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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1614-1622
Heterodimerization of the  and  Chains of the Interleukin-3
(IL-3) Receptor Is Necessary and Sufficient for IL-3-Induced
Mitogenesis
By
Paul C. Orban,
Megan K. Levings, and
John W. Schrader
From The Biomedical Research Centre, University of British Columbia,
Vancouver, BC, Canada.
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ABSTRACT |
The high-affinity receptor for interleukin-3 (IL-3) is a complex of
the IL-3-binding subunit ( IL-3) and a larger  chain c, or, in the mouse, c or its
close relative IL-3. There is evidence that the critical
event that initiates signaling is not the approximation of the
cytoplasmic domains of IL-3 and IL-3, but
is, rather, the formation of a - homodimer. Many of these studies
involved the analyses of receptor chimeras where the cytoplasmic
domains were derived from IL-3, c or
IL-3, and the extracellular domains were derived from
other cytokine receptors, such as the erythropoietin receptor (EpoR).
However, evidence that the EpoR may also associate with other receptors
clouds the interpretation of these experiments. Therefore, we
reevaluated the structure of the functional IL-3R using chimeric
receptors with extracellular domains derived not from members of the
cytokine-receptor family, but from CD8 or CD16. We show, by expression
of these chimeras in Ba/F3 or CTLL-2 cells, that mitogenic signals were
only generated by heterodimerization of the cytoplasmic domains of
IL-3 and IL-3. Homodimers of either IL-3 or IL-3, alone or in combination,
were nonfunctional. Furthermore, the ability of heterodimers to
stimulate mitogenesis correlated with their ability to induce tyrosine
phosphorylation of JAK-2. These data suggest that the physiological
activation of the IL-3R involves the generation of simple heterodimers
of IL-3 and IL-3.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE CYTOKINE-RECEPTOR FAMILY can be
divided into those which are homodimers of a single subunit (eg,
receptors for growth hormone and erythropoietin [Epo]), and those
which are heterodimers or higher-order oligomers made up of 2 or more
different subunits (eg, interleukin-4 [IL-4] and
IL-6).1,2 The receptors for IL-3, IL-5, and
granulocyte-macrophage colony-stimulating factor (GM-CSF) belong to the
latter group. Each of these cytokines binds specifically with low or
moderate affinity to a unique  chain termed, respectively, IL-3R,
IL-5R, and GM-CSFR. These complexes of the ligand and its
respective chain each bind with high affinity to a common beta
chain (c), which is required for activation of
intracellular signals.3 In the mouse, while IL-3R, IL-5R, and GM-CSFR and their ligands interact with the murine homolog of c, complexes of IL-3 and IL-3R can also
form a functional IL-3 receptor by interacting with an IL-3-specific
chain (IL-3) that is the product of a relatively
recent duplication of c.4,5
Activation of JAKs follows rapidly after binding of cytokines to their
receptors, and is essential for the initiation of intracellular signaling.6-8 Moreover, in some artificial systems in which
the cytoplasmic domain of a transmembrane protein was replaced by JAK2,
the induction of dimerization of the membrane-associated JAK was
sufficient to deliver a proliferative signal.9,10 These
observations suggest that ligand-induced dimerization or oligomerization of cytokine receptor subunits triggers signaling by
bringing together JAK molecules associated with the receptor subunits,
and allowing trans-phosphorylation and activation of JAKs.11
Experiments on several of the multi-subunit cytokine receptors
have suggested that dimerization of their respective long chains which,
like c, have multiple potential docking sites for
signaling molecules, is sufficient for the initiation of a
proliferative signal. Thus, it has been shown that homodimerization of
gp130,12,13 hc,14-17 the
IL-2R,18 or the IL-4R19,20 are sufficient for the initiation of biochemical and biological events. These observations are consistent with the paradigm of the IL-6R where the
function of the ligand-binding () chain is merely to induce dimerization of the chain, which mediates all the intracellular signaling functions.21 However, because these observations
have all involved overexpression of the receptor chains in cell lines, it is by no means clear that they accurately reflect physiological signaling processes. The situation in normal cells with smaller numbers
of receptors and different levels of signal transduction molecules may
be significantly different. In the case of IL-3 (or the closely related
IL-5 or GM-CSF), it is unclear whether signaling is mediated by simple
heterodimerization of the IL-3 and IL-3
chains, implying an active role for IL-3, or
alternatively, by analogy with the receptor for IL-6, is mediated by
homodimerization of the chain. Truncations of the relatively short
cytoplasmic domains of the IL-3R, IL-5R, and GM-CSFR do not
affect ligand binding, but, in contrast with results with the IL-6R
chain, abolish signaling17,22-24 (and P.C. Orban, K.B.
Leslie, unpublished data). Further, Stomski et
al25 reported finding disulfide-linked heterodimers of
human IL-3 and c after binding of IL-3.
However, there is other evidence that c homodimers might
be both necessary and sufficient for transduction of the mitogenic
signal of IL-3. The c exhibits extensive homology with
the Epo receptor (EpoR), which forms a homodimer in the active
state.26,27 Observations that a chimera composed of the
extracellular and transmembrane portion of the EpoR and the cytoplasmic
portion of the mouse IL-3 chain
(Epo/IL-3) conferred responsiveness to Epo in murine
IL-3-dependent cell lines28-31 have been interpreted as
showing that dimerization of the chain was sufficient for
signaling. Other evidence has come from the demonstration of the
existence of "preformed" dimers of the c in cell
lines32 as well as in primary leukemic cells.33
However, a simple homodimer of the EpoR may not constitute a functional
receptor, and may include additional subunits such as the steel ligand
factor receptor (c-kit) and c.34-37 Indeed, when cells are stimulated with Epo, c undergoes tyrosine
phosphorylation and is coimmunoprecipitated with the
EpoR.34,35 Moreover, when expressed in the T-cell line
CTLL-2, which does not express c, neither EpoR nor a
chimeric receptor consisting of the extracellular and transmembrane
portion of the EpoR together with the cytoplasmic portion of
IL-3 supported ligand-induced
proliferation.16,28,38 In contrast, however, when the
IL-3 and IL-3 were transfected into
CTLL-2 cells, they were capable of transducing a ligand-induced mitogenic signal.
To avoid the confounding possibility that the extracellular component
of a cytokine receptor chimera could qualitatively influence biochemical signaling, perhaps by recruiting additional receptor subunits to the receptor complex,34,36,37,39 we generated chimeras in which the cytoplasmic domains of mouse IL-3
and IL-3 were fused to extracellular domains derived
from proteins that were unrelated to cytokine receptors.
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MATERIALS AND METHODS |
Plasmids.
DNA manipulations were performed by standard methods. The plasmids
pSut1 and pCD226 containing the mouse IL-3 and mouse
IL-3 cDNAs, respectively, were the kind gift of Atsushi
Miyajima (University of Tokyo, Tokyo, Japan). The plasmid pCd16.tm7.syk
was the kind gift of Brian Seed (Massachusetts General Hospital,
Boston, MA), and the plasmid pMV7F1.2, containing the
human CD8 cDNA, was the kind gift of Dan Littman (The Skirball
Institute, New York, NY). The plasmids pXM and pXMR129C,
containing cDNAs encoding the wild-type and Cys-Cys mutated forms of
the mouse EpoR, were the kind gift of Harvey Lodish (The Whitehead
Institute, Cambridge, MA). All chimeric receptors were cloned into the
expression vector pME18 , which was derived from pSut1 by removing
the inserted cDNA. Polymerase-mediated amplification with Vent
polymerase (New England Biolabs, Mississauga, Ontario, Canada) was used
to incorporate restriction enzyme sites for subcloning and shuttling
components of receptor chimeras. The anti-sense oligo GGA ATT CTC AGG
CGT CTG TCA CAG GCG TCA CCTC was used to amplify and add an
EcoRI site to the 3' end of the cytoplasmic domain of
IL-3. Similarly, the antisense oligo GGA ATT CAG ACT CAG
CAC ACC TTC CCA GAC TGG CTAT was used to amplify and introduce an
EcoRI site the 3' end of IL-3, and the
anti-sense oligo TGA ATT CGT CCT AGG AGC AGG CCA CA to amplify and
introduce an EcoRI site to the 3' end of the EpoR.
For construction of the EpoR/IL-3 chimera, the
extracellular domain of the mouse EpoR was amplified using the sense
oligo TGA ATT CAT CAT GGA CAA ACT CAG GGT G and the anti-sense oligo CCC GAT ATC CAG GTC GCT AGC GGT CAG, adding an EcoRV site to
the 3' end. The complementary portion (transmembrane and
cytoplasmic domain) of the IL-3 sequence was amplified
using the sense oligo CCC GAT ATC GAC TGG GTG ATG CCC ACG adding an
EcoRV site, and the antisense oligo as above. After subcloning
and sequencing, chimeric cDNAs were formed by digestion with
EcoRV and ligation of the 2 subclones.
For construction of the IL-3/IL-3
chimera, the extracellular portion of the IL-3 cDNA was
amplified using the sense oligo CCC GAA TTC ATG GCC GCC AAC CTG TGG
CTC, which adds an EcoRI site for subcloning, and the
anti-sense oligo CCC GAT ATC CTT CAC AGG CAT CAC CTC, which adds an
EcoRV site to the 3' end. The amplified fragment was
subcloned, digested with EcoRV, and ligated to the complementary (transmembrane and cytoplasmic) portions of the IL-3 cDNA (see above).
To generate CD8 chimeras, sense oligos were designed to add
EcoRV sites immediately 5' of the transmembrane portion
of the cDNAs of IL-3 (CCC GAT ATC AAG ACA GCC TTG GTG
ACT TCA GTG), and the EpoR (CCC GAT ATC CCT CTC ATC TTG ACG CTG) and
the IL-3 (see above). Amplified fragments were cloned
and sequenced before ligation to the extracellular portion of the
CD8 cDNA, which has an endogenous EcoRV site immediately
5' of the transmembrane domain sequence.
CD16/7 chimeras were generated by replacing the portion encoding syk in
the plasmid pCd16.tm7.syk with the appropriate cytokine receptor
cytoplasmic domain. The IL-3 cytoplasmic domain was amplified using a sense oligo CAC CAT GGG ATC CAC GCG TAA GTC GCT GCT
CTA CCG CCTG to introduce an MluI site for fusion to the CD16/7
extracellular and transmembrane domains. Similarly, the IL-3 cytoplasmic domain was amplified using the sense
oligo GGA ATT CAC GCG TGT TTA TGG GTA CAG GACA to introduce an
MluI site. To create the CD16/7/EpoR chimera a
self-complementary oligo pair with SalI and BglII
compatible ends (sense: TCG ACG CGT TCC CAC CGC CGG ACT CTG CAG CAG AAG
ATC, and anti-sense: GAT CTT CTG CTG CAG AGT CCG GCG GTG GGA ACG CGT
CGA) was introduced into a Bluescript subclone (Stratagene, La Jolla,
CA) containing the cytoplasmic portion of the EpoR, which was digested
with XhoI and BglII, so as to introduce the
MluI site to the 5' end of the EpoR cytoplasmic domains.
Cell culture.
Ba/F3 cells and transfected clones were maintained in RPMI 1640 medium
(Stem Cell Technologies, Vancouver, BC, Canada), supplemented with 10%
fetal calf serum (FCS) (Intergen, Purchase, NY), 50 µmol/L 2-mercaptoethanol, and 2% (vol/vol) 10X concentrated WEHI
3B-conditioned medium as a source of IL-3. CTLL-2 cells and transfected
clones were maintained in Dulbecco's modified Eagle's medium (Stem
Cell Technologies), supplemented with 10% FCS, 50 µmol/L
2-mercaptoethanol, and 3% (vol/vol) IL-2-containing conditioned media
derived from AgX063 cells transfected with the murine IL-2
cDNA.40
Transfections and screening of protein expression.
Ba/F3 and CTLL-2 cells were washed twice in phosphate-buffered saline
(PBS) and 1 × 107 cells were resuspended in 800 µL
of transfection buffer (25 mmol/L HEPES, 0.75 mmol/L
Na2HPO4, 140 mmol/L KCl, 5 mmol/L NaCl, 2 mmol/L MgCl2, 0.5% Ficoll 400 [Pharmacia Biotech,
Uppsala, Sweden]). For each transfection, cells were mixed with 1 µg
of linearized pPGKNeo or pPGKPuro together with 15 µg of the
linearized expression vector of interest, and subjected to
electroporation using a Bio-Rad gene pulser (Bio-Rad, Mississauga,
Ontario, Canada) at 960 µF and 270 V. After transfection, the cells
were cultured in the appropriate media for 48 hours and then
transferred to selection media in 96-well plates. Individual clones of
neomycin- or puromycin-resistant clones were tested for expression of
the appropriate receptor chain by flow cytometry. Either OKT8 (American
Type Culture Collection [ATCC], Rockville, MD) or Leu2A (Becton
Dickinson, Mississauga, Ontario, Canada) were used to detect
cell-surface expression of human CD8. 3G8 (Cedar Lane, Hornby,
Ontario, Canada) was used to detect cell-surface expression of human
CD16. 9D3 (Alice Mui, DNAX, Palo Alto, CA) was used to
detect expression of IL-3. Rabbit polyclonal antiserum
raised against the extracellular domain of IL-3R was used to detect
expression of the IL-3R.41
Cell proliferation assays.
Proliferation of cells was assessed by either
[3H]-thymidine incorporation into de novo synthesized
DNA, or 3-[Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) incorporation. Cells were washed 3 times with HEPES-buffered saline solution (HBSS) supplemented with 2% (vol/vol) FCS. Cells were
plated at 1,000 cells/well in a Terasaki microtiter plate (Disposable
Products, Adelaide, SA, Australia) for [3H]-thymidine
incorporation or at 10,000 cells/well in a 96-well plate for MTT
assays. Chemically synthesized IL-3 (Ian Clark-Lewis, The Biomedical
Research Centre, Vancouver, BC, Canada) was used to assess maximal
proliferation of Ba/F3 cells. Recombinant murine IL-2 (Genzyme,
Cambridge, MA) was used to assess maximal proliferation of CTLL-2
cells. Recombinant human Epo was a kind gift from Dr Ross Hardison
(Pennsylvania State University, State College, PA). For
use in bioassays, the 3G8 (anti-CD16) antibodies were dialyzed against
RPMI to remove azide and were added in serial dilutions to cells. Cells
were incubated at 37°C for 40 hours, and then pulsed for a further
8 hours with either [3H]-thymidine at a final
concentration of 15 µCi/mL, or 0.75 µg/mL MTT (Sigma, Oakville,
Ontario, Canada). In the [3H]-thymidine uptake assays,
cells were harvested onto filters and counted in a scintillation
counter. In MTT assays, the reaction product was solubilized by
addition of 100 µL of 10% sodium dodecyl sulfate (SDS), 50%
dimethyl formamide (DMF), pH 4.5, and the plates were read at
OD550 using a BioTek plate reader (BioTek, Winooski, VT).
Cell-surface biotinylation.
Cells were washed twice in HBSS and resuspended at 2 × 107 cells/mL in HBSS supplemented with 0.8 mg/mL
Sulfo-NHS-Biotin (Pierce, Rockford, IL). After 15 minutes on ice, the
cells were washed 3 times with 50 mL of HBSS supplemented with 10% FCS
to quench the biotinylation reaction, followed by 1 wash in HBSS. The
cells were lysed in lysis buffer (20 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1% Nonidet P-40, 2 mmol/L EDTA, 1 µmol/L phenylmethylsulfonyl fluoride, 2 µg/mL leupeptin, 0.7 µg/mL pepstatin, 10 µg/mL
aprotinin, 10 µg/mL soybean trypsin inhibitor). Before specific
immunoprecipitation, lysates were "precleared" by absorption with
protein A-Sepharose (Pharmacia, Uppsala, Sweden). Chimeric receptors
with the extracellular domain of human CD8 were immunoprecipitated
with OKT8, followed by adsorption onto protein A-Sepharose. Beads were
washed 3 times with lysis buffer, boiled in SDS sample buffer, and the
eluate was subjected to SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) and immunoblotting. The surface-biotinylated material
precipitated with anti-CD8 was detected by immunoblotting with
streptavidin-conjugated horseradish peroxidase (HRP;
Calbiochem, La Jolla, CA).
Cell stimulations.
To analyze the biochemical effects of using anti-CD16 antibodies to
generate homodimers or heterodimers of the cytoplasmic domains of the
IL-3R or , the respective transfectants were placed in RPMI, 10%
FCS, 0.2% WEHI-3B for 16 hours, washed 3 times with serum-free RPMI
supplemented with 20 mmol/L HEPES (SFM), and incubated in SFM at 1 × 107 cells/mL at 37°C for 1 hour. The cells were
then left untreated as a control or stimulated with synthetic IL-3 (10 µg/mL) for 10 minutes, or anti-CD16 as follows. The monoclonal
anti-CD16 (3G8, 1 µg/mL) was allowed to bind to the cells on ice for
15 minutes, after which cells were washed once with SFM, and
resuspended in 1 mL of SFM containing 1 µg/mL secondary goat
-mouse Ig (DAKO A/S, Glostrup, Denmark). The cells
were then transferred to 37°C for 10 minutes and lysed in lysis
buffer supplemented with phosphatase inhibitors (1 mmol/L sodium
orthovanadate, 1 mmol/L sodium molybdate, 10 mmol/L sodium fluoride).
Lysates were then incubated with antisera against JAK-2 (Upstate
Biotechnology Inc, Lake Placid, NY), followed by adsorption onto
protein A-Sepharose. Beads were washed and boiled as above, and the
eluates were subjected to SDS-PAGE and immunoblotting with 4G10
(Upstate Biotechnology Inc) to detect tyrosine-phosphorylated JAK-2
and, after stripping, with antisera to JAK-2 to assess equivalency of loading.
| |
RESULTS |
Epo-mediated homodimerization of IL-3 leads
to mitogenesis in Ba/F3 cells.
Given that the discrepancies in the literature about the function of
homodimers of the cytoplasmic domain of IL-3 may relate to differences between clones of cell lines, we first repeated the
experiments of expressing full-length EpoR or the
EpoR/IL-3 chimera using our line of IL-3-dependent
Ba/F3 cells and IL-2-dependent CTLL-2 cells (see
Fig 1 for schematic diagrams of all
chimeric receptors used in this work). Like others28,31 we
observed that the EpoR/IL-3 chimera was capable of
transducing a proliferative response to Epo in Ba/F3 cells
(Fig 2). We noted, however, that untransfected Ba/F3 cells displayed some small but reproducible response to higher concentrations of Epo, consistent with the observations of Damen et al,42 and suggesting that Ba/F3
cells express some endogenous EpoR. Expression of the R129C mutant of the EpoR in Ba/F3 cells, which is expressed as a homodimer by virtue of
a Cys-Cys bond in the extracellular region,26 also conferred factor independence to BaF/3 cells (data not shown), as
reported by others.16,26 In contrast, when these various constructs were expressed in IL-2-dependent CTLL-2 cells, which do not
express any of the receptor chains known to be involved in Epo or IL-3
signaling, we failed to obtain clones that responded to Epo; nor, in
the case of the R129C mutant, did we obtain factor-independent CTLL-2
transfectants (data not shown). These differences between Ba/F3 and
CTLL-2 cells are consistent with previous
observations.16,28,38
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| Fig 1.
Schematic representation of the chimeric receptors used
in this study. (A) Receptors that are inducibly dimerized after
addition of a cytokine. The transmembrane (TM) segment is derived from
the same cytokine receptor as the cytoplasmic domain in each case. (B)
Receptors that exist as constitutive dimers by virtue of the hCD8
extracellular domain, which forms a disulfide-linked dimer. The
transmembrane portion is derived from the same cytokine receptor as the
cytoplasmic domain in each case. (C) Receptors that are inducibly
dimerized after addition of an antibody against CD16. The transmembrane
portion is derived from CD7.
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| Fig 2.
Ba/F3 cells transfected with the full-length EpoR or the
EpoR/IL3 chimera proliferate in response to Epo. Cells
were washed free of IL-3 and incubated with indicated concentrations of
Epo. After 40 hours, the cells were pulsed with MTT for a further 4 hours, and MTT reduction was measured as optical density at 550 nmol/L
(OD550) of solubilized cells. Data are plotted as the
percentages of the maximal response of cells cultured in IL-3. Error
bars represent SEM of triplicate samples. Similar results were obtained
with several individual clones.
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Homodimers of IL-3 fail to stimulate
mitogenesis in CTLL-2 cells.
One approach to determining whether the generation of simple dimers of
the IL-3 is sufficient to generate signals is to
investigate the activity of dimers of the cytoplasmic portion of
IL-3 formed when IL-3 brings together
IL-3 and a chimera of the extracellular portion of
IL-3 and the cytoplasmic domain of IL-3.
Clones of CTLL-2 cells were derived that expressed either full-length
IL-3 alone, full-length IL-3 alone, both
full-length chains together, or the full-length IL-3 and
the chimeric IL-3/IL-3 together. Expression of the exogenous receptor chains was assessed by flow cytometry (Fig 3). As shown in
Table 1, CTLL-2 clones that expressed both
full-length IL-3 and IL-3 proliferated in
response to IL-3, whereas clones that expressed either chain alone, the
IL-3/IL-3 chimera alone, or the
full-length IL-3 together with the
IL-3/IL-3 chimera, showed no growth in
response to IL-3. These data suggested that simple dimers of the
cytoplasmic domain of IL-3 were not sufficient for the
generation of mitogenic signals, at least in these cells.
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| Fig 3.
Flow cytometric analyses of expression of chimeric
receptors. CTLL-2 cells expressing both IL-3 and
IL-3/IL-3 (top), or IL-3
and IL-3 (bottom) were stained with antibodies against
the extracellular domain of IL-3 or IL-3.
Thin lines represent results with cells treated with fluorescein
isothiocyanate (FITC)-coupled secondary antibody alone; thick lines
represent results with cells incubated with anti-receptor antibodies
and then FITC-coupled secondary antibody. CTLL-2 cells bearing
IL-3 alone or IL-3 alone displayed levels
of expression similar to those shown for CTLL IL-3 + IL-3.
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Constitutive heterodimers of IL-3 and
IL-3, but not homodimers of
IL-3, stimulate mitogenesis in Ba/F3 and
CTLL-2 cells.
Given that the use of extracellular domains of growth factor receptors
in such chimeric proteins might be difficult to interpret because of
the possibility of the recruitment of cytokine-receptor family
subunits,34,36,37,39,43 we next generated chimeras in which
the extracellular regions of an unrelated cell-surface molecule, human
CD8,44,45 were fused with the transmembrane and
cytoplasmic portions of the respective IL-3 and Epo receptor chains
(see Fig 1). Human CD8 has been shown to spontaneously form
disulfide-linked dimers at the cell surface.45 Chimeric receptor chainsCD8/EpoR, CD8/IL-3, and
CD8/IL-3were each expressed in Ba/F3 cells, as
confirmed by flow cytometric analysis (Fig 4A). In addition, we derived Ba/F3 clones that coexpressed both the
CD8/IL-3 and CD8/IL-3 chimeras. To
confirm expression of both of these chimeras in the same clone, we
biotinylated the cell surface, lysed the cells, and immunoprecipitated
the chimeric proteins with an antibody against CD8. The presence of the
biotinylated chimeras in the anti-CD8 immunoprecipitates was assessed
by SDS-PAGE and blotting with streptavidin-conjugated peroxidase. As
shown in Fig 4B, biotinylated molecules corresponding to the predicted sizes of both chimeras were present.
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| Fig 4.
Heterodimerization of the cytoplasmic domain of
IL-3 and IL-3 is required to induce
mitogenesis in Ba/F3 cells. (A) Flow cytometric analyses confirming
expression of the CD8/IL-3R chimeric receptors in Ba/F3 cells. Thin
lines represent controls treated with FITC-coupled secondary antibody
alone; thick lines represent results with cells stained with CD8 and
FITC-coupled secondary antibody. The bottom righthand panel represents
the CD8 signal in cells expressing both CD8/IL-3 and
CD8/IL-3. (B) Immunoprecipitation of biotinylated
cell-surface molecules to confirm the expression of
CD8/IL-3 and CD8/IL-3 in doubly
transfected cells. Lane 1, control Ba/F3; lane 2, Ba/F3 transfected
with CD8/IL-3; lane 3, Ba/F3 transfected with
CD8/IL-3; lane 4, Ba/F3 transfected with
CD8/IL-3 and CD8/IL-3. (C)
[3H]-thymidine incorporation by transfectants incubated
in the absence of IL-3. Cells were cultured with or without IL-3 for 40 hours and then incubated for an additional 8 hours in the presence of
[3H]-thymidine. Data are plotted as the percent of
maximal incorporation observed in IL-3. Error bars represent the SEM of
triplicate samples.
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Ba/F3 cells expressing the CD8/EpoR chimera grew independently of
exogenous IL-3 or Epo (Fig 4C). This was despite the low levels of
expression of the chimeric CD8/EpoR protein as judged by our inability
to detect its expression by flow cytometry (Fig 4A). This observation
suggests that the CD8 domains formed disulfide-linked dimers as
predicted, and that the generation of simple dimers of the EpoR
cytoplasmic domain in these cells was sufficient for their
proliferation and survival. In contrast, neither the Ba/F3 cells
expressing CD8/IL-3 alone, nor those expressing
CD8/IL-3 alone, were capable of factor-independent
growth, though these clones continued to respond normally to IL-3. On
the other hand, all clones that expressed both the
CD8/IL-3 and CD8/IL-3 chimeras showed
factor-independent growth. Clones expressing both chimeric receptors
were obtained by transfecting a series of clones that expressed one
chimeric chain, and were thus not factor-independent with the second
CD8 chimera. Other clones were generated by cotransfection of parental
cells with both chimeric chains simultaneously. All clones expressing
one CD8 chimera could be rendered factor-independent by expression of
the alternate CD8 chimera. This result also showed that, while the
clones expressing the individual CD8 chimeras remained
factor-dependent, they were nonetheless fully capable of
factor-independent growth when transfected with both chimeras.
These experiments with CD8/IL-3 and
CD8/IL-3 chimeras were repeated in CTLL-2 cells with
identical results (Fig 5A through C). These
data clearly indicate that simple dimerization of IL-3, at least when expressed at the levels we observed, is not sufficient to
promote survival or mitogenesis in either cell line. In contrast, both
cell types reproducibly exhibited factor-independent growth when the
cytoplasmic domains of IL-3 and IL-3 were
heterodimerized. These results suggest that heterodimers of
IL3 and IL3 cytoplasmic domains are
significantly more efficient in initiating signaling than dimers of
IL-3.
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| Fig 5.
Heterodimerization of the cytoplasmic domain of
IL3 and IL3 induces mitogenesis in CTLL-2
cells. (A) Flow cytometric analysis confirming expression of the
hCD8 chimeric receptors. Thin lines represent results with cells
treated with FITC-coupled secondary antibody alone; thick lines
represent results with cells treated with CD8 and FITC-coupled
secondary antibody. (B) Immunoprecipitation of surface biotinylated
proteins confirming expression of both CD8/ and CD8/ in doubly
transfected cells as described in the legend to Fig 4B. Lane 1, parental CTLL-2; lane 2, CTLL-2 transfected with
CD8/IL-3; lane 3, CTLL-2 transfected with
CD8/IL-3; lane 4, CTLL-2 transfected with
CD8/IL-3 and CD8/IL-3. (C)
[3H]-thymidine incorporation of transfectants in the
absence of IL-2. Cells were cultured with or without IL-2 for 40 hours
and then incubated for an additional 8 hours in the presence of
[3H]-thymidine. Data are plotted as the percent of
maximal incorporation observed in IL-2. Error bars represent the SEM of
triplicate samples.
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Inducible heterodimers of IL-3 and
IL-3, but not homodimers of
IL-3, stimulate mitogenesis in Ba/F3 cells.
These data showed a requirement for heterodimerization of the
cytoplasmic domains of IL-3 and IL-3 and
contradicted a body of evidence suggesting that the dimerization of the
cytoplasmic domain of IL-3 is sufficient for mitogenic
signaling.28-31 Therefore, we investigated this question
using another system that involved the use of chimeras consisting of
the extracellular portion of the human CD16 molecule, the transmembrane
segment of human CD7, and the cytoplasmic portions of either the EpoR,
IL-3, or IL-3. In this system, the
chimeric proteins are expressed as monomers but dimerization can be
induced using a monoclonal antibody (MoAb) (3G8) specific for the
extracellular domain of CD16.10,46 These chimeras were
expressed alone or together in Ba/F3 cells, and expression was
confirmed by flow cytometry (Fig 6A).
Proliferative responsiveness to the MoAb to hCD16 (3G8) was assessed by
[3H]-thymidine incorporation (Fig 6C and D). Cells
expressing only CD16/7/IL3 or only
CD16/7/IL3 failed to respond to any concentration of the
dimerizing antibody. However, in keeping with the results obtained with
the CD8 chimeras, those cells that expressed both CD16/7/IL-3 and CD16/7/IL-3 exhibited
proliferative responses to the dimerizing antibody.
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| Fig 6.
Induction of heterodimerization of the cytoplasmic
domains of IL3 and IL3 is mitogenic in
Ba/F3 cells. Flow cytometric analysis confirming expression of (A) the
CD16/7 chimeric receptors. The bottom righthand panel represents the
CD16 signal obtained in cells expressing both CD16/7/ and
CD16/7/. (B) The expression of CD16/7/ and CD8/.
Thin lines represent results with cells treated with FITC-coupled
secondary antibody alone; thick lines represent results with cells
treated with CD16 and FITC-coupled secondary antibody. (C and D)
[3H]-thymidine incorporation of Ba/F3 cells transfected
with the indicated CD16/7 chimeras in response to addition of the
CD16 antibody, 3G8. Cells were cultured with increasing amounts of
3G8 for 40 hours and then incubated for an additional 8 hours in the
presence of [3H]-thymidine. Data are plotted as the
percent maximal [3H]-thymidine incorporation observed in
IL-3.
|
|
Coincident formation of homodimers of cytoplasmic domains of
IL-3 and IL-3
is not mitogenic.
It was conceivable that the mitogenic responses to the anti-CD16
antibody, observed in cells expressing both CD8/IL-3 and CD8/IL-3, reflected a requirement for signals generated
by the coincident formation of independent homodimers of
IL-3 or IL-3 cytoplasmic domains. To
investigate this possibility, we derived clones of Ba/F3 cells
expressing both CD8/IL-3 and CD16/7/IL-3. Expression of both receptor chimeras was confirmed by flow cytometry (Fig 6B). In these cells, CD8/IL-3 was constitutively
dimerized and anti-CD16 antibodies were used to generate homodimers of
the cytoplasmic domain of IL-3. These clones were
derived from the same CD8/IL-3-expressing clones that
generated factor-independent subclones when CD8/IL-3 was
also expressed (Fig 4C). However, addition of anti-CD16 did not induce
proliferation at any dose tested (Fig 6D).
Mitogenic effects of heterodimers of the cytoplasmic domains of
IL-3 and IL-3
correlate with tyrosine phosphorylation of JAK-2.
Transmission of a proliferative signal through the IL-3 receptor is
known to involve the tyrosine phosphorylation and activation of
JAK-2.6,11 Therefore, we assessed phosphorylation of JAK-2 after antibody-induced dimerization of chimeric receptors in Ba/F3 cells expressing various combinations of CD16/7 chimeras. Clones expressing a single CD16/7 chimera (IL-3 or
IL-3) were transfected with the alternate CD16/7
chimeras. Dimerization of CD16/7/IL-3 alone or
CD16/7/IL-3 alone failed to stimulate phosphorylation of
JAK-2 (Fig 7). However, when dimerization
of CD16 extracellular domains was induced in cells expressing both
CD16/7/IL-3 and CD16/7/IL-3,
phosphorylation of JAK-2 was clearly evident (Fig 7). These data
indicated that homodimerization of the cytoplasmic domains of either
IL-3 or IL-3 alone was ineffective in
activating JAK-2, and that only the generation of heterodimers of the
cytoplasmic domains of IL-3 and IL-3
resulted in tyrosine phosphorylation of JAK-2. Furthermore, activation
of JAK-2 in cells expressing the various chimeras correlated with their
ability to proliferate.
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| Fig 7.
Antibody-induced heterodimerization of the cytoplasmic
domains of IL-3 and IL-3 induces tyrosine
phosphorylation of JAK-2. Factor- and serum-starved cells were
incubated on ice with anti-CD16 antibodies for 10 minutes (16).
Cells were washed once with RPMI, and after the addition of secondary
goatmouse, the cells were stimulated at 37°C for
10 minutes. Alternatively, the cells were stimulated with IL-3 for 10 minutes or left unstimulated as a control ( ). Cell lysates were
subjected to immunoprecipitation (IP) with antibodies against JAK-2,
and the eluates were resolved by SDS-PAGE. The membranes were
immunoblotted (IB) with 4G10 (PY) to detect phosphorylation on
tyrosine.
|
|
| |
DISCUSSION |
We have shown that the transduction of mitogenic signals from the
murine IL-3 receptor requires the heterodimerization of the cytoplasmic
domains of the 2 receptor components IL-3 and IL-3. Our experiments were performed in murine cells
well characterized with respect to endogenous cytokine receptor
expression, and used murine cytokine receptor components to minimize
the likelihood of confounding molecular interactions. We obtained
qualitatively identical results using two different means of generating
dimers of the IL-3 and IL-3 cytoplasmic
domains (Figs 4 and 6). In the case of CD8, dimerization was achieved
via a single constitutive disulfide bond and, in the case of CD16,
through addition of a specific MoAb. The use in these chimeras of the
extracellular domains of CD8 or CD16, rather than those of a cytokine
receptor family member such as the EpoR, obviated the possibility of
recruitment of additional cytokine receptor subunits, which clouded the
interpretation of previously reported experiments with chimeric
receptors in which the extracellular domains were derived from subunits
of cytokine receptors. Significantly, the same results were obtained from experiments performed in IL-3-dependent Ba/F3 cells and in IL-2-dependent CTLL-2 cells (Figs 4 and 5).
We could find no circumstance in which homodimerization of either the
cytoplasmic domains of IL-3 or IL-3
resulted in growth. Thus, neither constitutive dimerization of the
IL-3 cytoplasmic region in a CD8/IL-3
chimera nor the anti-CD16-induced dimerization of
CD16/7/IL-3 supported proliferation of Ba/F3 or CTLL-2
cells (Figs 4C, 5C, and 6D). All of these cells expressed chimeras that were potentially functional, because derivatives of these clones transfected with respective CD8/IL-3 or
CD16/7/IL-3 chimeras proliferated spontaneously or in
response to anti-CD16 antibody. Likewise, in CTLL-2 cells, the
generation of IL-3 cytoplasmic domain homodimers by
addition of IL-3 to cells expressing IL-3 and an
IL-3/IL-3 chimera did not result in
IL-3-dependent proliferation (Table 1), whereas all clones we obtained
that expressed both IL-3 and full-length
IL-3 proliferated in the presence of IL-3. In all cases,
heterodimerization of the IL-3 and IL-3
cytoplasmic domains resulted in proliferation (Figs 4C, 5C, and 6D).
Moreover, the simultaneous presence in the same cells of homodimers of
the IL-3and IL-3 cytoplasmic domains was
insufficient for mitogenesis (Fig 6D).
Our findings that coexpression of CD8/IL-3 and
CD8/IL-3 results in mitogenesis suggest not only that
dimerization of the and cytoplasmic domains is required, but
also that simple heterodimers rather than higher-order oligomeric
complexes may be sufficient. Furthermore, dimerization of CD16 receptor
chimeras was induced by addition of an MoAb that can only bind to 1 epitope on each CD16 monomer. Thus, each divalent antibody can only
link 2 CD16/cytoplasmic domain receptor chimeras. Our data suggest that
all that is required of the extracellular domains for mitogenic signaling is that they generate a simple heterodimer. However, we
cannot exclude the possibility that the cytoplasmic domains of and
heterodimers drive the formation of higher-order multimers.
Importantly, we found that the ability of the various clones to grow
correlated with the tyrosine phosphorylation of JAK-2, the key step in
the biochemical events that lead from cytokine receptor activation to
proliferative responses.6,7,47 Thus, cell lines expressing
either 16/7/IL-3 or 16/7/IL-3 showed no detectable JAK-2 phosphorylation when antibody-mediated
homodimerization was induced by anti-CD16. In contrast, when the
complementary CD16/7 chimera was also expressed in these lines,
addition of anti-CD16 resulted in phosphorylation of JAK-2 (Fig 7).
Our data on the failure of homodimerized IL-3 to result
in growth (Table 1, Figs 4C, 5C, and 6D) or biochemical changes associated with growth (Fig 7) contrast with a series of studies reporting that the induction of homodimerization of the cytoplasmic domain of c or IL-3 resulted in
proliferation.15,16,28 However, in all these cases the
extracellular domains of the chimeric receptors were derived from
cytokine receptors. Our results and those of others35 show
that the EpoR, or chimeras that include the extracellular domain of
EpoR, and the cytoplasmic domain of c or
IL-3 function in Ba/F3 cells (Fig 2) but not in CTLL-2
cells (data not shown). Together, these data indicate a requirement for
an additional molecule, absent in CTLL-2 cells, for the function of the
EpoR. Thus, in Ba/F3 cells, we observed that the
EpoR/IL-3 chimera gave a mitogenic signal in the
presence of Epo, or when the chimera was constitutively dimerized by
virtue of an EpoR point mutation (Fig 2 and data not
shown).28,31 However, in CTLL-2 cells neither chimera was
functional (data not shown), suggesting that Ba/F3 cells express an
additional protein that was required for the function of chimeras with
EpoR extracellular domains and is not present in CTLL-2 cells.
Therefore, our results support the notion that chimeras of the
cytoplasmic domain of IL-3 and the extracellular portion
of the EpoR are functional only if additional membrane-associated molecules are recruited. As discussed, there is evidence that the
activated EpoR interacts functionally with other receptors not present
in CTLL-2 cells, including c-kit and c.34-37
There is one report of homodimerization of c leading to
mitogenesis in the likely absence of the recruitment of other subunits of the cytokine receptor family. Patel et al48 generated
chimeras of the cytoplasmic domains of c or GM-CSFR
using as the extracellular domains, the leucine-zipper portions from
c-fos and c-jun. In keeping with our observations, they showed that
heterodimers of the GM-CSFR and c cytoplasmic domains
delivered the strongest mitogenic signal. However, in contrast to our
results, they also observed a weak mitogenic signal from homodimers of
the chain, and a still weaker, but detectable, signal from chain homodimers.
What, aside from recruitment of other cytokine receptor subunits, might
explain the differences between our data, which imply that heterodimers
of the cytoplasmic domains of IL-3 and
IL-3 are necessary for transduction of proliferative
signals, and those that indicate that homodimers of c,
IL-3,28-31 and even
IL-3,48 can generate signals? The most
parsimonious explanation would be that at sufficient levels of
expression, in cells that also express at sufficient levels key
intracellular signaling proteins, homodimers might generate mitogenic
signals, but at lower levels of efficiency than / heterodimers.
In the simplest model, JAK-2 is associated to the same degree with both
IL-3 and IL-3, and homodimerization of
either IL-3 or IL-3 could then lead to
apposition and trans-activation of JAK-2. The generation of
mitogenic signals from the homodimers might depend on unphysiologically
high levels of expression present in transfected cell lines, and at
physiological levels of expression, only the generation of simple
heterodimers of IL-3 and IL-3 activate
signal transduction. It could additionally be postulated that steric
considerations, or differences in the affinity of JAK-2 for the
different dimers could mean that the relative efficiency of JAK-2
activation decreases in the hierarchy of
IL-3IL-3 >>
IL-3IL-3 > IL-3IL-3.
Several lines of evidence support an active role for the cytoplasmic
domain of IL-3 in signal transduction. First, deletion or mutation of the short cytoplasmic tails of the IL-3R, IL-5R, and
GM-CSFR chains abrogates signal transduction triggered by the
receptor ligand17,22-24,49 (and P.C. Orban, K.B. Leslie, unpublished data). In support of a direct role of these
subunits in recruitment and activation of JAK-2, Ogata
et al50 have shown that JAK-2 can bind to IL-5R.
Likewise, we have shown that the cytoplasmic domain of IL-5R binds
directly to JAK-2 (P. Orchansky, J.W. Schrader, manuscript in
preparation). Together with evidence that
c also binds JAK-2,51 these data fit well
with the model that both IL-3 and
IL-3 contribute to recruitment of 2 molecules of JAK-2
to a heterodimeric complex. The notion of a hierarchy of efficiency of
activation of JAK-2 of IL-3IL-3 >>
IL-3IL-3 > IL-3IL-3 correlates well with the elegant
experiments of Patel et al48 with overexpressed proteins.
We contend, however, that at the levels of receptor expression found in
primary cells, typically of the order of a few hundred high-affinity
receptors per cell, only IL-3IL-3
heterodimers generated by the presence of physiological ligand would
deliver sufficient signal to trigger mitogenesis.
| |
ACKNOWLEDGMENT |
We thank Sheila Ayres for technical assistance. We thank Harvey Lodish
for the EpoR cDNA, Atsushi Miyajima for the IL-3R and
IL-3 cDNAs, Dan Littman for the CD8 cDNA, Brian Seed
for the CD16/7 cDNA, Alice Mui for 9D3, and Ross Hardison for Epo.
| |
FOOTNOTES |
Submitted November 17, 1998; accepted May 4, 1999.
P.C.O. and M.K.L. contributed equally to this work.
Supported by the Medical Research Council of Canada and the Protein
Engineering Network of Centres of Excellence.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to John W. Schrader, MB, PhD, The
Biomedical Research Centre, 2222 Health Sciences Mall, University
of British Columbia, Vancouver, BC V6T 1Z3, Canada; e-mail:
john{at}brc.ubc.ca.
| |
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[Abstract]
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A. E. Frankel, J. Ramage, M. Kiser, R. Alexander, G. Kucera, and M. S. Miller
Characterization of diphtheria fusion proteins targeted to the human interleukin-3 receptor
Protein Eng. Des. Sel.,
August 1, 2000;
13(8):
575 - 581.
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ABSTRACT
INTRODUCTION