From the Department of Immunology, M. D. Anderson
Cancer Center, Houston, Texas 77030, the ¶ Gladstone
Institute of Virology and Immunology, San Francisco, California
94141-9100 and the Department of Medicine, School of Medicine,
University of California, San Francisco, California 94143, and the
Departments of Medicine and Cell Biology, Washington University
School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Signal transduction by the erythropoietin
receptor (EPOR) is activated by ligand-mediated receptor
homodimerization. However, the relationship between extracellular and
intracellular domain oligomerization remains poorly understood. To
assess the requirements for dimerization of receptor cytoplasmic
sequences for signaling, we overexpressed mutant EPORs in combination
with wild-type (WT) EPOR to drive formation of heterodimeric
(i.e. WT-mutant) receptor complexes. Dimerization of the
membrane-proximal portion of the EPOR cytoplasmic region was found to
be critical for the initiation of mitogenic signaling. However,
dimerization of the entire EPOR cytoplasmic region was not required. To
examine this process more closely, we generated chimeras between the
intracellular and transmembrane portions of the EPOR and the
extracellular domains of the interleukin-2 receptor Erythropoietin (EPO)1 is
essential for the survival, proliferation, and terminal differentiation
of erythroid progenitor cells (1-3). The erythropoietin receptor
(EPOR) belongs to the cytokine receptor superfamily and as such
contains the structural motifs of four cysteine residues with canonical
spacing and the sequence WSXWS in the extracellular domain
(4, 5). Although protein tyrosine phosphorylation is required for
cytokine signaling (6), the cytoplasmic regions of these receptors lack
intrinsic enzymatic activity. Signal transduction instead is mediated
by cytoplasmic protein tyrosine kinases of the JAK family (7). These
kinases are constitutively associated with receptor chains; residues in the membrane-proximal region of the EPOR cytoplasmic tail are required
for this association. Ligand binding results in homodimerization of
EPOR chains, which is critical for the initiation of intracellular signaling (8-12).
The role of homodimerization in the initiation of signaling is
highlighted by constitutively active variants of the EPOR chain (10,
13). In the three mutants described to date, R129C, E132C, and E133C,
amino acids in the extracellular domain are replaced by cysteines,
leading to the formation of intermolecular disulfide bridges and
constitutive signaling. In contrast, a number of other cytokine
receptors signal through the heterodimerization of asymmetric receptor
chains. For example, interleukin-2 (IL-2) binds and heterodimerizes the
IL-2R One of the earliest detectable signaling events induced by the EPOR is
the phosphorylation of cellular proteins on tyrosines. This
phosphorylation is thought to be mediated largely by JAK2, although
other protein tyrosine kinases may also be involved (16). Eight
tyrosine residues are present in the membrane-distal portion of the
EPOR cytoplasmic tail, and several of these have been identified as
docking sites for signaling molecules with phosphotyrosine binding
motifs, including STAT5, phosphatidylinositol 3-kinase p85 subunit, and
SHP-1 (17-21). Recruitment of these molecules to the EPOR/JAK2 complex
leads to the activation of several signaling cascades, most likely
through a change in subcellular localization, tyrosine phosphorylation,
and/or oligomerization. In addition, tyrosine-phosphorylated JAK2 may
interact directly with certain signaling molecules, including the
recently described CIS/JAB/SOCS family of proteins which may be
important for the down-regulation of signaling (22-26).
Although it is clear that ligand-induced dimerization of the EPOR
extracellular region is required to initiate signal transduction (9-12, 27), the functional requirements for signaling with regard to
the oligomerization of cytoplasmic tails are unknown. To address this
question, we coexpressed wild-type (WT) and mutant EPORs in the 32D
cell line. We selected cell lines in which the mutant receptors were
expressed at 10-fold higher levels than WT receptors to favor the
formation of mutant-WT heterodimers for further analysis (10). This
strategy allowed us to examine the relative capacities of heterodimeric
EPORs to support EPO-dependent cellular proliferation. The
roles of these cytoplasmic tails were then established definitively using a chimeric receptor system in which the identity of the extracellular domains forces only heterodimerization of cytoplasmic receptor chains. These two assay systems demonstrated that dimerization of the membrane-proximal region of the EPOR cytoplasmic tail is required for signal transduction, whereas a monomer of the
membrane-distal region is sufficient for receptor-specific signaling.
Thus, the distal portion of the activated receptor can function as a
monomeric scaffold to recruit signaling molecules that are critical for inducing cell proliferation.
Plasmids--
pME18S STAT5A was the generous gift of Dr. A. Mui.
The pEFBOS JAK2 expression vector was the kind gift of Dr. D. Wojchowski. The EPOR mutant 1-321 was generated by exonuclease
digestion using the Erase-a-base kit (Promega Corp., Madison, WI).
Dideoxy sequence analysis demonstrated that the EPOR 1-321 mutant
contains residues 1-320 of the WT EPOR plus an additional 14 amino
acids (EINNCTALTGAGGQ) at the carboxyl terminus which are not encoded
by the WT EPOR. These residues are the result of the mutagenesis
procedure and are followed by a termination codon. To construct the
EPOR mutant W282R (28), EPOR cDNA was digested with
PvuII and BamHI and ligated to a synthetic,
double-stranded oligonucleotide encoding the W282R point mutation. EPOR
mutants were subcloned into the mammalian expression vector pMEX as
described previously (10).
A modified form of the pEFBOS vector (29) containing the murine IL-3
signal sequence and the FLAG epitope tag was generously provided by
Doug Hilton and Clare McFarlane (Walter and Eliza Hall Institute,
Melbourne, Australia). To generate Cell Lines, Culture Conditions, and Transfections--
32D is a
well described IL-3-dependent myeloid cell line (30).
Stable transfectants expressing WT or mutant EPORs were maintained in
RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf
serum (FCS) and 5% conditioned medium from the WEHI 3B cell line
(RPMI/FCS/WEHI), the latter as a source of IL-3.
32Dn20 cells express the WT EPOR from the pXM vector (10). To establish
cell lines coexpressing WT and mutant EPORs, 32Dn20 cells were
electroporated (using a Bio-Rad Gene Pulser apparatus) with pMEX
containing mutant EPOR cDNAs, using conditions described previously
(10). Transfected cells were selected by growth in RPMI/FCS/WEHI
containing 500 µg/ml G418 (Life Technologies), and clonal cell lines
were isolated by limiting dilution. Expression of the mutant EPORs was
verified by EPOR immunoprecipitations from metabolically labeled cells
(11). 32D cell lines coexpressing
Growth Factor-dependent Proliferation
Assays--
The ability of the EPOR-expressing cell lines to confer
EPO-dependent proliferation was tested by short term
proliferation assays as described previously (10). To examine
IL-2-dependent proliferation in cell lines coexpressing
Antibodies, Immunoprecipitations, SDS-Polyacrylamide Gel
Electrophoresis, and Immunoblots--
Antiserum specific for the
amino- or carboxyl-terminal region of the murine EPOR has been
described previously (33). Anti-JAK2 serum and the anti-phosphotyrosine
antibody 4G10 were obtained from Upstate Biotechnology (Lake Placid,
NY). Antisera specific for STAT5 was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Cell lysates were prepared for
immunoprecipitation as described previously (10), using lysis buffer
containing 1% w/v Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, supplemented with 1 mM sodium vanadate, 2 mM
phenylmethysulfonyl fluoride, and 10 µl/ml aprotinin. Lysates were
cleared by centrifugation and incubation with protein A-agarose beads
and immunoprecipitations performed as described previously (10).
For IL-2 stimulation, cells were washed and cultured in serum-free RPMI
for 4 h at 37 °C and then stimulated with 10 nM
IL-2 for 8-10 min at room temperature. Proteins were
immunoprecipitated from cell lysates, separated by electrophoresis on
7.5% SDS-polyacrylamide gels, and transferred to nitrocellulose
filters. The filters were blocked in buffer containing 1% bovine serum
albumin (BSA), 1 × phosphate-buffered saline (PBS), and 0.2%
Tween 20 (BSA/PBS/Tween) for 60 min at room temperature. The filters
were incubated with 4G10 antibody (1:1,000 dilution in BSA/PBS/Tween)
at 4 °C overnight, washed twice (15 min each) in 1 × TBS
containing 0.2% Tween 20. Horseradish peroxidase-conjugated anti-mouse
secondary antibody (Amersham Pharmacia Biotech) (1:10,000 dilution in
BSA/PBS/Tween) was incubated with the filters for 60 min at room
temperature. Filters were washed as described above and were developed
with chemiluminescent reagents (Pierce Chemical Co.) according to the manufacturer's instructions.
To label proteins metabolically, cells were washed with
methionine/cysteine-free medium and then incubated in
methionine/cysteine-free medium supplemented with 500 µCi/ml
[35S]methionine and -cysteine for 2 h. Proteins were
immunoprecipitated, separated by SDS-PAGE, and the gels were
fluorographed, dried, and exposed to x-ray film.
Nuclear Extracts and Electrophoretic Mobility Shift
Assay--
48 h after transfection, cells were stimulated with the
appropriate cytokine at the indicated concentration for 10 min at 37 °C and then harvested. Nuclear extracts were prepared as
described previously (34, 35) in the presence of the following protease inhibitors: 0.5 mg/ml antipain, 0.5 mg/ml aprotinin, 0.75 mg/ml bestatin, 0.5 mg/ml leupeptin, 0.05 mg/ml pepstatin A, 0.5 mM phenylmethylsulfonyl fluoride, 1.4 mg/ml phosphoramidon,
and 0.5 mg/ml soybean trypsin inhibitor as well as 1 mM
sodium orthovanadate. Oligonucleotide probes encoding the Fc To determine the functional requirements for oligomerization of
the EPOR cytoplasmic tails, we coexpressed WT and cytoplasmic tail
mutant EPORs in 32D cells. In previous studies, truncation mutants
lacking the majority of sequences in the cytoplasmic domain of the EPOR
dominantly inhibited EPO-induced cellular proliferation when
coexpressed in excess with the WT receptor, indicating that these
mutants heterodimerize with the WT receptor and block signal transduction from the cell surface (10, 27). Therefore, the coexpression of WT EPOR and less severe truncation mutants should clarify whether a dimer of the full-length EPOR cytoplasmic tail is
required for receptor functions involved in cellular proliferation, or
if growth signaling can be activated through asymmetric configurations of the EPOR intracellular domains.
IL-3-dependent 32D cells that express the WT EPOR
proliferate in response to stimulation with either EPO or IL-3 (10). We constructed the EPOR mutants shown in Fig.
1A, and the ability of these
mutants to support EPO-dependent proliferation alone or in
combination with the WT receptor was assessed in 32D cells. When
expressed singly, the EPOR 1-257, EPOR 1-321, and EPOR W282R mutants
were displayed on the cell surface at levels comparable to WT EPOR
(600-1,700 sites/cell), indicating that the biosynthesis of these
mutants is relatively normal. In addition, these mutants have WT
affinity for EPO (Kd 200-400 pM) (data
not shown). The EPOR 1-257 and EPOR W282R mutants were unable to
support EPO-dependent proliferation when expressed alone
(Fig. 1A). Expression of the EPOR 1-321 mutant maintained
the viability of cells and permitted weak proliferation in only
saturating concentrations of EPO (Figs. 1A and
2).
and
c chains. These chimeras allowed us to assess more
precisely the signaling role of each receptor chain because only
heterodimers of WT and mutant receptor chimeras form in the presence of
interleukin-2. Coexpression studies demonstrated that a functional
receptor complex requires the membrane-proximal region of each receptor
subunit in the oligomer to permit activation of JAK2 but only one
membrane-distal tail to activate STAT5 and to support cell
proliferation. Thus, this study defines key relationships involved in
the assembly and activation of the EPOR signal transduction complex
which may be applicable to other homodimeric cytokine receptors.
INTRODUCTION
Top
Abstract
Introduction
References
and
c receptor chains (for review, see Refs. 14 and 15).
EXPERIMENTAL PROCEDURES
EPOR, the extracellular region of
the human IL-2R
chain (encoding residues 1-214 of the mature
protein) was amplified by the polymerase chain reaction and subcloned
into the modified pEFBOS vector, downstream of the FLAG epitope tag.
The transmembrane and cytoplasmic domains of the murine EPOR (encoding
residues 226-483 of the mature protein) were amplified by polymerase
chain reaction; this fragment was subcloned into the pEFBOS vector 3'
to the IL-2R
chain extracellular region. The resulting construct
(pEFBOS.S.FLAG
E) contains the IL-3 signal sequence at the amino
terminus, followed in-frame by the FLAG epitope tag, the IL-2R
extracellular region, and the EPOR transmembrane and cytoplasmic
regions. Additional amino acids (TR or VD) were introduced in between
the epitope tag and the IL-2R
extracellular domain, and the IL-2R
extracellular region and EPOR transmembrane domain, respectively, by
the subcloning procedure.
EPORs containing the 1-321 or W282R
mutations were generated by swapping restriction fragments within the
EPOR cytoplasmic domain. Sequences of the
EPOR constructs were
confirmed by dideoxy sequence analysis. The murine
c
cDNA was the kind gift of Dr. T. Kono. To construct the murine
c-EPOR chimera, the transmembrane and intracellular
domains of the EPOR were amplified by polymerase chain reaction (from
amino acid residue 252 of the mature protein to the stop codon) with
oligonucleotides inserting unique 5' (BsmI) and 3'
(XbaI) restriction sites. This fragment was used to replace the corresponding transmembrane and intracellular domains of murine
c, and the construct was verified by DNA sequencing. To
generate the
EPOR 1-321 chimera, the
KpnI-BglII fragment of
EPOR and the
BglII-XbaI fragment of
EPOR 1-321were cloned
into the KpnI and XbaI sites of pCMV4neo. The
sequence of this construct was confirmed by automated DNA sequencing.
Expression of chimeric proteins and EPOR was confirmed by COS-7 cell
transfection and immunoprecipitation experiments (see below).
EPOR and
EPOR were established
by two rounds of electroporation and selection. First, cells were
electroporated with pEFBOS.S.FLAG
E and pSV2NEO, and
stably transfected cells selected by growth in RPMI/FCS/WEHI containing
500 µg/ml G418. G418-resistant cells were then electroporated with
pBABE/Puro and pCMV/
EPOR or pCMV/
EPOR 1-321. Stably transfected
cells were selected by growth in RPMI/FCS/WEHI containing 5 µg/ml
puromycin and clonal cell lines were isolated by limiting dilution.
2A, a somatic mutant of the 2C4 fibrosarcoma cell line that
specifically fails to express JAK2 (31), was the generous gift of Drs.
G. Stark and I. Kerr. These cells were cultured as described previously
in Dulbecco's minimal essential medium supplemented with 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 700 µg/ml G418. These
cells were transfected using calcium phosphate as described previously
(32) and according to the manufacturer's instructions.
EPOR and
EPOR or
EPOR 1-321, cells were washed three times
with RPMI/FCS and then plated in RPMI/FCS supplemented with IL-2 at the
indicated concentrations (see figure legends). Cells were passaged for
up to 2 weeks in IL-2-containing media. The culture density was
maintained under 106 cells/ml by dilution in fresh medium.
Viable cells, as judged by trypan blue dye exclusion, were counted
every 2-3 days, and total cell numbers were graphed to determine
doubling times. All cell lines used in this study were strictly growth
factor-dependent and died after 24 h in RPMI/FCS.
RI or
IRF-1 STAT response elements were end labeled with polynucleotide
kinase (Boehringer Mannheim) and [
-32P]ATP (Amersham
Pharmacia Biotech). DNA binding studies were performed with
105 cpm probe, 2 µg of poly(dI-dC) and 10 µg of nuclear
proteins as described (34, 35). For supershift analyses, the nuclear extract was preincubated with antibody for 45 min on ice before the
addition of the radiolabeled probe. Purified rabbit IgG specific for
STAT5 (C-17) was obtained from Santa Cruz Biotechnology.
RESULTS
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Fig. 1.
EPO-dependent proliferation and
receptor expression levels in cells expressing WT and mutant
EPORs. Panel A, schematic diagrams of WT and mutant
EPORs are shown. The position of conserved cysteines and
WSXWS (WSAWS in the EPOR) motif in the extracellular region
and conserved Box 1 and Box 2 motifs in the cytoplasmic region are
shown. The locations of the transmembrane (TM) domain,
residue Arg-129, and residue Trp-282 are also shown.
EPO-dependent proliferation was determined for cell lines
expressing WT or mutant receptors alone or cells coexpressing WT and
mutant receptors. Cells were assayed for growth in 10 µm/ml EPO over
a 3-day period. The level of cell growth is expressed as a percentage
of the total cell number in parallel cultures containing IL-3 (for
details, see "Experimental Procedures"). Panel B, cells
expressing WT EPOR alone (WT) or coexpressing WT and 1-257,
WT and 1-321, or WT and W282R were metabolically labeled, and proteins
were immunoprecipitated from detergent cell extracts with antiserum
specific for the amino terminus (WT+1-257, WT+1-321) or carboxyl
terminus of the EPOR (WT, WT+W282R). Immunoprecipitated proteins were
separated by SDS-PAGE, and gels were fluorographed and exposed to x-ray
film. Computer-generated images of the autoradiograms are shown;
DeskScan and Canvas software were used to generate the images. The
positions of WT (WT EPOR, arrowhead) and mutant EPORs are
indicated. WT and W282R comigrate on SDS-PAGE (as indicated).
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Fig. 2.
EPO-dependent proliferation of
32D cell lines expressing WT and mutant receptors. 32D cells
expressing only WT, 1-321, or W282R, or coexpressing WT and 1-321 or
WT and W282R were assayed for growth in concentrations of EPO ranging
from 0.005 to 10 µm/ml, over a 3-day period. The level of
proliferation is expressed as a percentage of the total cell number in
parallel cultures containing IL-3 (for details, see "Experimental
Procedures"). Panel A, growth of cells expressing WT,
1-321, or coexpressing WT and 1-321. Panel B, growth of
cells expressing WT, W282R, or coexpressing WT and W282R.
To generate coexpressing cells, a 32D cell line that expresses low levels of WT EPOR (32Dn20) was transfected with the mutants shown in Fig. 1A. Multiple clonal lines coexpressing WT and mutant EPORs were isolated. To determine the relative levels of WT and mutant EPOR expression in each cell line, proteins were metabolically labeled with [35S]methionine and -cysteine, immunoprecipitated with antiserum specific for the EPOR, and separated by SDS-PAGE. We selected cell lines in which the mutant receptors are expressed at 5-10-fold higher levels than the WT EPOR for further analysis (Fig. 1B). At this ratio of expression, EPOR 1-257 functions readily as a dominant inhibitor of WT EPOR-mediated proliferation (10).
To test the ability of these mutants to inhibit dominantly proliferation mediated by the WT receptor, cells coexpressing WT and mutant EPORs were cultured in media containing 10 units/ml EPO or IL-3 for 3 days. Interestingly, the truncation mutant EPOR 1-321, which was unable to support a significant level of EPO-dependent proliferation when expressed on its own, failed to block proliferative signaling (Figs. 1A and 2). However, a full-length form of the EPOR containing the single point mutation W282R in the cytoplasmic tail exerted potent dominant inhibitory effects (Figs. 1A and 2). To determine the EPO dose-response profiles of clonal cell lines coexpressing the WT receptor and EPOR 1-321 or EPOR W282R, we assayed the growth of these cell lines in various concentrations of EPO. For each mutant receptor, three independently isolated clonal lines were characterized, and the results from representative cell lines are shown in Fig. 2. The EPOR W282R mutant dominantly inhibited cellular proliferation at all concentrations of EPO tested, whereas the EPOR 1-321 mutant did not affect the dose-dependent proliferation of these cells in response to EPO (Fig. 2).
Together, these results suggest that dimerization between WT and EPOR
1-321 receptors can stimulate the signaling pathways leading to
cellular proliferation, whereas dimerization of WT and EPOR W282R is
insufficient for mitogenic signaling. However, by this approach the
precise molecular configuration of the cell surface receptor complexes
could not be regulated definitively. Therefore, we sought to explore
these findings further in a system in which the cell surface receptor
configuration could be defined specifically. We replaced the
extracellular domain of the EPOR with the extracellular domains of
IL-2R and
c because it has been demonstrated
definitively that IL-2-induced heterodimerization of these two chains
is necessary and sufficient to initiate signal transduction (36-38). A
EPOR chimera, containing the extracellular domain of the IL-2R
chain fused to the transmembrane and cytoplasmic tail of EPOR, was
constructed; for ease of detection, this construct was tagged with the
FLAG epitope at its NH2 terminus. A WT
EPOR chimera was
similarly constructed (although lacking the epitope tag) (for a
schematic of these chimeras, see Fig.
3A). Transient transfection
assays in COS-7 cells were used to confirm protein expression (data not
shown). The signaling function of these chimeras was assessed initially
by transient cotransfection in the
2A cell line. This cell line is a
somatic mutant of the 2C4 fibrosarcoma cell line which is specifically
deficient in JAK2 protein expression (31); this cell line also does not
express IL-2R
,
c, and STAT5 (data not shown). Because
there are two functional isoforms of STAT5 (STAT5A and STAT5B) which
seem to be almost completely interchangeable with regard to
cytokine-dependent activation (39-42), for simplicity we
used one of these isoforms, STAT5A, in our transfection experiments. Reconstitution of ligand-inducible STAT5 activation required the simultaneous transfection of cDNAs encoding
EPOR,
EPOR, JAK2, and STAT5A as well as stimulation of the cells with IL-2 (Fig. 3B). The identity of the induced DNA-binding complex was
confirmed by supershift analysis using an antibody specific for STAT5
(data not shown). Elimination of either receptor chain or the
associated kinase resulted in a loss of signal, demonstrating that each
of these components was required for STAT5 activation (Fig.
3B). Furthermore, the requirement for JAK2 demonstrated that
these chimeras couple appropriately to the kinase associated with the EPOR (43).
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Next, we introduced two of the mutant EPOR tails we have described,
EPOR 1-321 and EPOR W282R, into the context of these chimeras. Replacement of EPOR WT with the
EPOR 1-321 truncation did not significantly alter STAT5 induction by IL-2 through the
EPOR-
EPOR heterodimer (Fig. 4). In contrast,
dimerization of the
EPOR W282R mutant and
EPOR led to barely
detectable activation of STAT5. We believe that the minimal level of
STAT5 activation observed with the
EPOR W282R chimera reflects the
extreme sensitivity of the assay system. Nonetheless, the level of
STAT5 activation is reduced greatly compared with either the
EPOR or
EPOR 1-321 mutant upon pairing with the
EPOR.
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To extend these results, we examined the effects of the EPOR 1-321
truncation mutant on signaling. The pairing of
EPOR WT with
EPOR
1-321 resulted in STAT5 induction that was indistinguishable from the
pairing of two WT receptors. In contrast, the homodimerization of EPOR
1-321 cytoplasmic tails resulted in barely detectable STAT5
activation, in agreement with results obtained from 32D cells
expressing only EPOR 1-321 (data not shown). Finally, the pairing of
EPOR 1-321 with
EPOR W282R resulted in virtually no STAT5
activation (Fig. 4).
To analyze the proliferative potential of heteromeric EPOR complexes,
clonal 32D cell lines coexpressing EPOR and
EPOR, or
EPOR and
EPOR 1-321, were established. To verify that a proliferative response was caused by transfected receptor subunits and not by the
murine
c chain that is expressed endogenously in 32D
cells, clonal lines expressing
EPOR alone were also derived. These
cells were unable to proliferate or survive (data not shown), even in saturating concentrations (1.0 or 10 nM) of IL-2.
Coexpression of
EPOR and
EPOR or
EPOR and
EPOR 1-321
rendered 32D cells responsive to IL-2 (Fig.
5A), and the doubling time of
each clonal line was relatively unchanged over a 100-fold range of
ligand concentrations (e.g. media supplemented with 0.1, 1.0, or 10.0 nM IL-2; data not shown). Both JAK2 and STAT5
were efficiently tyrosine-phosphorylated in IL-2-stimulated cells
coexpressing
EPOR and
EPOR or coexpressing
EPOR and
EPOR
1-321, but not in cells expressing
EPOR alone (Fig. 5, B
and C). Thus, IL-2-mediated heterodimerization of
EPOR
and
EPOR, or
EPOR and
EPOR 1-321, generates functional
receptors that are capable of delivering long term proliferative
signals. In contrast,
EPOR alone, or
EPOR and the endogenous
murine
c chain, does not form functional receptor
complexes in response to IL-2. These results demonstrate definitively
that cellular proliferation can be stimulated by heterodimers
containing full-length EPOR and EPOR 1-321 cytoplasmic regions.
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DISCUSSION |
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The results presented herein demonstrate that dimerization of the entire EPOR cytoplasmic region is not required for the signal transduction pathways leading to cellular proliferation. When expressed on its own, the EPOR 1-321 mutant bound JAK2 and stimulated EPO-dependent JAK2 tyrosine phosphorylation (data not shown). This result indicates that oligomerization of the membrane-proximal portion of the EPOR cytoplasmic tail, which contains the binding site for JAK2 (43), is sufficient for JAK2 activation. JAK2 activation may occur via a trans-autophosphorylation mechanism, similar to the activation mechanism of other tyrosine kinases (45-49). Despite activation of JAK2, the EPOR 1-321 mutant, which lacks all eight cytoplasmic tyrosine residues, failed to support cell proliferation at physiological concentrations of ligand. This result is in agreement with the findings of others, who have demonstrated that sequences in the distal portion of the cytoplasmic tail play a critical role in receptor signal transduction for growth (19, 20, 28, 50, 51). However, the EPOR 1-321 mutant did not dominantly inhibit signal transduction by the WT EPOR. These findings suggest that the primary role of EPOR dimerization is to oligomerize and activate EPOR-associated JAK2, leading to the initiation of intracellular signaling events that require the presence of elements within the distal portion of the EPOR cytoplasmic tail. In this context, a single full-length receptor tail is sufficient within the dimeric receptor complex to engage this signaling machinery.
The initial findings using coexpressed EPOR variants were strongly
suggestive but were complicated by our inability to define precisely
the receptor complexes formed by the binding of EPO. To drive receptor
subunit heterodimerization in the absence of the potential artifact of
receptor homodimers, we generated two asymmetric chimeras in which the
extracellular domain of EPOR is replaced with the equivalent portions
of IL-2R and
c. Coexpression of these two molecules
along with JAK2 permitted IL-2-mediated STAT5 induction, highlighting
the importance of cytoplasmic tail dimerization in signal transduction.
Substitution of either receptor chain (but not both) with the
truncation mutant 1-321 led to levels of STAT5 induction which were
indistinguishable from the levels induced by a receptor complex
containing two WT receptor chains. These data demonstrate that only one
full-length receptor chain is required to initiate an EPOR-specific
signaling program. Furthermore, the fact that a receptor complex
containing two truncated receptors fails to activate STAT5 efficiently
emphasizes the functional importance of the membrane-distal portion of
the EPOR tail. Finally, the observation that EPOR 1-321 and EPOR W282R
heterodimers fail to signal demonstrates that two functional JAK2
binding sites are required in addition to a single full-length
cytoplasmic tail.
The IL-2-dependent proliferation of 32D cells expressing
EPOR and
EPOR, or
EPOR and
EPOR 1-321, confirmed that
asymmetric EPOR complexes are functional. The downstream EPOR signaling
molecules, JAK2 and STAT5, likewise were activated efficiently by IL-2
in coexpressing cell lines but not in cells expressing
EPOR alone. Therefore, dimerization of the EPOR is required to activate JAK2, which
directly or indirectly leads to phosphorylation of EPOR cytoplasmic
tyrosine residues. Once phosphorylated, a monomer of the distal portion
of the EPOR cytoplasmic region can function to recruit and activate the
signal transduction proteins essential for cellular proliferation.
However, several questions remain regarding the structure of the functional EPOR complex. Previous studies have shown that dimerization of the EPOR stimulates the signals leading to erythroid cell survival, proliferation, and differentiation (8, 10, 11, 44). Crystallographic structural analysis of the EPOR extracellular region complexed to a peptide agonist demonstrated directly the dimeric nature of the receptor, and biochemical experiments have shown that EPO promotes dimerization of the EPOR extracellular region (9, 12, 52). However, a dimer of the EPOR may be the minimal complex required for signaling, and higher order oligomers may form the functional receptor complexes on the cell surface. Further biochemical direct analysis of the EPOR on the membrane of living cells is required to determine the oligomeric nature of the activated receptor complex.
In addition, several lines of evidence suggest that EPOR subunit dimerization is necessary but not sufficient to stimulate signal transduction. First, we show here in two systems that W282R blocks signaling from the WT EPOR even though W282R is expressed on the plasma membrane, binds ligand, associates with JAK2, and contains all eight tyrosines in the cytoplasmic region (data not shown (28, 43)). Second, a double mutant EPOR, R129C/W282R, which contains W282R as well as the R129C substitution that promotes EPO-independent disulfide-linked receptor dimerization on the cell surface (11), is inactive in cell proliferation assays,2 whereas cells expressing an EPOR with the R129C mutation alone proliferate in the absence of any added growth factor (11, 33). These findings imply that the nonfunctional W282R mutant must alter either the relative orientations of the receptor cytoplasmic regions or the alignment of JAK2 molecules in the receptor dimer. In addition, EPO-mimetic peptides that bind to and dimerize the EPOR extracellular domain are not always sufficient to activate intracellular signal transduction (53). Thus, it appears that JAK2 molecules and/or the cytoplasmic regions of the EPOR must be brought into the correct orientation in the receptor dimer to create a competent, activated receptor complex. Some degree of flexibility may be allowed in the structure of the EPOR dimer, however, because recent studies suggest that there may be structural differences when the EPOR is activated by a peptide agonist, by the R129C mutation, or by EPO (9, 54).
We have described previously a model for heteromeric cytokine receptor
signaling which distinguishes receptor chains based on two essential
functions (55). One receptor chain appears to specify the signaling
program that is unique to the particular receptor complex. The second
receptor subunit does not contribute significantly to the specificity
of signal transduction but rather triggers receptor activation. This
structure/function arrangement applies to several members of the
receptor family which share the c chain as a trigger
chain (55-57). The present findings indicate that signaling through
the homodimeric EPOR is governed by similar structure/function
principles. In the EPOR configurations generated for the present
studies, the full-length EPOR drives specificity, and the EPOR 1-321
mutant functions as the trigger chain. Thus, in this homodimeric
receptor as in heteromeric receptors, a single phosphorylated receptor
tail is sufficient for the engagement of the downstream signaling
machinery. In the native EPOR complex, each receptor chain likely
functions as a trigger chain for its partner subunit, leading to the
efficient initiation of signal transduction by the homodimeric complex.
Further studies will be required to test the generalizability of this
model to other homodimeric receptor complexes.
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ACKNOWLEDGEMENTS |
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We thank Ruth Busche and Hong Lu for excellent technical assistance. We also thank Dr. Alice Mui for the STAT5A cDNA, Dr. Daniel Wojchowski for the JAK2 cDNA, Dr. Bing Su and Chiron Corp. for the gifts of IL-2, and Dr. Doug Hilton and Clare McFarlane for the modified pEFBOS vector. We acknowledge the generous gift of cell lines from Drs. George Stark and Ian Kerr. We thank Drs. Harvey F. Lodish and Rebecca G. Wells for advice during the course of these studies and for comments on the manuscript, Dr. Bradley W. McIntyre and members of his laboratory for assistance with scanning and image analysis, and Dr. Joan Egrie (Amgen Corporation) and Dr. Francis Farrell (R. W. Johnson Pharmaceutical Research Institute) for generous gifts of recombinant EPO. We thank John Carroll, Heather Livesay, Stephen Gonzales, and Neile Shea for assistance in the preparation of this manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the Kleberg Foundation and National Institutes of Health Grant CA-77447 (to S. S. W.), by grants from the James S. McDonnell Foundation and Abbott Laboratories (to G. D. L.), and by National Institutes of Health Grant GM-54351 and a grant from the J. David Gladstone Institutes (to M. A. G.). Preliminary work (by S. S. W.) was supported by National Institutes of Health Grant HL32262 and a grant from the Arris Pharmaceutical Corporation to Harvey F. Lodish.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ The first two authors contributed equally to this work.
** Scholar of the James S. McDonnell Foundation.
To whom correspondence should be addressed: Gladstone Institute
of Virology and Immunology, P. O. Box 419100, San Francisco, CA
94141-9100. Tel.: 415-695-3775; Fax: 415-826-1514; E-mail: mgoldsmith{at}gladstone.ucsf.edu.
2 S. S. Watowich, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: EPO, erythropoietin; EPOR, erythropoietin receptor; IL-2, IL-3, interleukin-2, interleukin-3; IL-2R, interleukin-2 receptor; WT, wild-type; CMV, cytomegalovirus; FCS, fetal calf serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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