(Received for publication, August 13, 1996, and in revised form, November 22, 1996)
From the Institute of Biochemistry, Rheinisch-Westfälische Technische Hochschule Aachen, 52057 Aachen, Germany, and ¶ Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Chemistry, Molecular Biology Unit, University of Ghent, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
We established a system of receptor chimeras that
enabled us to induce heterodimerization of different cytoplasmic tails. Fusion constructs were created that are composed of the extracellular parts of the interleukin-5 receptor and
chains, respectively, and the transmembrane and intracellular parts of gp130, the signal transducing chain of the interleukin-6 receptor complex. In COS-7 transfectants we observed a dose-dependent
interleukin-5-inducible STAT1 activation for which the presence of both
the
and the
chain chimera was needed. No STAT activity was
detected if one of the cytoplasmic tails of the receptor complex was
deleted, indicating that STAT activity resulted from a receptor dimer
rather than from higher receptor aggregates.
We further investigated whether dimerization of STAT1 depends on the juxtaposition of two STAT recruitment modules in a receptor complex. We show that a receptor dimer with only a single STAT1 docking site was still able to lead to STAT1 activation. This indicates that the formation of a paired set of STAT binding sites in a receptor complex is not the prerequisite for STAT factor dimerization. Our findings are discussed in view of alternative STAT dimerization models.
A detailed analysis of interferon
(IFN)1 signaling events first provided
insight into a general signaling mechanism, the Jak-STAT pathway, by
which many cytokines lead to an altered pattern of gene expression. Jak
(nus
inase) refers to a family of
cytoplasmic tyrosine kinases that comprises four known members in
mammals: Jak1, Jak2, Jak3, and Tyk2 (1). STAT (
ignal
ransducers and
ctivators of
ranscription) refers to a family of transcription factors
with seven known members (2). Cytokine-induced dimerization of receptor
components leads to the activation of Jaks, which are constitutively
associated with the cytoplasmic parts of the respective receptors. One
substrate of the Jaks is the receptor itself. Upon phosphorylating
specific tyrosine residues of the cytoplasmic tail of the receptor STAT
factors and other proteins with "matching" SH2 domains can be
recruited to the receptor where they become activated by tyrosine
phosphorylation. Subsequently, the STATs dissociate from the receptor
and translocate as homo- or heterodimers to the nucleus where they bind
to enhancer elements of target genes and influence transcriptional
activity (2, 3).
The events leading to STAT factor dimerization are not well understood.
Due to receptor dimerization, many cytokines lead to the formation of a
paired set of STAT docking sites, e.g. IFN signals
through a homodimer of STAT1 that binds to
-interferon-activated sequence elements of IFN
-regulated genes (4, 5). A single tyrosine
residue (Tyr-440) in the
chain of the IFN
receptor serves as a
docking site for STAT1 (6-8). Since IFN
receptor
chains
dimerize upon binding of IFN
(9), phosphorylation of Tyr-440 forms
two juxtaposed docking sites for latent STAT1. Dimerization of STAT1
monomers phosphorylated on a specific tyrosine residue (Tyr-701) might
be favored by the presence of another phosphorylated STAT1 monomer in
the near proximity (7). In the present study we tested whether the
formation of a STAT1 homodimer depends on the presence of two STAT1
docking sites in a dimerized receptor. For this reason we established a
system of receptor chimeras based on the extracellular parts of the
human interleukin-5 (IL-5) receptor
and
chains that enabled us
to induce heterodimerization of different cytoplasmic tails. We show
that a receptor dimer with only a single STAT1 docking site is still
able to lead to activated STAT1 dimers, indicating that close
juxtaposition of STAT binding sites in a receptor complex is not the
prerequisite for STAT factor dimerization.
Human IL-5 was expressed in Sf9 insect cells and
purified as described previously (10). 125I-hIL5 was
prepared with the IODOGEN iodination agent (Pierce) as described (11).
The eukaryotic expression vector pSVLgp130 was generously supplied by
Dr. T. Taga (Osaka, Japan). For flow cytometry the monoclonal antibody
16-4 specific for the human IL-5R chain2
and the monoclonal antibody S-16 specific for human
c chain (Santa
Cruz Biotechnology, Santa Cruz, CA) were used. Phycoerythrin-labeled goat anti-mouse Ig (Fab
)2 was obtained from Dianova
(Hamburg, Germany).
For amplification of
the cDNA encoding the extracellular part of the human IL-5R
chain and human
c the primers
ccg
ccaccATGgTCATCGTGGCGCATG (
sense),
cg
ATTTCCCACATAAATAGGTTG (
antisense),
gc
gccaccATGGTGCTGGCCCAGGGGCTG (
sense), and
cg
GGTGTCCCAGGAGCGCGCC (
antisense) were used.
Uppercase letters indicate cDNA sequence; underlined are restriction sites for XhoI, EcoRI, and
XbaI, which were added to facilitate cloning. The IL-5R
chain sense primer was designed to provide a "better" Kozak
consensus sequence. Therefore a point mutation was introduced, leading
to a valine instead of an isoleucine residue at position 2 of the
signal peptide of IL-5R
/gp130. Polymerase chain reactions were
performed with 20 ng of plasmid DNA, 20 pmol of each primer, and 1 unit
of Vent polymerase (New England Biolabs). The polymerase chain reaction
products were digested with XhoI/EcoRI or
XbaI/EcoRI, respectively, and inserted into
pSVLgp130 cut with the same enzymes, thus replacing the cDNA for
the extracellular region of gp130 with the one of the IL-5R
or
chain. The resulting plasmids were named pSVL-IL-5R
/gp130 and
pSVL-IL-5R
/gp130, respectively. Deletion constructs
IL-5R
/gp130
cyt and IL-5R
/gp130
cyt lack the gp130
cytoplasmic region apart from the first three residues (NKR). Due to
the cloning procedure, they contain additionally three vector-encoded
residues (IQT) before termination. The construction of chimeric
molecules Eg (EpoR/gp130), Eg/
B, and Eg/Tyr-
440 has been
described (12). Eg/
B includes only the box 1 and 2 regions of gp130
and therefore lacks the five carboxyl-terminal tyrosine residues.
Eg/Tyr-
440 contains additional 7 amino acid residues from the human
IFN
receptor (including Tyr-440). Eg/
B and Eg/Tyr-
440 end with
a FLAG epitope. From those expression constructs
EcoRI/BamHI fragments were inserted into
EcoRI/BamHI-digested pSVL-IL-5R
/gp130 and
pSVL-IL-5R
/gp130, thereby replacing the intracellular part of
gp130.
Simian kidney cells (COS-7, ATCC CRL 1651) were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Eggenstein, Germany) supplemented with 10% fetal calf serum, 100 mg/liter streptomycin, and 60 mg/liter penicillin. Cells were grown at 37 °C in a water-saturated atmosphere with 5% CO2. 107 COS-7 cells were transiently transfected with 20-30 µg of plasmid DNA by electroporation (Gene Pulser, Bio-Rad). Electroporations were performed at 960 microfarads and 230 V. Cells were harvested 48-72 h post transfection.
Electrophoretic Mobility Shift Assay (EMSA)Nuclear
extracts were prepared as described (13). Protein concentrations were
measured with the Bio-Rad protein assay. A double-stranded mutated SIE
oligonucleotide from the c-fos promoter (m67SIE: 5-GAT CCG
GGA GGG ATT TAC GGG GAA ATG CTG-3
) (14) was labeled by filling in 5
protruding ends with the Klenow enzyme, using
[
-32P]dATP (10 mCi/ml, 3,000 Ci/nmol). Nuclear extract
(2.5 to 5 µg of protein) was incubated with about 10 fmol (5,000 cpm)
of probe in gel shift incubation buffer (10 mM HEPES, pH
7.8, 1 mM EDTA, 5 mM MgCl2, 10%
glycerol, 5 µM dithiothreitol, 0.7 µM
phenylmethylsulfonyl fluoride, 0.1 mg/ml of poly(dI-dC), and 1 mg/ml
bovine serum albumin) for 10 min at room temperature. The protein-DNA
complexes were separated on a 4.5% polyacrylamide gel containing 7.5%
glycerol in 0.25-fold TBE at 20 V/cm for 4 h. Gels were fixed in
10% methanol, 10% acetic acid, and 80% water for 30 min, dried, and
autoradiographed.
COS-7 cells were released from the dishes by
treating them with phosphate-buffered saline, 10 mM EDTA at
37 °C for 10 min. Approximately 106 cells were incubated
with monoclonal antibody 16-4 or S-16 for 30 min, followed by
treatment with the secondary antibody (goat anti-mouse IgG
phycoerythrin-conjugated F(ab)2 fragment, Dianova, Hamburg, Germany). Fluorescence was measured on a FACScan (Becton Dickinson).
Upon transfection approximately
105 COS-7 cells were seeded into 24-well plates and
incubated for 2 days until they reached 80-90% confluence. Cells were
washed twice with cold binding medium (0.2% bovine serum albumin, 20 mM HEPES buffer (pH 7.0) in Dulbecco's modified Eagle's
medium). 200 µl of binding medium with 125I-IL-5 (10 nM and 2-fold dilutions thereof) were added for 2 h on
ice in the presence or absence of 100-fold excess of cold IL-5. Cells
were washed three times with cold phosphate-buffered saline containing
1 mM MgCl2, 0.1 mM
CaCl2, and 0.2% bovine serum albumin. They were lysed in 1 M NaOH overnight before cell-associated radioactivity was
measured in a counter. Specific binding was calculated as the
difference between the binding in the absence (total binding) and
presence (nonspecific binding) of unlabeled IL-5. The dissociation constant (Kd) was calculated by Scatchard analysis
of the binding data.
For the establishment of the
system of heterodimeric receptor chimeras, we first made hybrid
molecules composed of the extracellular parts of the human
interleukin-5 receptor (IL-5R) and
chains, respectively, and
the transmembrane and the intracellular regions of gp130, the signal
transducing receptor chain for IL-6-type cytokines. Upon transfection
of COS-7 cells with expression plasmids for IL-5R
/gp130 and
IL-5R
/gp130, surface expression of the chimeric molecules could be
observed in a fraction of the cells. Mock-transfected COS-7 cells were
not stained with antibodies recognizing human IL-5R
or
chains
(Fig. 1A). Affinity of these chimeric
constructs is similar to that seen with wild type IL-5 receptors
expressed in COS-1 cells (15, 16); IL-5R
/gp130 bound IL-5 with a low affinity (Kd = 1 nM), and coexpression
of IL-5R
/gp130 led to a 2-fold increase of binding (Fig.
1B).
IL-5 induces STAT activation in COS-7 cells
coexpressing IL-5R/gp130 and IL-5R
/gp130 chimeras. A,
surface expression of the chimeric molecules. COS-7 cells cotransfected
with expression plasmids for IL-5R
/gp130 and IL-5R
/gp130 or with
the vector pSVL were incubated with monoclonal antibodies specific for
the IL-5R
or
chain or with medium (= control) and consequently with phycoerythrin-conjugated-anti-mouse Ig F(ab
)2.
Surface staining was measured by flow cytometry. In each panel the
percentage of positive cells is indicated. B, IL-5 binding
to chimeric receptors: affinity conversion upon expression of the
IL-5R
/gp130. Scatchard plot of 125I-IL-5 binding to
COS-7 cells expressing IL-5R
/gp130 alone (
) or in combination
with IL-5R
/gp130 (
). The derived affinities (Kd) are 1 nM (IL-5R
/gp130) and 500 pM (IL-5R
/gp130 + IL-5R
/gp130). C, STAT
activation increases dose-dependently with IL-5
concentration. COS-7cells cotransfected with expression plasmids for
IL-5R
/gp130 and IL-5R
/gp130 were incubated with increasing
amounts of IL-5 for 30 min. STAT activity in nuclear extracts was
measured in an EMSA using the m67SIE probe. The retarded band
corresponds to a homodimer of STAT1 (12). D, coexpression of
the IL-5R
- and the IL-5R
-chimera is needed for an IL-5-inducible STAT activation in COS-7 transfectants. COS-7 cells expressing the
indicated chimeric receptors were stimulated with IL-5 (160 ng/ml),
erythropoietin (Epo, 7 units/ml), or left untreated. STAT activity in
nuclear extracts was measured in an EMSA using the m67SIE probe.
Dimerization of gp130 induced by cytokines (17) or agonistic antibodies
(18) leads to rapid activation of STAT factors in a variety of cell
lines (19, 20). We now tested whether the heterodimeric chimeric
receptors have signaling ability. COS-7 transfectants expressing
IL-5R/gp130 and IL-5R
/gp130 were stimulated (30 min, 37 °C)
with increasing amounts of recombinant human IL-5; nuclear extracts
were prepared and tested in an EMSA. Stimulation with 4 ng of IL-5/ml
already led to a signal, and the intensity of the gel shift bands
increased dose-dependently until at 80 ng/ml a maximum was
reached (Fig. 1C). As shown in Fig. 1D, the shifted band had the same mobility as but a lower intensity than the
one observed upon homodimerization of EpoR/gp130 chimeras which have
previously been shown to lead to STAT1 activation in COS-7-transfectants (12, 21). Importantly, expression of either the
IL-5R
/gp130 or the IL-5R
/gp130 chimera alone did not lead to
IL-5-inducible STAT activation (Fig. 1D). Therefore, both
the
- and the
-chain chimera contribute to the signaling ability of the receptor complex.
The stoichiometry of the components of the IL-5
receptor is not clear (see "Discussion"). The simplest model of a
functional IL-5 receptor system is a dimer comprising one chain and
one
chain, but further receptor chain multimerization cannot be excluded (e.g. Fig. 2A, left
panel, see also Ref. 22). Therefore, we investigated whether the
observed STAT activity might have resulted from receptor aggregates
(see Fig. 2A, right panel).
We generated constructs (IL-5R/gp130
cyt, IL-5R
/gp130
cyt)
with a deletion of the whole cytoplasmic region of gp130 and coexpressed them with the "complementary" full-length constructs (IL-5R
/gp130 or IL-5R
/gp130, respectively). However, no
IL-5-inducible STAT activation was observed (Fig. 2B),
although these cotransfectants bound IL-5 with high affinity (data not
shown). We therefore conclude that the STAT activity observed in COS-7
cells expressing IL-5R
/gp130 and IL-5R
/gp130 is unlikely to
result from higher receptor aggregates. Thus, the IL-5R-based chimeras
are suitable to study signaling of asymmetric receptor dimers.
We have shown previously that a
"tyrosine module" from the interferon receptor mediated a
specific activation of STAT1 when fused to the membrane proximal box
1/2 region of gp130 (12, 21). In that study chimeric receptors with the
extracellular part of the erythropoietin receptor were applied which
homodimerized upon stimulation with erythropoietin.
We then replaced the extracellular region of the erythropoietin
receptor by the IL-5R and
chains resulting in constructs IL-5R
/Tyr-
440 and IL-5R
/Tyr-
440 carrying a 7-amino acid
tyrosine module of the IFN
receptor distal from the box 1/2 region
of gp130. The constructs IL-5R
/
B and IL-5R
/
B contain the
gp130 box 1/2 region but no STAT recruiting tyrosine module. IL-5
stimulation of the two Tyr-
440 constructs led to a strong STAT1
activation (Fig. 3A), whereas no STAT1
activation was observed upon dimerization of the two
B constructs
(Fig. 3D). Importantly, dimerization of a single Tyr-
440
with a
B construct resulted in a clear STAT1 activation (Fig. 3,
B and C). Therefore, a single tyrosine module in
a cytokine receptor complex is sufficient for STAT1 activation. This
observation indicates that close contact of STAT factors by
juxtaposition of STAT binding sites at dimerized receptors is not
necessary for dimerization.
It was our intention to study whether a single STAT module in a
receptor complex is sufficient to achieve STAT activation. For this
purpose, an "asymmetric" receptor complex was created with chimeric
receptors based on the extracellular parts of the IL-5 receptor. The
IL-5 receptor complex is composed of the ligand-binding IL-5R subunit and the common
receptor chain (
c) that is shared with
the
receptors for IL-3 and GM-CSF and functions as an affinity converter (Ref. 15; reviewed in Ref. 23). Members of this receptor
family have already been successfully employed for the construction of
chimeric molecules by others (24-26). In this study we generated
fusion constructs of the extracellular parts of the IL-5R
and
chain, respectively, and the transmembrane and intracellular region of
gp130, the signal transducing chain of the IL-6 receptor. In COS-7
transfectants expressing the IL-5R
/gp130 and the IL-5R
/gp130 chimera, we observed an IL-5-inducible STAT activation (Fig. 1). Although "STAT activation" is certainly only one of several
possible experimental read-outs to characterize the chimeras
functionally, it indicates that they can be used to mimic signaling
events involving gp130 dimers just like chimeric receptors that
homodimerize upon ligand binding (e.g. via the extracellular
region of receptors for erythropoietin, granulocyte colony-stimulating
factor, epidermal growth factor, or neurotrophin-3; Refs. 12, 27, and
28).
Active receptor complexes depend on the presence of both IL-5R and
chimeric molecules. Expression of the IL-5R
/gp130 alone did not
yield an IL-5-inducible signal. This was expected since cross-linking
analyses as well as studies in solution indicated that IL-5, although
itself a homodimer, binds only to one IL-5R
chain (29-32).
Similarly, a chimeric molecule consisting of the extracellular domain
of murine IL-5R
and the transmembrane and intracellular regions of
c could mediate proliferation of transfectants only in the presence
of a functional
c chain (25).
The simplest model of the active IL-5 receptor complex consists of a
heterodimer of one and one
chain, but there are some reports
that members of the IL-3/IL-5/GM-CSF receptor family might undergo
further multimerization. 1) The cytoplasmic tail of the
c chain has
signaling capacity when dimerized upon triggering of chimeric receptor
constructs (25, 33, 34); 2) constitutively active mutants of the
c
chain could be isolated (35-37); 3) it was suggested that a
ligand-bound active receptor complex might normally contain a
c
chain dimer (37); and 4) the occurrence of ligand-independent
c
homodimers has recently been reported (38). In addition, unusual
affinities were observed when GM-CSF receptor
and
subunits were
expressed in COS-1 cells. These data have been discussed in view of
possible complex variability (39).
We therefore expressed IL-5R/gp130 together with an IL-5R
/gp130
construct lacking the whole cytoplasmic region (and vice versa) in COS-7 cells. If multimerization would occur, the
proximity of at least two cytoplasmic tails could be expected to result in the activation of Jaks and STATs. However, since such receptor complexes did not signal, we conclude that, at least under our experimental conditions, the STAT activity we observed upon IL-5 stimulation is very likely to result from an
/
receptor dimer rather than from higher receptor aggregates. We cannot exclude, however, the possibility that potential multimers do not signal due to
an "incorrect" orientation of their cytoplasmic tails.
The measured affinities of the IL-5R/gp130 chimeric receptors are
comparable to those of the wild type IL-5 receptor in COS-1 transfectants (15, 16). However, this affinity is lower than the one
observed on HL-60 eosinophilic cells (11). Maybe additional, so far
unknown receptor components contribute to the high binding affinity in
these cells. A potential ""-subunit of the GM-CSF receptor has
recently been suggested to be present in certain cell types (37). We
cannot rule out the possibility that the lack or the presence of
unknown receptor components in the COS-transfectants might also have
influenced the signals we observed. Additionally, we cannot exclude a
potential cross-talk between different receptor systems (such as
described for the stem cell factor receptor and the erythropoietin
receptor; Ref. 40) that might contribute to the responses we
observed.
The mechanisms leading to STAT activation are not well understood. Although STAT activation independent of receptor tyrosine residues has been described (41, 42), the importance of tyrosine residues within the cytoplasmic tail of cytokine receptors has been demonstrated in many reports (e.g. Refs. 6, 12, 28, and 43-45).
The following models for STAT activation at receptors phosphorylated on
tyrosine residues have been discussed (Fig. 4).
Cytokine receptors, usually containing multiple potential binding sites
for STAT factors, dimerize upon ligand stimulation. After STAT binding
to their receptor docking sites followed by their tyrosine
phosphorylation, the dimerization process might be favored by the
presence of other phosphorylated STAT factors in close proximity. The
"second" STAT monomer of the dimer could be recruited from a
phosphotyrosine residue nearby on the same receptor chain.
Alternatively, it might have been bound to the other chain of the
receptor dimer before (Fig. 4, upper part). Support for this
model comes from the observation that IFN receptor
chains
point-mutated at the tyrosine residue critical for STAT1 binding
(Tyr-440 in the human or Tyr-420 in the murine receptor) act as
dominant-negative mutants when overexpressed in homologous cells (46).
This effect could be explained by a model in which the IFN
-induced
formation of dimerized STAT1 binding sites was the prerequisite for
consequent STAT factor dimerization (see also discussion in Ref.
7).
From our studies, however, we conclude that the presence of dimerized STAT recruitment sites in an active receptor complex is not the prerequisite for STAT activation. We rather show that one "tyrosine module" is sufficient to achieve STAT activation.
This finding can be reconciled with other models for STAT activation.
One possibility would be that the second STAT factor binds to a
phosphorylated STAT monomer already bound to the tyrosine phosphorylated receptor and then becomes phosphorylated (Fig. 4,
middle part). The finding that there is no STAT1
phosphorylation in STAT2-deficient fibrosarcoma cells upon stimulation
with IFN could be explained by this model (47). However, the
formation of the STAT1/2 heterodimer might be a special case, since it
has been demonstrated that STAT1 has a rather high affinity for the phosphorylated peptide of STAT2 (7). According to analogous peptide
binding studies, it seems unlikely that STAT1 is also recruited by a
receptor-associated tyrosine-phosphorylated STAT1 molecule.
It is also possible that phosphorylated STATs dissociate from the receptor, a process that in analogy to the situation of p56lck (48) might be encouraged by alteration of the selectivity of the SH2 domain to target tyrosine phosphopeptides (see discussion in Ref. 49). Phosphorylated monomers would then form dimers in the cytoplasm (Fig. 4, lower part).
A recent study with proteins expressed in insect cells suggested that phosphorylation of the STATs by Jaks might include a transient STAT-Jak association via the STAT-SH2 domain. It was further shown that only one STAT monomer of a dimer needed to be phosphorylated for complex formation in vitro (50) (not included in Fig. 4). It remains to be shown to what degree this reflects signal transduction events in a cytokine-stimulated cell.
Our study demonstrated that ligand-induced STAT1 activation can occur even if a receptor complex contains only a single STAT recruitment module. Based on these results it will be of interest to find out whether there is a general mechanism of STAT activation and to delineate its molecular details.
We thank Dr. Peter Freyer for help in figure preparation, Marcel Robbertz for photographs, and Claude Haan for help in binding data analysis. We thank Boehringer Mannheim for the generous gift of recombinant human erythropoietin.