From the
Interleukin-6 (IL-6) triggers the formation of a high affinity
receptor complex constituted by the ligand-binding subunit IL-6
receptor
Interleukin-6 (IL-6)
IL-6
stimulates responsive cells by promoting the sequential association of
two transmembrane glycoproteins with distinct functional
properties
(6) . The
Random mutagenesis of hIL-6R
Deciphering which are the contact surfaces between hIL-6R
We have recently reported the
construction of a three-dimensional model of hIL-6 and its
superimposition onto the hGH structure in the context of the
h(GHbp)
shgp130 cDNA was obtained as a
5`-HindIII-3`-EcoRV fragment coding for a soluble
form of human gp130 (amino acids 1-605) by polymerase chain
reaction using plasmid pBS130BES
(27) as a template. The
3`-oligonucleotide was engineered in order to add a c-myc tag
immediately upstream of the stop codon
(30) . The amino acid
sequence of the C terminus of shgp130 was the following:
E
With respect to the AB2 loop, hIL-6R
In addition,
two shorter AB2 loop variants (Mut-6 and Mut-7), carrying the
equivalent regions of the tenth domain of tenascin
(24) or part
of an immunoglobulin constant domain
(25) , respectively, were
generated with the idea to eliminate altogether AB2 loop contact points
with gp130 (). Two other IL-6R
To verify the production of soluble hIL-6
receptors, the medium of metabolically labeled transfected cells was
immunoprecipitated with the non-neutralizing anti-hIL-6R
We next determined the affinity of the various
shIL-6R
Point mutations in the E2 strand and the AB2 loop do not
substantially affect the affinity for hIL-6, except for Mut-1, where a
slight but reproducible decrease (from 2.0 to 4.3 nM) was
observed (). Interestingly, when substitutions in the E2
strand are present together with mutations in the AB2 loop (Mut-4),
full hIL-6 binding activity is restored (). On the
contrary, all variants in which the complete AB2 loop had been
substituted (Mut-5 to Mut-8) displayed a strongly decreased ability to
interact with the cytokine (). Since the receptor mutants
with the weakest apparent affinity for hIL-6, namely Mut-5 to Mut-7,
were the same mutants that showed the lowest levels of expression
(compare Fig. 2and ), we believe that the
substitution of the entire AB2 loop might cause severe and unpredicted
conformational changes, which alter folding and/or stability of the
molecule.
The baculovirus-produced receptors were first
tested in coimmunoprecipitation experiments. The use of purified
proteins allowed us to quantify the amount of receptor used and to
compare the behavior of the wild type and Mut-4 over a wide range of
hIL-6 concentrations. The results, shown in Fig. 5, were in good
agreement with those previously obtained with COS-7 cell-produced
receptors. Interestingly, along the entire hIL-6 curve, the amount of
bound gp130 was lower for Mut-4 than for wild-type shIL-6R
Human HepG2 cells were treated
with increasing concentrations of hIL-6 (from 2 to 200 pM)
together with a fixed amount of wild-type and mutant receptors (100
nM). As shown in Fig. 6A, we observed that
while shIL-6R
Interestingly, combining substitutions in the E2 strand and
the AB2 loop as it was done generating variant Mut-4 does not totally
abolish interaction with gp130, in line with its being only a partial
antagonist. There are several explanations to this finding. First, not
having tested all the possible substitutions at positions 280 and 281
and not having mutated all residues of the AB2 loop, we cannot exclude
that a more systematic approach would lead to a full antagonist like
the one recently discovered for hGHbp
(45) . In this latter case,
however, one has to consider that, the hGH receptor being a homodimer,
mutation of Asp-152 in the center of the interface between the two
hGHbp molecules
(13) simultaneously affects both receptor
chains, which in our case would correspond to mutagenizing hIL-6R
Disregulated production of hIL-6 has been proposed as playing a
pathogenetic role in the development of multiple myeloma and autoimmune
diseases
(1, 3, 4, 5) . Generating hIL-6
antagonists is thus believed to be a potentially useful therapeutic
approach. In this framework, the properties of Mut-4 open up new
possibilities of blocking IL-6 activity. Initial attempts to counteract
IL-6 in vivo both in murine models and in human clinical
trials with the use of neutralizing monoclonal antibodies gave rise
sometimes to enhanced IL-6 effects as a consequence of the decreased
clearance of the cytokine complexed with
antibodies
(50, 51) . Although the mode of action of
soluble IL-6R
Mut-4 partially antagonizes
both hIL-6-induced early signaling, monitored as APRF activation
(15-min assay), and biological activity, assessed as induction of
haptoglobin production (20-h assay). However, antagonism was more
pronounced in the short-term assay. Since we have measured different
biological responses that are not known to be linked to each other, it
is possible that the transcription of the haptoglobin gene is not
strictly dependent on APRF activation. Alternatively, we cannot exclude
that the lower efficiency of long-term inhibition is due to a slow
release of hIL-6 from preformed complexes with Mut-4. The generation of
shIL-6R
Mutated residues are in boldface. The wild-type
sequence is shown for comparison. For each mutant, where no change is
indicated, the wild-type sequence is present. The apparent affinity for
hIL-6 of each soluble receptor produced in COS-7 cells was determined
as described under ``Experimental Procedures.'' The results
shown are the means of at least two separate experiments.
The enzyme-linked immunosorbent assay for the
detection of haptoglobin in HepG2 culture medium was conceived and
optimized by Dr. R. Laufer, whose help we gratefully acknowledge. We
also thank Dr. A. Tramontano, Dr. M. Sollazzo, and J. Clench for
critically reading the manuscript; P. Neuner for the synthesis of
oligonucleotides; and M. Emili for graphical work.
(IL-6R
) and the signal-transducing
chain
gp130. Since the cytoplasmic region of IL-6R
is not required for
signal transduction, soluble forms of IL-6R
(sIL-6R
) show
agonistic properties because they are still able to originate
IL-6
sIL-6R
complexes, which in turn associate with gp130. A
three-dimensional model of the human IL-6
IL-6R
gp130
complex has been constructed and verified by site-directed mutagenesis
of regions in shIL-6R
(where ``h'' is human) anticipated
to contact hgp130, with the final goal of generating receptor variants
with antagonistic properties. In good agreement with our structural
model, substitutions at Asn-230, His-280, and Asp-281 selectively
impaired the capability of shIL-6R
to associate with hgp130 both
in vitro and on the cell surface, without affecting its
affinity for hIL-6. Moreover, the multiple substitution mutant
A228D/N230D/H280S/D281V expressed as a soluble protein partially
antagonized hIL-6 bioactivity on hepatoma cells.
(
)
elicits a variety
of biological responses on different target cells
(1) . While the
physiological production of IL-6 regulates B-cell proliferation and
maturation, T-cell activation, and the production of acute-phase
proteins in liver during inflammation
(2) , the disregulated
production of this cytokine is believed to play a crucial role in the
pathogenesis of multiple myeloma, post-menopausal osteoporosis, and
autoimmune diseases
(3, 4, 5) .
subunit of the IL-6 receptor
(IL-6R
) specifically recognizes the cytokine at low affinity
(10
M)
(7) . The complex formed by
IL-6 and IL-6R
is in turn able to interact with the extracellular
region of the
chain, namely gp130, which has no intrinsic IL-6
binding properties, but is required for the generation of high affinity
(10
to 10
M)
IL-6-binding sites
(8) . While the integrity of the
intracytoplasmic region of gp130 is absolutely required for
IL-6-dependent signal transduction (9), the intracellular region of the
IL-6R
chain is dispensable (10). As a result, soluble forms of
IL-6R
, constituted by the entire extracytoplasmic region of the
protein, do not behave as IL-6 antagonists, but still bind to the
cytokine and mediate its function through interaction with
membrane-bound gp130
(11) .
identified distinct sets of residues that are either involved in hIL-6
binding or in hgp130 recognition (12). These findings suggest that it
might be possible to generate receptor variants with antagonistic
instead of agonistic properties by introducing mutations that still
allow formation of the hIL-6
shIL-6R
complex, but selectively
impair its association with hgp130 in a high affinity receptor complex.
Such engineered molecules might be of potential therapeutic value for
the treatment of diseases in which hIL-6 plays a pathogenetic
role
(1, 2, 3, 4, 5) .
and
hgp130, but also between hIL-6 and its receptors, can be attempted, on
a predictive base, by molecular modeling. A suitable template for
initiating modeling of the hIL-6
hIL-6R
hgp130 complex
is the crystallographic structure of hGH bound to the extracellular
region of its homodimeric receptor
(13) . This is supported by
the following observations. First, structural predictions strongly
suggest that hIL-6 shares a common four-helix bundle topology with
hGH
(14, 15) . Second, hGHbp, hIL-6R
, and hgp130 all
belong to the hematopoietin family of receptors, characterized by the
presence of a conserved 220-amino acid-long region called the
cytokine-binding domain (CBD), which is responsible for the interaction
with the cytokine and is predicted to fold as a tandem repeat of
immunoglobulin-like
barrels
(16) . Third, the hGH system
shows rather close functional analogy to the hIL-6 system since the
generation of the biologically active hGH
h(GHbp)
complex proceeds through the association of an initial
hGH
hGHbp complex with a second hGHbp
molecule
(17, 18) . The x-ray structure of the
hGH
h(GHbp)
complex revealed that the hormone serves
as a bridging ligand that contacts the two receptors with distinct
binding sites (named sites 1 and 2) located on opposite sides of the
molecule
(13) . Receptor dimerization is further stabilized by a
direct interaction between the C-terminal subdomains of their
respective CBDs
(13) .
receptor complex; this approach allowed us to
identify a patch of amino acid residues on hIL-6 involved in the
interaction with hgp130
(15, 19) . In this paper, we
present a further advancement of the model, which describes the
structure of the complex of hIL-6 bound to the CBDs of hIL-6R
and
hgp130. The model also predicts residues in hIL-6R
involved in the
interaction with hgp130. Their mutagenesis gave rise to shIL-6R
variants with reduced binding to the extracellular portion of hgp130
both in vitro and on the cell surface. Finally, when the
mutant showing the most impaired interaction with hgp130 in vitro was tested on cells, it partially inhibited hIL-6 activity.
Molecular Modeling
Modeling of the hIL-6R
and hgp130 CBDs was initiated by inscribing their amino acid sequences,
according to a preliminary sequence alignment against hGHbp, onto the
structural template provided by the corresponding hGHbp molecules
within the hGH
h(GHbp)
complex (Protein Data Bank code
2HHR)
(15, 20) . This alignment was based on conserved
residues such as the four cysteines predicted to form disulfide
bridges, prolines in the interdomain linker, the WSXWS
motif
(21) , and the pattern of hydrophobic residues within the
strand regions. Assignment of buried and solvent-accessible
positions was accomplished using a complete model of the
hGH
h(GHbp)
complex generated from the available
C-
coordinates with the WHATIF software package
(22) . In a
subsequent step, unfavorable contacts and cavities within the
hydrophobic core of the two subdomains were minimized by optimization
of side chain conformations, and where necessary, the packing was
improved, shifting parts of the sequence within the
strands
regions. The connecting loop regions containing the majority of
insertions and deletions were modeled either manually or using the loop
search procedure as implemented in the INSIGHT software
package
(23) . For generation of the AB2 loop variants of
hIL-6R
, we used either the available x-ray structures of
hGH
h(GHbp)
(Protein Data Bank code 2HHR)
(13) and tenascin (Protein Data Bank code 1TEN)
(24) , the
latter superimposed manually onto the hIL-6R
CBD model, or
structurally similar segments from an immunoglobulin constant domain
(Protein Data Bank code 1FDL)
(25) and the staphylococcal
nuclease (Protein Data Bank code 2SMN)
(26) , selected from the
Protein Data Bank protein structure data base
(20) , through the
loop search procedure of INSIGHT
(23) . Amino acid sequences for
the receptor chains correspond to the SWISSPROT data bank codes
GHR_HUMAN (hGHbp) and IL6R_HUMAN (hIL-6R
) or to code A36337
(hgp130) from the PIR protein sequence data bank.
Plasmid Construction and Site-directed Mutagenesis of
shIL-6R
shIL-6R cDNA was obtained as a
5`-EcoRI-3`-XbaI fragment by polymerase chain
reaction using the hIL-6R
complete cDNA as a template
(27) .
The 3`-primer was designed in order to introduce an artificial TAG stop
codon at amino acid 324 preceded by a sequence coding for six
histidines. The fragment generated was then introduced into the
expression vector pcDNAI (Invitrogen) to obtain plasmid pC6FRH. Plasmid
pC6FRH was used as a template to obtain receptor mutants by polymerase
chain reaction
(28, 29) . All the mutant cDNAs were
cloned into pcDNAI. Their identity was verified by sequencing. For the
expression of soluble IL-6 receptors in insect cells, the coding
sequences of histidine-tagged wild-type shIL-6R
and Mut-4 were
excised from pcDNAI and introduced as a
5`-BamHI-3`-XbaI-filled fragment into pBlueBacIII
baculovirus transfer vectors (Invitrogen) previously restricted with
BamHI-HindIII blunt-ended to obtain plasmids
pBsIL-6RH and pBMut-4H.
FEEQKLISEEDL-Stop. To express the protein in insect
cells, the cDNA was inserted as a
5`-HindIII-filled-3`EcoRV fragment into pBlueBacIII
to obtain the transfer plasmid pBsGPm.
Cytokine Production and Purification
Recombinant
human IL-6 and recombinant human OM were produced and purified as
described previously
(31, 32) . The specific bioactivity
of hIL-6 was 2 10
units/mg of protein as assessed
by 7TD1 cell growth assay
(33) .
I-IL-6
(900-1200 Ci/mmol) was purchased from Amersham Corp.
Expression of shIL-6R
COS-7 cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum plus
glutamine and antibiotics at 5% CO Mutants in COS-7
Cells
. Cells (2.5
10
) were seeded in 100-mm tissue culture dishes and
transfected with 2 µg of the various shIL-6R
expression
vectors using the DEAE-dextran technique
(34) . 16 h after
transfection, cells were replated in 100-mm dishes and grown in
complete medium at 37 °C. After 72-96 h, the medium was
collected, centrifuged, and used for coimmunoprecipitation experiments
and binding analysis. To monitor the expression level of each mutant,
2.5
10
transfected COS-7 cells were replated in
35-mm dishes and, 48 h after transfection, metabolically labeled with
[
S]methionine for 4 h. The supernatants were
immunoprecipitated with anti-human IL-6R
monoclonal antibody
I6R1/9.G11
(27) and protein A-Sepharose 4 Fast Flow (Pharmacia
Biotech Inc.).
Binding Analysis
Unlabeled ligand competition
assays were performed essentially as described previously
(27) .
Briefly, appropriate amounts of cell supernatants, previously
determined in titration experiments and to which imidazole was added up
to 5 mM (final concentration), were mixed with 20-40
pMI-IL-6 and increasing concentrations of
unlabeled cytokine. The total volume of the binding reaction was 500
µl. Under equilibrium conditions, 40 µl of
Ni
-nitrilotriacetic acid-agarose (QIAGEN Inc.) were
added, and incubation was prolonged for 1 h. Bound ligand
(``resin-associated'') was separated from free ligand
(supernatant) by centrifugation through a cushion of 30% sucrose in
PBS. All steps were performed at 4 °C. Normalization of the amount
of receptor used in binding experiments was achieved as follows. 10% of
the transfected cells were used to monitor the production of each
receptor by immunoprecipitation of metabolically labeled supernatants
and densitometric analysis of gels similar to the one shown in
Fig. 2
, using Image software Version 1.22 (National Institutes of
Health, Bethesda, MD). The remaining 90% of the cells from the same
transfection were used as a source of unlabeled receptor in binding
experiments. 30 ± 10 µl of culture medium were utilized in
the case of wild-type shIL-6R
. For each mutant, the quantity of
supernatant used was adjusted for its expression level with respect to
wild-type shIL-6R
. The total volume of the binding reaction was
kept constant (500 µl) by adding supernatant from untransfected
COS-7 cells. In the case of Mut-5 to Mut-7, the culture medium was also
concentrated 5-10-fold using Centricon-10 (Amicon, Inc.). The
final concentration of
I-IL-6 was 400-800
pM. The apparent affinity of the various soluble hIL-6
receptors for hIL-6 was determined after Scatchard transformation of
the results
(27) . Analysis of binding data and curve fitting was
done using UltraFit software (Biosoft
).
Figure 2:
Expression level of shIL-6R mutants
in the medium of transiently transfected COS-7 cells. COS-7 cells
transfected with expression plasmids carrying wild-type (W.T.)
or mutant (Mut-1 to Mut-8) cDNA encoding shIL-6R
were
metabolically labeled with [
S]methionine. 250
µl of conditioned supernatants were immunoprecipitated with 1
µg of anti-hIL-6R
monoclonal antibody I6R1/9.G11 and protein
A-Sepharose. Immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis followed by autoradiography. In control experiments,
cells were transfected with the empty expression vector (controllane). One representative experiment is
shown.
Confluent monolayers of A375 human melanoma cells
grown in 24-well tissue culture dishes were preincubated with 500
µl of binding buffer (Dulbecco's modified Eagle's
medium plus 50 mM Hepes, pH 7.2, 3% bovine serum albumin, 0.2%
sodium azide) for 45 min at 23 °C and then washed with PBS.
Normalized aliquots of conditioned COS-7 cell medium were added to each
well in duplicate, together with 1 nMI-IL-6 Binding on A375
Cells
I-IL-6,
and incubated for 90 min at 23 °C (see also legend to
Fig. 4
). Normalization was performed by determining for each
receptor the quantity of conditioned medium capable of retaining 6 fmol
of
I-IL-6 on Ni
-nitrilotriacetic
acid-agarose as described under ``Binding Analysis.'' The
total volume of the binding reaction was kept constant (200 µl)
throughout different wells. Nonspecific binding was measured in the
presence of a 200-fold molar excess of unlabeled IL-6. At the end of
the incubation time, monolayers were washed twice with cold binding
buffer and solubilized with 200 µl of 1 N NaOH. Cell-bound
radioactivity was measured in a
-counter.
Figure 4:
Mut-1 to Mut-4 are impaired in their
association with membrane-bound gp130. Confluent monolayers of human
A375 cells (3 10
cells/well) in 24-well tissue
culture plates were incubated for 90 min with 1 nM
I-hIL-6 alone (column1) or in
combination with equivalent amounts of wild-type (W.T.;
column2) and mutant (columns 4-7)
receptors. After washing steps, cell-bound radioactivity was determined
as described under ``Experimental Procedures.'' In a
competition experiment (column3), cells were
incubated with 1 nM
I-hIL-6, wild-type
shIL-6R
, and 1 µM unlabeled OM. Data are expressed as
specific binding. One representative experiment is shown. For details,
see also ``Experimental
Procedures.''
Production of Soluble Receptor Chains in Insect
Cells
Production of soluble hIL-6 receptors in insect cells was
achieved using the MaxBac baculovirus expression system
(Invitrogen) following the manufacturer's instruction with minor
modifications. High Five
cells (Invitrogen) were used for
protein production. The culture supernatants were harvested at 36 h
post-infection, dialyzed against PBS, and directly loaded on a
Ni
-nitrilotriacetic acid-agarose column. After
washing steps with PBS, 8 mM imidazole, both wild-type
shIL-6R
and Mut-4 were eluted in PBS, 80 mM imidazole.
Purified proteins were dialyzed against PBS. Receptor purity was
estimated to be >90% as judged by silver staining of
SDS-polyacrylamide gel. Soluble gp130 was produced in insect cells
following the same procedure. For in vivo labeling of shgp130,
High Five
cells were infected with the shgp130 recombinant
virus. 24 h later, cells were incubated for 1 h in methionine-free
Grace's medium and metabolically labeled with 100 µCi/ml
[
S]methionine in the same medium for 4 h at 27
°C. The supernatant, containing
S-shgp130, was
harvested and used in immunoprecipitation experiments.
Coimmunoprecipitation of shgp130 and shIL-6R
was performed at 4
°C as described
(15, 27) .
Nuclear Extract Preparation and Electrophoretic Mobility
Shift Assays
Nuclear extracts from HepG2 cells were prepared as
described
(35) . For gel retardation analysis, nuclear extracts
were incubated with a P-labeled double-stranded
oligonucleotide containing the serum-inducible element of c-fos in its superactive form as described
(36) . DNA-protein
complexes were loaded on 5% polyacrylamide gel containing 0.5
Tris borate/EDTA and 2.5% glycerol and electrophoresed at 130 V for 3
h. The gels were dried and autoradiographed.
Induction of Haptoglobin Synthesis and Protein
Determination
HepG2 human hepatoma cells were grown in minimum
essential medium with nonessential amino acids (Life Technologies,
Inc.) supplemented with 10% fetal calf serum. Induction of haptoglobin
synthesis was achieved essentially as described
(37) with minor
modifications. HepG2 cells were plated in 24-well cell culture plates
at a density of 1 10
cells/cm
and left
to reach confluence. Confluent monolayers were washed with PBS, starved
for 1 h in minimum essential medium without fetal calf serum and
nonessential amino acids, and subsequently treated for 20 h in 250
µl of serum-free minimum essential medium with hIL-6 and its
soluble receptors. The amount of haptoglobin secreted in the culture
medium was monitored by an enzyme-linked immunosorbent assay developed
at the Istituto di Ricerche di Biologia Molecolare P. Angeletti by Dr.
R. Laufer. 96-Well microtiter plates (Nunc Maxisorp) were coated with
goat polyclonal serum anti-human haptoglobin (Sigma) (100 µl in
each well of a 1:1000 dilution in 50 mM sodium carbonate, pH
9.4). Following a blocking step with 200 µl of TBSMT (5% non-fat
dry milk in Tris-buffered saline, 0.05% Tween 20), 50 µl of an
appropriate dilution (1:50 or 1:100) of cell culture medium in TBSMT
were added to each well and incubated for 1 h at room temperature.
Plates were washed, and 50 µl of rabbit polyclonal serum anti-human
haptoglobin (Sigma) (1:1000 dilution in TBSMT) were added. After 1 h of
incubation, wells were washed, and bound haptoglobin was revealed by
sequential incubation with alkaline phosphatase-conjugated donkey
polyclonal serum anti-rabbit IgG (Pierce) (1:1000 dilution in TBSMT)
and enzyme substrate. Absorbance at 405 nm was quantitated using a
microplate reader (Labsystems Multiskan
). Human
haptoglobin (Fluka) was used as a reference reagent to obtain a
standard curve. Dilutions of cell culture media were chosen in order to
obtain absorbance values in the range of linearity of the standard
curve.
RESULTS
Computer-assisted Modeling of the Human
IL-6
As a first step toward
the modeling of the hIL-6 receptor complex, we aligned the CBDs of
human IL-6RIL-6R
gp130 Complex
and gp130 with that of hGHbp (Fig. 1A),
taking advantage of conserved residues such as the WSXWS
motif, prolines in the interdomain linker, and the four cysteines
predicted to form disulfide bridges. The sequences of the CBDs of
hIL-6R
and hgp130 were thus inscribed onto the structural
templates from the hGH
h(GHbp)
complex, where the
hGHbp that binds hGH at site 1 (helix D) corresponds to hIL-6R
,
and the hGHbp bound at site 2 (helixes A and C) corresponds to hgp130.
In a subsequent step, the packing of buried side chains was optimized,
and finally, the loop regions including insertions and deletions were
modeled (for details, see ``Experimental Procedures''). The
resulting final alignment (shown in Fig. 1C) reflects
the most satisfactory internal packing. Two important observations can
be made. 1) Deletions and insertions occur only within loop regions;
and 2) the relative orientation of the two subdomains is likely to be
identical in hGHbp, hIL-6R
, and hgp130 because there are no
insertions or deletions between subdomains 1 and 2, and also the
PXPP motif at the beginning of subdomain 2 is conserved.
Therefore, as in both hIL-6R
and hgp130 CBDs the
strand
structure and subdomain orientation were predicted to be identical to
those of hGHbp, we were encouraged to combine their three-dimensional
models with that of hIL-6
(15) and to orient the three molecules
as in the hGH
h(GHbp)
complex, with hIL-6R
corresponding to hGHbp1 and hgp130 corresponding to hGHbp2. The final
model, shown in Fig. 1B, predicts that, similar to the
hGH
h(GHbp)
complex
(13) , the dimerization
interface between hIL-6R
and hgp130 is constituted by the AB2 loop
and the beginning of the E2 strand of their CBDs (for nomenclature, see
legend to Fig. 1). While mutations in the E2 strand of
hIL-6R
have already been reported to decrease the interaction of
the hIL-6
hIL-6R
complex with gp130
(12) , the sequence
alignment shown in Fig. 1C predicts that the AB2 loops
of both hIL-6R
and hgp130 are shorter than that of hGHbp by four
residues, pointing toward a possible different role of these regions in
the two systems. To test our model, we decided to generate targeted
mutations in the AB2 loop of hIL-6R
either individually or in
combination with substitutions in the E2 strand.
Figure 1:
Sequence
alignment of the CBDs of hIL-6R, hgp130, and hGHbp and schematic
view of the computer-predicted model of the human
IL-6
IL-6R
gp130 complex. A, comparison of the
extracellular domain topology of GHbp (left), IL-6R
(middle), and gp130 (right). The central element, the
common CBD with two subdomains (openboxes), is
present in IL-6R
and gp130 together with additional domains.
FNIII, fibronectin III. B, schematic MOLSCRIPT (52)
representation of the three-dimensional model of hIL-6 interacting with
the CBDs of hIL-6R
and hgp130. The three components of the complex
are oriented analogously to the hGH
h(GHbp)
complex
structure (13), with hIL-6 serving as a bridging ligand enabling
hIL-6R
and hgp130 to dimerize. The predicted interface between the
two receptors is shown in more detail, with hIL-6R
residues 228
and 230 (AB2 loop) and residues 280 and 281 (E2 strand) indicated as
spheres. C, multiple sequence alignment of
hIL-6R
, hgp130, and hGHbp (see ``Experimental
Procedures'').
strand regions are boxed. Regions
implied by the hGH
h(GHbp)
complex x-ray structure
(13) to be involved in cytokine recognition (loop regions AB, EF, and
BC2 and the interdomain linker) are indicated (#) in addition to
regions participating in receptor dimerization (E2 strand and AB2 loop)
(*).
Mutagenesis of the Presumptive hIL-6R
We generated a double substitution of
residues 280-281 in the E2 strand, which, as previously
mentioned, were already shown to be required for hIL-6hgp130
Dimerization Interface
hIL-6R
binding to hgp130
(12) . According to our model, these residues
correspond to hGHbp Tyr-200 and Ser-201, both of which are involved in
hGHbp homodimer formation
(13) . His-280 was changed to Ser and
Asp-281 to Val. These substitutions were predicted to be compatible
with the conformational constraints and unable to establish specific
contacts with the neighboring gp130 residues Lys-241, Thr-285, and/or
Glu-275.
mutants
previously generated at the end of the loop (Arg-232 and Trp-233)
showed no decrease in biological activity
(12) . We decided
therefore to mutate Asn-230 either singly (Mut-2) or in combination
with Ala-228 (Mut-3) (). To facilitate charge repulsion
with the predicted facing Asp-288 in hgp130, both residues were
substituted with Asp. We also constructed a mutant carrying all four
substitutions (A228D/N230D/H280S/D281V, Mut-4) (). Of the
remaining residues, Pro-231 was not mutated because its conservation in
the hIL-6R
sequence from various species (data not shown)
suggested that it plays an important structural role in the
conformation of the loop. Arg-229 was also excluded because it
corresponds to Gly in murine IL-6R
(38); since murine IL-6R
is able to functionally interact with human gp130
(10) , this
position is unlikely to be an important contact point.
mutants, containing
longer versions of the AB2 loop taken either from hGHbp
(13) or
from an unrelated protein
(26) , were also generated (Mut-5 and
Mut-8, respectively). Both variants were predicted to lead to steric
clashes and/or charge repulsion ().
Expression Level and hIL-6 Binding Properties of
shIL-6R
All mutants were generated in the context
of a cDNA encoding a soluble form of human hIL-6R Mutants
, with a
C-terminal stretch of six histidine residues immediately upstream of an
artificial termination codon introduced at amino acid 324. Wild-type
and mutant cDNAs were expressed in COS-7 cells (see ``Experimental
Procedures'').
monoclonal antibody I6R1/9.G11
(27) , which is directed against
the N-terminal Ig-like domain of hIL-6R
.
(
)
As shown in Fig. 2, a protein with an apparent
molecular mass of
53 kDa was immunoprecipitated from the culture
medium of transfected cells. The signal was specific since it was not
detected when an empty expression vector was used (Fig. 2,
controllane). Mut-5 to Mut-7 were constantly
produced in lower amounts, suggesting that the corresponding mutations
might affect either stability or secretion. On the contrary, the
expression level of the other five variants was comparable to that of
the wild type.
variants for hIL-6 using a technique that exploits
C-terminal histidine tagging (27). A fixed amount of labeled cytokine
was incubated with the histidine-tagged soluble receptor, and
self-competition was carried out by adding increasing concentrations of
unlabeled cytokine. Under equilibrium conditions, a metal-chelating
resin was added; receptor-bound (resin-associated) counts were
separated from free radioactivity by centrifugation. The values
obtained were plotted in a typical self-displacement curve, and the
apparent affinity was determined after Scatchard transformation of the
data
(27) . Using this technique, an apparent
K
value for IL-6 of COS-7-produced
wild-type shIL-6R
was obtained (2.0 ± 1
10
M) (), which is close to
that previously determined with standard techniques
(39) .
Substitution Mutants in Both the AB2 Loop and the E2
Strand Have Impaired Interaction with hgp130
We selected the
mutants showing a hIL-6 binding affinity similar to that of wild-type
shIL-6R and evaluated their interaction with shgp130 by
coimmunoprecipitation in the presence of hIL-6 using suitable
monoclonal antibodies
(15, 27, 40, 41) .
S-Labeled shgp130 (produced in insect cells; see
``Experimental Procedures'') was incubated with
I-hIL-6 (as an internal standard for immunoprecipitation)
and aliquots of transfected COS-7 cell culture medium containing either
native or mutant receptors. After in vitro binding,
hIL-6
shIL-6R
shgp130 complexes were immunoprecipitated
with anti-hIL-6R
monoclonal antibody I6R1/9.G11 and resolved by
SDS-polyacrylamide gel electrophoresis (see also legend to
Fig. 3
, A and B). The results are shown in
Fig. 3A. As expected, substitutions at the beginning of
the putative E2 strand (Mut-1) strongly reduced the association of the
hIL-6
shIL-6R
complex with the shgp130 molecule
(Fig. 3A, lane4). However, also the
single mutation at position 230 in the AB2 loop (Mut-2) had decreased
interaction with shgp130 (lane2), which was
substantially unaffected by the addition of a second negatively charged
residue (Mut-3) in near proximity (lane3). Finally,
the quadruple mutant Mut-4 displayed the lowest ability to
coimmunoprecipitate
S-shgp130, thus suggesting an additive
effect of the two pairs of mutations located in different regions of
the receptor (lane5).
Figure 3:
shIL-6R mutants (Mut-1 to Mut-4) have
a reduced capability to coimmunoprecipitate
S-shgp130.
A represents the results of a coimmunoprecipitation assay.
Conditioned COS-7 culture medium containing either wild-type
shIL-6R
(W.T.; lane1) or selected
mutants (Mut-1 (lane4), Mut-2 (lane2), Mut-3 (lane3), and Mut-4 (lane5)) was mixed with 2 µg of anti-hIL-6R
monoclonal antibody I6R1/9.G11 and 30 µl (packed volume) of protein
A-Sepharose for 4 h at 4 °C. After washing steps, the Sepharose
beads were incubated for 12 h with 5 nM
I-hIL-6
and 100 µl of
S-shgp130. Immunoprecipitates were
electrophoresed on SDS-12% polyacrylamide gel. The dried gel was
subjected to autoradiography at -80 °C (exposure time
= 36 h). B shows the portion of the gel (the same as in
A) corresponding to migrated
I-hIL-6 after 1 h
of exposure at room temperature.
We next tested receptor
variants for their ability to interact with membrane-bound gp130.
Binding experiments were performed on A375 human melanoma cells, which
are known to display more hgp130 molecules than hIL-6R on their
surface
(42) . Specific binding of
I-hIL-6 to A375
monolayers was strongly enhanced in the presence of wild-type
shIL-6R
(Fig. 4). The increase was competed by excess
unlabeled OM, an IL-6-related cytokine that directly binds, at low
affinity (K
10
M), hgp130 and competes for its association with the
hIL-6
hIL-6R
complex
(27) . On the contrary, only a
minor increase in specific binding was detected when cells were
challenged with
I-hIL-6 plus receptor derivatives
(Fig. 4). Interestingly, Mut-1 and Mut-4 were the most defective
variants also in this assay.
Mut-4 Is a hIL-6 Antagonist
The results shown
above indicated Mut-4 as the receptor mutant with the most affected
interaction with hgp130 and full hIL-6 binding activity. To study its
biological activity, the production of Mut-4 and of wild-type
shIL-6R was scaled up by using the baculovirus expression system.
Both His-tagged proteins were produced at high levels (>5 mg of
protein/liter of medium) and purified from the supernatant of infected
High Five
insect cells (see ``Experimental
Procedures'').
, and
this was maintained even at the highest cytokine concentration (100
nM).
Figure 5:
Evaluation of the ability of wild-type
shIL-6R and Mut-4 produced in insect cells to coimmunoprecipitate
S-shgp130. 2 µg of purified wild-type shIL-6R
(W.T.; lanes 1-5) or purified Mut-4 (lanes
6-10), both produced in insect cells, were mixed with 100
µl of
S-shgp130 and increasing concentrations of
hIL-6. Immunoprecipitations with 5 µg of anti-hIL-6R
monoclonal antibody I6R1/9.G11 were performed as described in the
legend to Fig. 3.
To verify the potential antagonism of Mut-4 for hIL-6
activity, we first tested its ability to interfere with early steps in
the IL-6 signal transduction pathway. The assay we used was the
activation of transcription factor APRF/STAT3 in human hepatoma
cells
(35, 36) . Acquisition of DNA binding by APRF is
dependent on tyrosine phosphorylation and takes place within minutes of
stimulation
(43, 44) .
increased the sensitivity of the cells to hIL-6
(compare lanes 2-4 with 5-7), Mut-4 not
only lacked agonistic activity, but instead inhibited IL-6-dependent
APRF activation (compare lanes 2-4 with
8-10). To test if antagonism was specific for hIL-6,
Mut-4 was added to HepG2 cells induced with OM, which is also known to
efficiently induce APRF phosphorylation and binding
(43) . As
shown in Fig. 6B, Mut-4 did not affect signaling by OM.
Figure 6:
Mut-4 specifically inhibits
hIL-6-dependent APRF activation in HepG2 human hepatoma cells. A shows the hIL-6 antagonistic activity of Mut-4 on HepG2 cells
compared with the agonistic properties of wild-type shIL-6R. HepG2
cells were treated for 15 min with increasing concentrations of hIL-6
alone (lanes 2-4) or together with a fixed amount of
purified wild-type shIL-6R
(W.T.; lanes
5-7) or purified Mut-4 (lanes 8-10). 5 µg
of nuclear extracts were used to monitor the binding of APRF to the
serum-inducible element probe by electrophoretic mobility shift assay
(see ``Experimental Procedures''). The dried gel was exposed
for 36 h. B demonstrates that Mut-4 does not inhibit
OM-dependent APRF activation in HepG2 cells. Nuclear extracts were
prepared from HepG2 cells treated (15 min) with 200 pM IL-6 or
OM alone (lanes2 and 5, respectively) or in
combination with 100 nM wild-type shIL-6R
(lanes3 and 6) or Mut-4 (lanes4 and
7). Electrophoretic mobility shift assay was performed as
described for A, but the gel was exposed for only 12
h.
We next tested whether Mut-4 might inhibit hIL-6 ability to induce
the production and secretion of the acute-phase protein haptoglobin in
the same cells. HepG2 cells were treated for 20 h with IL-6 (200
pM) alone or in combination with increasing concentrations of
wild-type and mutant receptors. At the end of the incubation time, the
medium was collected and assayed for the presence of secreted
haptoglobin (see ``Experimental Procedures''). As expected,
the addition of wild-type shIL-6R to hIL-6 induced a
dose-dependent increase (Fig. 7). On the contrary, Mut-4
antagonized hIL-6 bioactivity in a dose-dependent fashion, with a 50%
inhibition achieved at the highest concentration tested (100
nM) (Fig. 7). Also in this case, the effect was specific
since neither the native nor the mutant receptor interfered with the
OM-induced production of haptoglobin (data not shown).
Figure 7:
Mut-4
down-modulates hIL-6-induced synthesis of haptoglobin in HepG2 cells.
HepG2 cells were cultured for 20 h with 200 pM hIL-6 and the
indicated amounts of either wild-type (W.T.) shIL-6R
(blackbars) or Mut-4 (shadedbars). In control experiments, cells were either
untreated (whitebar) or incubated with 200
pM hIL-6 alone (hatchedbar). The amount of
secreted haptoglobin was measured with an enzyme-linked immunosorbent
assay as described under ``Experimental Procedures.'' Means
± S.D. of three separate experiments are shown. No effect on
haptoglobin production was observed when cells were challenged with
soluble receptors only (data not shown).
DISCUSSION
In this paper, we present a computer-assisted
three-dimensional model of the interaction between hIL-6 and its two
receptor chains, based on the x-ray structure of the
hGHh(GHbp)
complex (13), and use it to selectively
introduce mutations into the hIL-6R
chain that modify its
biochemical and biological properties. Residues in both the putative
AB2 loop and the E2 strand of the hIL-6R
CBD are identified as
being involved in the interaction with hgp130. To date, residues in the
E2 strand and the AB2 loop in the receptor-receptor interface have been
demonstrated to be involved at the structural level only for
homodimeric hGHbp
(13) . This finding has been recently
confirmed, from the functional point of view, by the isolation of a
natural mutant of hGHbp (D152H) unable to homodimerize (45). Our
results demonstrate that the same regions are also involved in
stabilizing a heterodimeric receptor (hIL-6R
hgp130). It is
thus tempting to speculate that participation of the AB2 loop and the
E2 strand in receptor association is a common rule for both homodimeric
complexes, like those assembled by GH, erythropoietin, and granulocyte
colony-stimulating factor, and heterodimeric receptors, like those for
IL-3, IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor
(16).
and hgp130 at the same time. Another important difference arises from
the fact that hgp130, in contrast with homodimeric hGHbp, is able to
interact with a variety of receptors such as leukemia inhibitory factor
receptor
and ciliary neurotrophic factor receptor
and
probably also with the specific OM and IL-11 receptor
(6) . This
suggests that the gp130 interface might be more flexible and able to
establish several low specific contacts with its dimerizing partner
receptor, rendering the dimerization interface tolerant to mutations.
Finally, our model for the hIL-6
hIL-6R
hgp130 complex
is compatible with the results obtained in several mutagenesis studies
on both hIL-6 and hIL-6R
(12, 46, 47), but is probably incomplete
given the observation that hgp130 undergoes a covalent dimerization as
a final step in the formation of biologically active receptor
complexes
(48) . The possibility is thus open that hIL-6R
might bind two distinct hgp130 chains via separate surfaces and that
our mutagenesis has only identified one of them. This idea is in line
with the identification by site-directed mutagenesis of two distinct
sites on hIL-6 that are both responsible for the interaction with
hgp130
(19, 49) . Further mutagenesis of hIL-6,
hIL-6R
, and hgp130 and the determination of the stoichiometry of
the receptor complex in vitro for wild-type and mutant
molecules will be required to clarify this important issue.
antagonists should be identical to that of
monoclonal antibodies, it cannot be ruled out that IL-6
soluble
IL-6R
complexes might be cleared from the body with kinetics
faster than that of immune complexes.
mutants with a completely abolished binding to hgp130 and
the analysis of their biological activity should demonstrate if the
strategy outlined in this paper can be effectively used to fully
inhibit hIL-6 in vivo.
Table: List of amino acid substitutions
introduced in the context of predicted AB2 loop and E2 strand mutants
(Mut-1 to Mut-8) and binding affinity of shIL-6R variants
for hIL-6
, IL-6 receptor
(the prefixes
``h'' and ``s'' represent human and soluble,
respectively); hgp130, human gp130; shgp130, soluble human gp130; hGH,
human growth hormone; hGHbp, hGH-binding protein; CBD, cytokine-binding
domain; OM, oncostatin M; PBS, phosphate-buffered saline; APRF,
acute-phase response factor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.