From the
Interleukin-4 (IL-4) is a pleiotropic cytokine eliciting various
responses in target cells upon binding to its receptor. Activation of
the IL-4 receptor probably involves interaction of the ligand with both
the IL-4 receptor
Interleukin-4 exerts its activity on target cells by interaction
with at least two membrane-bound receptor chains, i.e. the
IL-4R
In order
to investigate the interactions between hIL-4 and the two hIL-4
receptor components, we have established a hIL-4-responsive murine
myeloid precursor cell line. A chimera of the extracellular domain of
hIL-4R
Statistical evaluations of proliferation and radioligand binding
data were done using the computer program GraFit (Erithacus Software).
pKCR-4G
along with the selection plasmid pSV2neo
(15) was introduced
into FDC-P1 cells by electroporation. After selection in the presence
of G418, a resistant clone was stained with an hIL-4R
The unexpected result was that
proliferative activities of individual hIL-4 mutants were drastically
different in the two cellular systems. As illustrated in
Fig. 6A, mutants Y124D and S125D acted like wild type
hIL-4 on FDC-4G cells, whereas variant R121D as well as double and
triple hIL-4 mutants containing the R for D exchange at position 121
displayed no significant activity. Double mutant Y124D/S125D showed an
intermediate, partial agonist phenotype.
Human IL-4 receptor
In order to generate a
hIL-4-responsive cell line, we have expressed a chimera of hIL-4R
The
stoichiometry of the IL-4 receptor complex has not yet been definitely
settled. By functionally expressing a hIL-4R
FDC-4G cells show a similar growth behavior in response
to mIL-4 and hIL-4 regarding both intensity and duration. Since hIL-4
operates by means of a hybrid receptor system laking the intracellular
domain of hIL-4R
We finally used the FDC-4G line as a cellular
read-out system allowing for the molecular analysis of interactions
between hIL-4, hIL-4R
It is
interesting to note that the two most critical amino acids of hIL-4 for
signaling in FDC-4G and TF-1 cells, respectively (Arg
The skillful technical assistance of C. Müller is
gratefully acknowledged. We thank Dr. W. Sebald for generous support,
Drs. W. Ostertag and W. Müller for providing us with cell lines,
Dr. C. Laker for advice on cell culture during the initial stages of
the study, C. Söder and H. Spengler for recombinant hIL-4 mutant
proteins and GM-CSF, Dr. P. Reusch for anti-hIL-4R
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit (IL-4R
) and the common
chain
(c
). Although human and murine IL-4 receptor
chains are
specific for IL-4 from the same species, murine c
can form a
signal-competent complex with human IL-4R
(hIL-4R
) and human
IL-4 (hIL-4). We have generated a hIL-4 responsive murine myeloid cell
line (FDC-4G) expressing a chimera comprising the extracellular domain
of human IL-4R
and the intracellular domain of human granulocyte
colony-stimulating factor receptor (hG-CSFR). This hybrid receptor was
shown to form a complex with hIL-4 and the murine c
-chain.
Biological activities of human IL-4 variants on murine FDC-4G cells and
on the human erythroleukemic cell line TF-1 displayed a strikingly
different pattern. Single amino acid replacements at two different
positions in the C-terminal helix of hIL-4, the region of the
previously defined ``signaling site,'' lead to an inverse
agonist/antagonist behavior of the resulting cytokines in the two
cellular systems. From these findings we conclude that upon formation
of the activated IL-4 receptor complex murine and human c
interact
with hIL-4 in a geometrically different fashion.
(
)
subunit (formerly termed IL-4R)
(1) , and the common receptor
chain (c
)
(2, 3) , both of which are members of the hematopoietin
receptor superfamily
(4) . The induction of productive IL-4
receptor complex formation by binding of the ligand is not yet
understood; however, mutational analysis of hIL-4 indicated that two
distinct structural determinants of the cytokine are important for this
process
(5, 6) . Amino acids located within helixes A
and C of the hIL-4 molecule have been shown to be essential for the
interaction with hIL-4R
, whereas three positions in the C-terminal
helix have proven crucial for signaling. Replacement of residues
Arg
, Tyr
, or Ser
yielded high
affinity partial agonists or antagonists of hIL-4 in cellular assays
employing human B or T cells
(6) , most probably by interfering
with a productive interaction of hIL-4 and c
. hIL-4 does not
detectably bind to murine IL-4R
(1, 7) . Several
groups have shown, however, that human IL-4R
can confer hIL-4
responsiveness to murine lymphoid cells when expressed by gene
transfer, thus indicating that murine c
is able to form a
signaling competent receptor complex with hIL-4 and hIL-4R
(1, 8, 9, 10, 11) .
fused to the cytoplasmic portion of hG-CSFR was expressed
in factor-dependent FDC-P1 cells and shown to become associated with
murine c
upon binding of hIL-4. Subsequently the formation of
productive receptor complexes resulting in cell proliferation was
studied using mutant variants of hIL-4. Comparison of the results with
those obtained in a parallel set of experiments employing the
hIL-4-responsive human cell line TF-1 revealed a different activity
pattern of hIL-4 variants. These findings are discussed with respect to
the putative interaction of hIL-4 with murine and human c
.
RNA Isolation, cDNA Synthesis, and Molecular
Cloning
Total RNA was isolated from 1 g of fresh human placenta
(obtained from Dr. J. Martius, Department of Gynecology, University of
Würzburg) according to the guanidinium
thiocyanate-phenol-chloroform extraction method
(12) , and used
as a template for cDNA synthesis which was performed using Superscript
Reverse Transcriptase (Life Technologies, Inc.) following the
manufacturer's instructions. The cDNA clone providing the
extracellular domain of hIL-4R has been described previously
(5) . Oligonucleotide synthesis, polymerase chain reactions, and
other enzymatic manipulations of DNA were done following standard
procedures
(13) . For transfection experiments SV40-based
expression vector pKCR
(14) and neomycin resistance plasmid
pSV2neo
(15) were employed. Other reagents and enzymes were
from Boehringer (Mannheim, Germany), Fermentas (Vilnius, Lithuania),
Amersham-Buchler (Braunschweig, Germany), and Merck (Darmstadt,
Germany).
Cell Culture, Transfections, and Flow
Cytometry
Both the murine myeloid precursor cell line FDC-P1
(16) and the human erythroleukemic cell line TF-1
(17) were described previously. Cells were routinely grown in
DMEM, 8% FCS (FDC-P1) or RPMI 1640, 8% FCS (TF-1). Media were
supplemented with 5% culture supernatant of mIL-3 producing
X63Ag8-653 BPV mIL-3 cells
(18) (FDC-P1) or recombinant
GM-CSF basically produced as described for hIL-4
(19) at a
final concentration of 5 n
M (TF-1). Transfection of FDC-P1
cells was performed by electroporation using a Easyjectelectroporator (Eurogentec). 3.5
10
cells
were washed and resuspended in 400 µl of DMEM, 5% FCS containing 50
µg of pKCR derivative and 2 µg of pSV2neo and subsequently
subjected to an electric pulse of 250 V at 1500 microfarads. Cells were
then kept in DMEM, 8% plus mIL-3 for 48 h before adding G418 to a final
concentration of 1 mg/ml. After cultivation in 24-well plates for 2
weeks (medium was renewed twice), G418-resistant cell clones were
propagated in 75-ml flasks and further screened for receptor expression
by flow cytometry. For cytometric identification of hIL-4R
expression on transfectants, 10
cells were stained with
monoclonal antibody X2/45 (final concentration 400 n
M)
directed against the extracellular domain of hIL-4R
(20) in 100 µl of PBS for 20 min at 4 °C, incubated with
100 µg/ml fluorescein (dichlorotriazinyl amino
fluorescein)-conjugated goat-anti mouse IgG Fc
fragment-specific
(Dianova) and analyzed using a Coulter Epics Elite flow cytometer with
a 488-nm laser.
Radioligand Binding Analysis
Iodination of
recombinant hIL-4 was done as described
(21) , specific activity
of the radiolabeled cytokine (0, 5 µCi/pmol) was determined by
competition binding measurements employing a solid phase binding assay
based on the recombinant extracellular domain of hIL-4R
(6) . Saturation binding experiments were performed after a
standard protocol
(22) . Briefly, samples of 2
10
cells were incubated with varying concentrations of radioligand
in a volume of 200 µl at 4 °C for 2 h. Cells were then
separated from unbound radioactivity by centrifugation through a
silicon oil layer, and bound label was determined using a
counter
(Beckman). Unspecific binding of radioligand as measured in a parallel
experiment, including at least 100-fold excess of unlabeled ligand was
subtracted before plotting the data.
Chemical Cross-linking of Radiolabeled hIL-4 to Cells and
Immunoprecipitation of Receptor Complexes
Samples of 3
10
cells were rinsed twice with RPMI medium and incubated
in 500 µl of RPMI, 2% bovine serum albumin containing 1 n
M
I-labeled hIL-4 (0.5 µCi/pmol) at 4 °C for 1 h.
Cells were then washed twice with ice-cold RPMI, treated with 1 m
M disuccimidyl suberate (Pierce) in PBS at 4 °C for 30 min,
washed with PBS, and subsequently lysed in 500 µl of lysis buffer
(50 m
M Tris-HCl, pH 7.5, 140 m
M NaCl, 1 m
M EDTA, 0, 5% Nonidet P-40, 17 m
M Na
P
O
, 2 m
M phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, 100
µg/ml leupeptin). After an incubation at 4 °C for 30 min the
cell lysates were frozen at -70 °C for 12 h and thawed on
ice. Insoluble material was pelleted by centrifugation at 15,000
g for 15 min. Supernatants were gently rotated for 1 h
at 4 °C after addition of either 1 ng of anti-hIL-4R
antibody
X14/38,
(
)
or 0.5 µl of ascites fluid
containing anti-mc
antibody TUGm3
(2) (a gift from Dr. K.
Sugamura, Tohoku University School of Medicine, Sendai, Japan).
Precipitation of immunocomplexes was performed by centrifugation at
2,000
g for 2 min after an additional incubation for 1
h at 4 °C with 12 µl of anti-mouse IgG- (or anti-rat
IgG)-agarose slurry (Sigma). The agarose pellets were washed once in
lysis buffer, once in 100 m
M Tris-HCl, pH 8.0, 0.5
M LiCl, and finally boiled for 5 min in 2
Laemmli buffer.
Pellets were then removed by centrifugation and supernatants subjected
to SDS-polyacrylamide gel electrophoresis (6%). After fixing in 12.5%
acetic acid, 12.5% 2-propanol for 30 min, gels were dried and
subsequently scanned for radioactivity using a PhosphorImager
(Molecular Dynamics).
Cytokines, Cell Proliferation Assays, and Data
Processing
Wild type hIL-4 and mutants R121D, Y124D, and S125D
have been described previously
(5, 6) . Double and
triple mutants R121D/Y124D, R121D/S125D, Y124D/S125D, and
R121D/Y124D/S125D, which have been constructed and purified in the same
way and are described in detail elsewhere
(20) . As a source of
murine IL-4 the culture supernatant (10%) of an mIL-4 secreting 3T3
transfectant cell line (provided by Dr. W. Müller, Institute of
Genetics, University of Cologne) was used. Cytokine-induced
proliferation of FDC-4G and TF-1 cells was measured either
spectrometrically by means of blue formazan formation from
3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide-tetrazolium (MTT) or by [H] thymidine
incorporation into de novo synthesized DNA as described
previously
(23) . In both cases cells were washed in medium
twice to remove mIL-3 or hG-CSF and incubated with 100 or 200 µl of
medium containing various concentrations of IL-4 or IL-4 variants for
24 h (FDC-4G cells) or 72 h (TF-1 cells), followed by incubation with
MTT or [
H]thymidine for 4 h, respectively.
Generation of the Murine Myeloid Cell Line FDC-4G
Expressing a Chimera of hIL-4R
A hybrid gene
comprising the first 232 codons of the human hIL-4R and hG-CSFR
gene (encoding
the signal peptide and the extracellular domain)
(1) fused to
codons 605 through 813 of the hG-CSFR gene
(24) (encoding the
transmembrane and intracellular domain) was constructed in two
consecutive steps. First a BamHI/ BclI fragment was
amplified from hIL-4R
cDNA by polymerase chain reaction with the
5`-primer STA12 (5`-CTAAGGATCCATGGGGTGGCTTTGCTCTG-3`) and the 3`-primer
KHF8 (5`-AT-GCTGATCAAAGCTTCGATATCTTCTCGAGGTGCTGCTC-GAAGGGCTC-3`) and
ligated into the unique BamHI site of the expression vector
pKCR
(14) . Primer KHF8 introduced unique restriction sites for
both XhoI and HindIII into the plasmid. They were
subsequently used to clone the hG-CSFR gene fragment which was
amplified from human placenta total RNA with the primers KHF9
(5`-GAAGATCTCGAGATCATCCTGGGCCTG-3`) and KHF10
(5`-GAAGATCTGATATCTA-CTCGAGAAGCTTCTAGAAGCTCCCCAG-3`). The resulting
construct pKCR-4G encodes a mature hybrid receptor protein of 417 amino
acids length, including two codons (Leu-Glu) equivalent to the
XhoI site at the fusion position (see Fig. 1).
specific
monoclonal antibody and was analyzed for surface expression of
hIL-4R
by flow cytometry. The cells from this clone, termed
FDC-4G, displayed an approximately 10-fold increase in fluorescence
when compared with control cells (Fig. 2), thus clearly
indicating a hIL-4R
-positive phenotype.
Figure 2:
Cytometric analysis of G418-resistant
transfectant cell clone FDC-4G. FDC-P1 ( I) and FDC-4G cells
( II) were stained with anti-IL-4R mAb X2/45 and
fluorescein-conjugated anti-mouse IgG and subsequently analyzed by flow
cytometry as described under ``Material and
Methods.''
FDC-4G Cells Have Acquired Proliferative Responsiveness
to Human Interleukin-4
We next examined whether the
G418-resistant transfectant clone FDC-4G had become responsive to
hIL-4. FDC-P1 cells have been shown to respond to murine IL-4 by
transient proliferation
(25) ; therefore we used mIL-4 as a
control in the proliferation tests. Fig. 3shows that although
FDC-P1 cells were only responsive to mIL-4, transfectant clone FDC-4G
also proliferated in a hIL-4-dependent manner. This was consistent with
the cytometry data and confirmed that this cell line functionally
expresses the hIL-4/hG-CSFR hybrid receptor.
Figure 3:
Proliferative response of FDC-P1 and
FDC-4G cells to mIL-3, mIL-4, and hIL-4. FDC-P1 cells as well as clone
FDC-4G derived from a transfection experiment with the
hIL-4R/hG-CSFR hybrid receptor expression construct were tested
for cell proliferation by the MTT method as described under
``Materials and Methods.'' Formation of formazan dye was
determined spectrometrically after 24-h incubation without cytokine
stimulation ( white bars) or in response to mIL-3 (undiluted
culture supernatant of mIL-3 producing X63Ag8-653 cells;
dotted bars), mIL-4 (undiluted culture supernatant of mIL-4
producing 3T3 cells; striped bars), and hIL-4 (2 n
M;
black bars).
Since the G-CSFR
can transmit a sustained growth signal into factor-dependent FDC-P1
cells when activated by ligand-induced dimerization
(26, 27) , we examined if hIL-4 could replace mIL-3 to
allow for permanent culture of FDC-4G cells. Cultivation tests over a
wide range of hIL-4 concentrations (up to 10 µ
M) showed
that hIL-4, like mIL-4, can prevent cell death for only 72-96 h
(data not shown).
Ligand Binding Characteristics of the hIL-4R
We next wished to
measure the affinity for hIL-4 of the signaling-competent 4G hybrid
receptor as well as the number of ligand binding sites per cell.
Therefore the saturation binding experiment shown in Fig. 4was
performed. The K/hG-CSFR
Hybrid in the Murine Cellular Background
was found to be 158
p
M, which is in the same range as was determined for human
peripheral T cells
(5) . A saturation of hIL-4 binding to FDC-4G
cells was observed at a concentration of 69 p
M bound
radioligand. This value is equivalent to 3,500 hIL-4 binding
sites/cell.
Figure 4:
Saturation binding of
I-hIL-4 to FDC-4G cells. Samples of 2
10
FDC-4G cells were incubated with increasing concentrations of
radiolabeled hIL-4. Bound label was measured as described under
``Materials and Methods.'' The inset shows a
Scatchard plot of the data. Saturation was reached at a bound ligand
concentration of 69 p
M (=3,500 binding sites/cell);
K was determined to be 158
p
M.
The hIL-4R
Chemical
cross-linking of radiolabeled hIL-4 to FDC-4G cells followed by
immunoprecipitations using antibodies against IL-4R components was
performed in order to define the composition of the operative receptor
complex. As shown in Fig. 5, monoclonal antibody X14/38 directed
against the extracellular domain of hIL-4R/hG-CSFR Hybrid and Murine c
Chain
Form a hIL-4 Binding Receptor Complex on FDC-4G Cells
predominantly
precipitates a radiolabeled complex with a molecular mass of
approximately 75 kDa. This band originates from hIL-4 cross-linked to
the hIL-4R
/hG-CSFR hybrid receptor, since it corresponds to the
expected size of the glycosylated chimera and does not appear in
control precipitations from FDC-P1 cells (data not shown) and TF-1
cells. The latter instead show a dominant band (approximately 155 kDa)
which can be attributed to a conjugate of iodinated hIL-4 and
hIL-4R
. Presence of the extracellular domain of hIL-4R
in
both complexes could also be proven by probing Western blots of
immunoprecipitations with mAb X14/38 (data not shown).
Figure 5:
Immunoprecipitation of
I-hIL-4- cross-linked receptor complexes from FDC-4G
cells and TF-1 cells. After chemical cross-linking of radiolabeled
hIL-4 to FDC-4G cells or TF-1 cells, immunoprecipitation using the
indicated antibodies followed by polyacrylamide gel electrophoresis was
performed as described under ``Material and Methods.''
Lanes 1, 2, and 3, immunoprecipitations from lysates
of TF-1 cells and FDC-4G cells, respectively, with anti-hIL-4R
antibody X14/38. Lane 4, control precipitation from a lysate
of FDC-4G cells without receptor antibody added. Lane 5,
immunoprecipitation from a lysate of FDC-4G cells with anti-mc
antibody TUGm3. Lane 2 shows a shorter exposure of an
identical sample as in lane 3.
As a second
component, murine common chain could be identified in the hIL-4
binding receptor complex on FDC-4G cells. In addition to the hIL-4/4G
conjugate, a weaker band running at approximately 80 kDa was
precipitated by anti-hIL-4R
antibody. Due to its proximity to the
intense 4G receptor band, it was only resolved on short exposures. This
band showed the same migration behavior as the hIL-4/hc
conjugate
from TF-1 cells,
(
)
and, together with the 4G
hybrid derived band, appeared prominently when
I-hIL-4
cross-linked proteins of FDC-4G cells were immunoprecipitated with
mc
-specific mAb TUGm3. Moreover, both hIL-4R
- and
mc
-specific antibodies were capable of precipitating a molecular
species of approximately 140 kDa in size, which most likely represents
the ternary complex hIL-4/4G/mc
.
Biological Activities of hIL-4 Signaling Mutants on the
Murine Cell Line FDC-4G and the Human Cell Line TF-1 Are Strikingly
Different
In order to address the activation of the 4G hybrid
receptor during ligand binding, we subjected FDC-4G cells to
proliferation experiments using a set of hIL-4 mutant proteins. It has
been shown previously that hIL-4 variants with amino acid positions
Arg, Tyr
, and Ser
,
respectively, exchanged for aspartic acid were affected in their
signaling properties on hIL-4-responsive human cells
(5, 6, 28) . Therefore we employed variants
R121D, Y124D, and S125D
(6) as well as the double and triple
mutants R121D/Y124D, R121D/S125D, Y124D/S125D, and R121D/Y124D/S125D
(20) to these tests. The TF-1 cell line expressing the authentic
human IL-4R was used as a reference.
Figure 6:
Proliferative activity of hIL-4 and hIL-4
mutants on FDC-4G cells and TF-1 cells. FDC-4G cells ( A) and
TF-1 cells ( B) were incubated with the concentrations of hIL-4
or hIL-4 mutant indicated. Cell proliferation was determined by
[H]thymidine incorporation after 24 h for FDC-4G
or 72 h for TF-1.
, hIL-4 wild type;
, hIL-4 Y124D;
,
hIL-4 R121D;
, hIL-4 S125D; ▾, hIL-4 Y124D/S125D;
,
hIL-4 R121D/Y124D;
, hIL-4 R121D/S125D;
, hIL-4
R121D/Y124D/S125D.
It has been demonstrated that hIL-4 Y124D behaves as a high affinity
antagonist when assayed in proliferation tests with both human
peripheral T cells
(5) and TF-1 cells
(28) . Our results
with TF-1 (Fig. 6 B) were consistent with these findings. In
contrast to FDC-4G cells, TF-1 cells respond to Y124D only to a minimal
extent (partial agonist activity <5% of wild type). R121D, which was
unable to induce FDC-4G proliferation, had some 30% partial agonist
activity in the TF-1 system. All double and triple mutants were devoid
of any detectable proliferative activity.
An Amino Acid Replacement at Position Arg
Competition experiments were performed in order to
address whether hIL-4 variants with the R121D mutation were inactive on
FDC-4G cells because of impaired binding to the heterologous hIL-4R
derivative expressed on these cells or due to antagonist properties of
the mutant ligands. Fig. 7shows that R121D as well as the double
and triple mutants R121D/Y124D, R121D/S125D, and R121D/Y124D/S125D
competitively inhibited hIL-4-induced proliferation of FDC-4G cells.
The results obtained with Y124D confirmed that this variant, despite
being a potent hIL-4 antagonist for TF-1 cells
(28) , has
agonist activity on FDC-4G cells comparable with that of wild type
hIL-4.
Renders hIL-4 Variants Antagonists in FDC-4G
Cells
Figure 7:
Competitive inhibition of hIL-4-induced
proliferation of FDC-4G cells by hIL-4 mutants. Cell proliferation was
measured by [H]thymidine incorporation in the
presence of a constant concentration of hIL-4 wild type (2 n
M)
and increasing concentrations of hIL-4 mutants as indicated.
,
hIL-4 Y124D;
, hIL-4 R121D;
, hIL-4 R121D/Y124D; ▾,
hIL-4 R121D/S125D;
, hIL-4
R121D/Y124D/S125D.
chain has been shown to render
murine IL-4-reactive cells responsive to hIL-4 when expressed after
gene transfer
(8, 9, 10, 11) . Human and
mouse IL-4R
bind exclusively to IL-4 from the same species in
vitro (7) , hence IL-4R
obviously mediates species
specificity of the activated IL-4 receptor. Functional IL-4R, however,
includes at least one additional receptor component that was found to
be identical with the interleukin-2 receptor
chain or c
(2, 3) . hIL-4 responsiveness of murine cells due to
expression of hIL-4R
thus implies that murine c
can
accommodate both mIL-4 and hIL-4.
and hG-CSGR in FDC-P1 cells. The initial reason for taking this
approach was our interest in the functional roles of individual IL-4R
subunits. It is only beginning to become understood what particular
contributions the cytoplasmic parts of IL-4R
and c
make to an
IL-4-specific cellular response. Insight into the function of
cytoplasmic portions of the IL-4R in the course of signaling is as yet
limited. Cytoplasmic sequence elements of hIL-4R
have been
correlated with functional importance in hIL-4 induced proliferation
(8, 11, 29) . It has been shown that a small
sequence motif in the intracellular domain of IL-4R
is critical
for interaction with insulin receptor substrate 1 (IRS-1) protein
(29) , a molecular contact which probably (among other
consequences) leads to an activation of phosphoinositol 3-kinase and
apparently links the IL-4R to a signaling pathway with similarities to
a set of reactions initiated by the activated insulin receptor. Two
phosphotyrosine-containing peptides derived from the intracellular
domain of hIL-4R
were found to directly interact with the
IL-4-inducible transcription factor IL-4 Stat
(30) . In
addition, association of members of the JAK kinase family with the IL-4
receptor complex was demonstrated
(31, 32) . Recent
results obtained on the IL-2R and (in less detail) the IL-4R system
indicate an interaction of JAK-3 with c
and strongly suggest a
binding of JAK-1 to IL-4R
(33, 34, 35) .
Both kinases become phosphorylated as a consequence of ligand-induced
receptor activation, whereas only JAK-3 is activated.
/hG-CSFR chimera we
anticipated a system yielding information about oligomerization
processes during hIL-4 receptor activation, in particular about a
possible involvement of hIL-4R
homodimerization. G-CSFR has been
shown to homodimerize upon ligand binding, and moreover, the
intracellular domain of G-CSFR was used experimentally to transmit a
homodimerization signal of a fused extracellular human growth hormone
receptor domain into FDC-P1 cells, resulting in sustained human growth
hormone-dependent cell proliferation
(12, 18) . The
failure of hIL-4 to maintain sustained growth of FDC-4G cells makes a
participation of hIL-4R
homodimerization in formation of the
activated hIL-4R appear very unlikely. Together with our chemical
cross-linking data it rather confirms the notion that hIL-4 binding
leads to an assembly of hIL-4R
and c
(here: murine c
)
into a functional receptor complex, an event which is fundamental for
signaling.
, this raises the question what common
intracellular mechanisms are involved in both cases. The cytoplasmic
portion of G-CSF receptor shares a membrane-proximal box of sequence
homology with both IL-4R
and IL-2 receptor
subunit
(36) . For IL-2R
, a stretch of amino acids comprising this
motif was found to be essential for interaction with JAK-1 kinase
(32) . As for IL-4, IL-2, and several other cytokines, tyrosine
phosphorylation of Stat family members and other proteins has also been
observed upon G-CSF-induced activation of its cognate receptor,
probably due to JAK kinase activity
(37) . Our recent work
indicates that stimulation of FDC-4G cells by hIL-4 results in
phosphorylation of the 4G hybrid receptor.
(
)
We
suggest that by interacting with their extracellular domains, hIL-4
brings the intracellular domains of 4G receptor and mc
into close
proximity to each other. The serine-rich membrane proximal sequence
motif of the hG-CSFR component is associated with a JAK kinase which in
turn activates mc
-associated JAK-3 kinase. As a result of this
event a mitogenic signal is generated by the artificial receptor
subunit combination which resembles that one produced by the natural
murine IL-4R complex.
, and c
in the course of receptor
activation, thereby extending previous mutational analysis of hIL-4
(5, 6) . The most surprising results of this study were
obtained when the proliferative response of FDC-4G cells and TF-1 cells
to signaling-deficient hIL-4 variants were compared. These data
indicate that the participation of human or murine c
in the
formation of a productive hIL-4/hIL-4 receptor complex is not
equivalent. Instead, requirements of hc
and mc
, respectively,
for the integrity of particular amino acid side chains in helix D of
hIL-4 differ in a characteristic fashion. Variant hIL-4 R121D displayed
no detectable biological activity on murine FDC-4G cells expressing a
hIL-4R
/G-CSFR chimera and behaved as an antagonist in competition
experiments with wild type hIL-4. In contrast, when assayed with the
human cell line, it showed about 40% wild type activity. For variant
hIL-4 Y124D an almost reciprocal activity profile in the two cell types
was observed. Although proliferation of FDC-4G cells was induced by
Y124D to an extent indistinguishable from that of wild type hIL-4, the
variant cytokine had only a minimal residual activity on TF-1.
and
Tyr
), are separated by one helical turn of helix D, which
results in an aligned spatial location of their side chains
(38) (see Fig. 8 A). Since sequence comparison
reveals a shortened C terminus by four amino acids of mIL-4 compared
with hIL-4 (Ref. 7, see also Fig. 8 B), it is tempting to
speculate that the interaction interface of mIL-4 is shifted by one
helical turn equivalent. It has been reported that deletion of the
three C-terminal amino acids of mIL-4 result in a variant cytokine with
antagonist properties
(7) . In the light of the findings
presented in this study, it would be interesting to analyze mIL-4 Q116D
and mIL-4 Y119D, the murine homologs of hIL-4 R121D and hIL-4 Y124D,
respectively, with regard to their activity on murine cells. We cannot
exclude the possibility that murine and human c
have different
functional characteristics in IL-4 signaling. In the IL-2 receptor
system, human IL-2R
heterodimer is capable of binding IL-2,
whereas murine
is not
(39) . Biochemical studies on
the interaction of IL-4 and c
are important to address whether
such differences also exist regarding the IL-4R.
Figure 8:
Structural situation of hIL-4 amino acids
presumably interacting with c. A, solution structure of
hIL-4 after (38) showing the spatial locations of amino acid side
chains involved in signaling. B, amino acid alignment of the C
termini of mIL-4 ( top) and hIL-4 ( bottom). Sequence
identities are indicated, and amino acid positions of hIL-4 probably
interacting with c
as well as their murine counterparts are
highlighted.
, the IL-4
receptor
subunit; hIL-4, human interleukin-4; hIL-4R, human
interleukin-4 receptor; mIL-4, murine interleukin-4; mIL-3, murine
interleukin-3; hIL-4R
, human interleukin-4 receptor
subunit;
c
, common receptor
chain; hc
, human common receptor
chain; mc
, murine common receptor
chain; hG-CSFR,
human granulocyte-colony-stimulating factor receptor; GM-CSF, human
granulocyte/macrophage colony-stimulating factor; IL-2, interleukin-2;
IL-2R
, interleukin-2 receptor
and
chain; DMEM,
Dulbecco,'s modified essential medium; FCS, fetal calf serum;
G418, Geniticin; MTT,
3-{4,5-dimethythiazol-2-yl}-2,5-diphenyl-tetrazolium
bromide-tetrazolium; 4G, hIL-4R
/hG-CSFR hybrid receptor; IgG,
immunoglobulin G; PBS, phosphate-buffered saline.
antibodies, Dr.
K. Sugamura for anti-c
antibodies, Dr. A. Duschl and B. Schnarr
for valuable discussions on immunoprecipitation, Dr. J. Martius for
human placenta, and W. Hädelt for oligonucleotide synthesis and
DNA sequencing.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.