as B1-B7 within a FNIII domain. The
alignments are facilitated by the similar sequence lengths and
conserved characteristic residues of IFNGR-1 and the N- and C-terminal
halves of IFNAR-1. Sequence alignments were similar with GCG version
9.0 and with ClustalW. (Expected loops are designated by the flanking
-strands: e.g. the loop connecting
-strands 4 and 5 is
denoted "L4-5.") As commonly observed within families of
homologous proteins, the "core"
-sheet structure is well
conserved, and much of the variability arises in the interstrand loops,
particularly the loops L2-3 and L4-5 in SD2 and SD4 of IFNAR-1. In
our homology model of BoIFNAR-1, the positions of the cysteines in the
IFNAR-1 are within a reasonable distance for the formation of four
disulfide bonds homologous to those found in IFNGR-1. Similarly,
individual
-strands of IFNAR-1 aligned closely with those of
IFNGR-1. Thus, the predicted core
-sheet should be quite reliable,
with some ambiguity as to the precise location or orientation of
residues in the loops.
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Fig. 1.
Alignment of human IFNGR-1 with bovine and
human IFNAR-1 sequences. The alignment was performed independently
for the N-terminal and C-terminal halves of IFNAR-1. The -strands,
identified by the IFNGR-1 crystal structure are underlined
and denoted by B1-B7 for each subdomain, were used to
predict
-strands in the IFNAR-1 receptors.
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-strands
(31, 33, 62, 64, 65). The search for functionally important residues
was aided by information about ligand/receptor interactions in other
members of the cytokine receptor families, and by our three-dimensional
homology model (27, 30, 37-39, 61, 66).
2 to cells by 1- to 2-fold over COS cells
transfected with empty vector (Table I;
see also Ref. 49, Figs. 3-5; and Ref. 48). Thus, any excess endogenous
(simian) IFNAR-2 does not make a substantial contribution to ligand
binding. Cell surface expression levels of different constructs were
compared through a common FLAG epitope. We thereby demonstrated that
high binding and low binding IFNAR-1 variants express at comparable
levels (Table I). Previously we had shown for both BoIFNAR-1 and
HuIFNAR-2 that the affinity and ligand specificity of the receptors
expressed on COS cells is essentially the same as their affinity and
specificity when produced as purified fusion proteins of the receptor
extracellular domain with an IgG Fc domain (11,
23).2 Thus, the low
background IFN binding contributed by endogenous COS cell receptors
(<5000 receptors/cell) does not significantly contribute to the
observed high levels of IFN binding, which is attributable to the high
level expression of ectopic BoIFNAR-1. However, because the
Simian-derived COS cells are biochemically responsive to human IFNs
through their endogenous receptors, this system is useful only for
ligand binding assays. Finally, the expressed receptors on the cell
surface are likely to be correctly folded, because misfolded proteins
in eukaryotic cells are generally not efficiently presented on the cell
surface; thus, questions about protein misfolding, common with
bacterial expression systems, are largely mitigated when the proteins
are demonstrated to express efficiently on a eukaryotic cell
surface.
Alanine mutations created in BoIFNAR-1
-strands 4 and 5; the suffix "H" refers to human homologue mutants).
2 ranged from the high levels equivalent to (or occasionally higher than) parental BoIFNAR-1, to receptors whose binding was low and
indistinguishable from COS background (Table I, Fig. 2). However, all receptors were present
on the cell surface at levels similar to parental (70-140% of
BoIFNAR-1), as monitored by flow cytometric detection of a common FLAG
epitope (55) at the N terminus (the FLAG epitope was previously shown
to have no effect on ligand binding (49)). The expression of BoIFNAR-1 gave very high levels of ligand binding as shown previously;
unfortunately, like parental BoIFNAR-1, many mutants with strong
binding did not approach saturation so that Kd
values could not be accurately estimated. Therefore, results are
compared as the percent binding relative to the unaltered BoIFNAR-1
control at the maximal IFN input (1.3-4 nM). For
comparison, mock-transfected and HuIFNAR-1-transfected COS cells were
shown to bind an average of 3 and 6% of the level of COS cells
transfected with BoIFNAR-1 (Table I).
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Fig. 2.
Binding of
[32P]IFN- 2 to COS cells
expressing BoIFNAR-1 mutants. A variety of mutants are presented,
as is the parental BoIFNAR-1 construct. IFN bound is from 100 µl of a
binding reaction containing ~1 × 105 cells.
75% while retaining high
cell surface expression. Mutants with low binding of IFN were
consistently expressed at levels comparable to parental BoIFNAR-1 and
other high binding mutants (Table I).
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Fig. 3.
Binding of
[32P]IFN- 2 to COS cells
expressing single alanine substitutions with strong effects on IFN
binding. Bovine IFNAR-1 parental is presented along with human
IFNAR-1 and mock-transfected COS-1 cells, and individual residues that
retained only 0-25% of the original parental binding when changed to
alanine. These critical mutations include mutant 2L2-3W, 2B3F,
2L4-5Y, and 3L3-4W. Sample size is the same as in Fig. 2.
2 binding.
2. All of these
residues are conserved in the human and murine IFNAR-1. This was
surprising, because the initial experiments assumed that differences in
ligand binding between human and BoIFNAR-1 would reflect a small number
of residues that differed between them. We therefore examined some
loops in SD2 and SD3 with significant sequence differences between
HuIFNAR-1 and BoIFNAR-1, by substituting clusters of residues
found in HuIFNAR-1 for the homologous residues in BoIFNAR-1 (Table
II; Fig. 4). The human
homologue mutants in SD2 (identified by
the suffix "H"), 2L4-5H and 2L6-7H, caused small or insignificant
decreases in ligand binding (31 and 13%, respectively). However, when
both of these homologous substitutions were engineered into the same
mutant (2L4-5/6-7H), the binding of IFN-
2 was significantly
decreased by 70% of that seen with BoIFNAR-1 while retaining
expression equivalent to the BoIFNAR-1 control. Similarly in SD3, the
homologue cluster mutations 3L3-4:2H and 3L5-6:2H, produced decreases
of 21 and 0%, respectively, whereas the combination of both homologous
substitutions produced a larger, but still modest, decrease in binding
of about 48%, slightly more that seen with 3L3-4:2H alone. However,
when combinations of three human cluster substitutions ("Bo3H",
composed of 2L6-7H, 3L3-4:2H and 3L5-6:2H) or all four human
clusters ("Bo4H") were placed in SD2 and SD3, these multiple
mutants decreased ligand binding by a minimum of 75% of the original
binding. Thus, the substitution of homologous amino acids found in
HuIFNAR-1 in any one of these loops produced small or insignificant
decreases in ligand binding, whereas several combinations of changes
led to very strong decreases in ligand binding, approaching a level
similar to HuIFNAR-1.
Human substitution mutations created in BoIFNAR-1
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Fig. 4.
Binding of
[32P]IFN- 2 to COS cells
expressing BoIFNAR-1 with clusters of residues from HuIFNAR-1.
Sample size is the same as in Fig. 2.
DISCUSSION
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ABSTRACT
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DISCUSSION
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2 with 1-10 nM affinity, and binds a broad selection of
other type I IFNs (23). Based on subdomain substitutions between the
human and bovine homologues, this strong binding, relative to the weak ligand binding by human IFNAR-1, was shown to reside predominantly in
the two central subdomains, SD2 and SD3 (49). Here, through a series of
alanine and homologue substitution mutations, we have identified
residues in SD2 and SD3 of BoIFNAR-1 that strongly affect the binding
of HuIFN-
2. The strongest decreases in binding came from the
substitution of any of five hydrophobic residues, conserved in bovine,
human, and murine IFNAR-1. Smaller effects came from mutating charged
residues proximal to those hydrophobics.
View larger version (76K):
[in a new window]
Fig. 5.
Homology model of IFNAR-1 with alanine
mutagenesis results. The model of bovine IFNAR-1 was
generated using the coordinates of human IFNGR-1 provided by S. E. Ealick and M. R. Walter, as described under "Experimental
Procedures." Red, single alanine substitutions producing
decreases of 75%. Blue, alanine cluster mutations
producing decreases in ligand binding of 0-57%. Orange,
alanine cluster mutations producing decreases in ligand binding of
75%.
Most mutations resulted in little significant change in binding of
IFN-2 (Table I; Fig. 5, blue). Where cluster mutations showed strong decreases in binding, the localization was generally refined to a single residue, which decreased IFN binding by
80% of
native BoIFNAR-1 (red). Several clusters, which produced
large decreases in IFN binding, were not refined to single residues (orange).
Alanine mutagenesis identified five hydrophobic residues that
individually decreased the binding of HuIFN-2 by 82-96% while retaining cell surface expression levels of 69-125% of parental receptor. The homology model presents the functionally important aromatic residues Trp132, Phe139,
Tyr160, and Trp253 as well exposed on the
receptor surface, easily accessible for protein interactions. The three
residues in SD2 (Trp132, Phe139,
Tyr160) are localized to a common region.
Trp253, in SD3, is on the protein surface and can be
aligned in this extended view with the residues of SD2 to define a
common binding surface for IFNs. In contrast, Tyr141
appears buried beneath Phe139 and Tyr160,
forming extensive
-strand contacts within SD2. Therefore
Tyr141, although not directly involved in ligand binding,
is likely to be required for the proper presentation of
Phe139 and Tyr160. Each of these critical
hydrophobic residues is conserved in the human and murine IFNAR-1 homologues.
Two other alanine clusters involving residues
113EDK115 (2L1-2) and
157ETV159 (2L4-5:2) produced decreases of
IFN-2 binding of
90% (orange) (Fig. 5).
157ETV159 (ENI in HuIFNAR-1 and INS in murine)
is proximal (4-14 Å) to both Phe139 and
Tyr160 and could be involved in either positioning them or
in direct ligand interactions. The highly charged
113EDK115 cluster is near the interface with
SD3 and is conserved in human and murine IFNAR-1. This could be
involved in important contacts with SD3 or with IFNs. Mapping within
these clusters was not further refined.
The conservation of the critical large hydrophobic residues in bovine, human, and murine IFNAR-1 was surprising, because the strategy originated in our studies of bovine/human IFNAR-1 chimeras and had presumed that the key residues would be those that differ between bovine and human IFNAR-1. However, like IFNAR-1, IFNGR-1 and growth hormone receptor demonstrate the importance of aromatic residues (predominantly W and Y) combined with charged residues (K, R, E, and D) packing against the critical aromatics in forming a binding interface (65, 67, 68). In BoIFNAR-1, the aromatic/charged pairs found appear to be Asp128/Trp132, Arg136/Phe139/Arg140, Glu157/Tyr160/Glu162/Asp163, Phe241/Lys243, and Lys252/Trp253/Lys254. Therefore, we hypothesize that the conserved aromatic residues are the foci of the receptor/ligand interface and that the clusters of residues surrounding the key hydrophobic residues modulate ligand binding and distinguish the low affinity human from the high affinity bovine IFNAR-1. These surrounding residues may also help differentiate the binding affinities of the diverse type I ligands.
This hypothesis led us to substitute homologous human residues, which
were predicted from the three-dimensional model to be proximal to the
conserved critical hydrophobic residues. Substituting individual
clusters of human residues (2L4-5H or 2L6-7H, 3L3-4:2H or 3L5-6:2H)
for their homologous bovine residues produced minimal or no changes in
IFN-2 binding (Table II). However, various combinations of two to
four homology clusters produced a cumulative decrease of 50-80% in
ligand binding. These effects are consistent with our hypothesis that
residues surrounding the critical aromatic residues may, when taken
together, be responsible for the differences between human and bovine affinities.
Because of its low affinity for IFN, HuIFNAR-1 is a poor target for
structure/function studies. What little is known about the ligand
binding site of HuIFNAR-1 has been derived from indirect studies. Two
neutralizing mAbs against HuIFNAR-1, EA12 (50) and 4A7 (40), seem to
recognize epitopes that are difficult to map to a single domain. The
epitopes for two other neutralizing anti-HuIFNAR-1 mAbs have been
identified. The high affinity mAb 2E1 appears to recognize epitopes
within the non-contiguous sequences 244HLYKWK249 and
291EEIKFDTE298, with strong contributions to
antibody binding attributed to Lys249, Glu291,
and Asp296 (40). This epitope includes Trp248,
the human homologue of our critical bovine Trp253 (Fig.
6). The epitope for the anti-human
IFNAR-1 64G12 mAb (67) that neutralizes the activity of all tested
human type I IFNs on human cells has been localized to a sequence in
SD1, 62FSSLKLNVY70 (41, 42). However, our
mutations in the homologous sequence (62FSSVELENVF71) in BoIFNAR-1 produced only
insignificant to moderate effects on binding of IFN- 2 (Fig. 6).
This is consistent with our observations and those of Kumaran et
al. that SD1 plays at most a minor role in the species specificity
of human versus murine IFN binding (49, 52). Because the
three-dimensional model places the 64G12 epitope within 9-17 Å of the
critical Trp132 residue in SD2, we speculate that mAb 64G12
sterically occludes Trp132 and any neighboring residues
involved in IFN binding.
|
On the three-dimensional IFNAR-1 model, the epitopes for these two
independently produced neutralizing mAbs (40-42, 50, 67) are proximal
to and located on the same face of IFNAR-1 as the residues that we
identify in BoIFNAR-1 as being critical to the binding of IFN-2;
i.e. in both BoIFNAR-1 and HuIFNAR-1 there is one face of
IFNAR-1 that is involved in ligand binding. Thus, our mutagenesis study
of BoIFNAR-1 significantly enhances the interpretation of the currently
available studies of HuIFNAR-1 and provides focus for future studies of
HuIFNAR-1.
In conclusion, in BoIFNAR-1 we have identified five large
hydrophobic residues, conserved in the human and murine homologues, whose substitution with alanine decreases ligand binding by 82-96%. Several clusters of amino acids, proximal to the important aromatic residues, have additionally been identified whose coordinated substitution by alanine produces large decreases in binding of human
IFN-2. The critical residues of BoIFNAR-1 map to the same face and
are proximal to amino acids independently identified as part of
epitopes for anti-HuIFNAR-1 mAbs that efficiently neutralize IFN
activity and block IFN binding, supporting the relevance of the current
results to the human IFNAR-1.
The most straightforward interpretation of our binding, domain
swapping, and alanine mutagenesis data (23, 48, 49) is that the single
nanomolar affinity site for HuIFN-2 on BoIFNAR-1 primarily, but not
exclusively, involves ligand interactions with SD2 and SD3. The finding
that ligand binding appears focused in SD2 and SD3 is novel in terms of
previous models (39, 43, 69) and presents a unique pattern of
ligand-receptor interactions for the cytokine superfamily. Although
confirmation of any model must await a crystal structure, this
extensive analysis of BoIFNAR-1 enables the focused analysis of the
human IFNAR-1.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Mark R. Walter and Steven Ealick for sharing unpublished coordinates of IFNGR-1; Dr. Prem Yadav (UMDNJ-Robert Wood Johnson Medical School) and Dr. Michael Karpusas (Biogen, Inc.) for great help in homology modeling; Drs. Bruce Daugherty, Julie DeMartino, and Youmin Weng (Merck, Inc.) for assistance with mutagenesis; Christina DeCoste (Environmental and Occupational Health Sciences Institute and Cancer Institute of New Jersey Flow Cytometry Core Facility) for flow cytometry, and the DNA Sequencing and Synthesis Facility (Robert Wood Johnson Medical School and Cancer Institute of New Jersey).
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FOOTNOTES |
---|
* This work was supported in part by Grant 35-99 from the Foundation of University of Medicine and Dentistry of New Jersey (to J.A.L.) and by the Department of Molecular Genetics & Microbiology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by United States Public Health Services-National
Institutes of Health Training Grant IH AI07403-06, by funds from the
Department of Molecular Genetics & Microbiology, and by a Molecular
Genetics & Microbiology Departmental Honors Fellowship.
§ To whom correspondence should be addressed: Dept. of Molecular Genetics & Microbiology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Ln., Piscataway, NJ 08854. Tel.: 732-235-5224; Fax: 732-235-5223; E-mail: langer@umdnj.edu.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M009663200
2 J. A. Langer, unpublished data.
3 M. R. Walter, personal communications.
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ABBREVIATIONS |
---|
The abbreviations used are:
IFNAR, type I
interferon receptor;
IFN, interferon;
HuIFN, human IFN;
IFNGR-1, interferon- receptor;
SD1-4, subdomains 1 through 4;
BoIFNAR, bovine IFNAR;
DMEM, Dulbecco's modified Eagle's medium;
SOE, splice
overlap extension;
HA, hemagglutinin;
PBS, phosphate-buffered saline;
mAb, monoclonal antibody.
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