 |
INTRODUCTION |
Activation of the EpoR1
elicits multiple intracellular signals that ultimately lead to cell
division and differentiation of erythroid progenitor
and precursor cells. One primary signaling event following receptor
activation is the phosphorylation of certain cellular proteins on
tyrosine residues (1, 2). Interaction of Epo with its receptor results
in the activation of the cytoplasmic tyrosine kinase JAK2 and in the
phosphorylation of the intracellular domain of EpoR (3-5). Tyrosine
phosphorylated residues on EpoR then constitute binding sites for other
intracellular proteins that become eventually tyrosine phosphorylated.
Other tyrosine kinases are also activated in response to Epo such as
c-Fes (6), Lyn (7), and Syk (8). Tyrosine phosphorylated
proteins may be recruited directly to the EpoR via their Src homology
domains or indirectly through adaptor or scaffold proteins. Several
tyrosine kinase substrates phosphorylated upon Epo activation have been identified including STAT5 transcription factor (9), SHP-2 tyrosine
phosphatase (10), Shc (11), phospholipase C-
1 (12), Vav (13), c-Cbl
(14), IRS-2 (15), GAB-1 (16), and CrkL (17). Yet the relations between
all the components involved in Epo signaling as well as the
identification of their respective targets are only partially
elucidated, and EpoR may activate other additional signal transduction pathways.
In an attempt to characterize substrates for epidermal growth factor
(EGF) receptor, Fazioli et al. (18) developed an expression cloning strategy for cDNAs encoding EGF receptor substrates. The approach relied on batch purification of an entire set of putative substrates, achieved by immunoaffinity chromatography using
anti-phosphotyrosine antibodies (19, 20). Antisera generated against
the entire pool of purified proteins were subsequently used for the
screening of cDNAs expression libraries. We applied this
methodology to identify new cDNAs encoding signaling proteins
involved in Epo activation, either tyrosine phosphorylated proteins or
proteins bound to these proteins. In the present work we report that
one of these cDNAs encodes G
2, the
2
subunit of heterotrimeric GTP-binding proteins, or G proteins.
G proteins traditionally associate with G protein-coupled receptors
(GPCRs) that contain seven membrane-spanning domains. G proteins
function as intermediates that couple cell surface receptors to
intracellular effectors. Heterotrimeric G proteins are made of three
polypeptides: an
subunit that binds and hydrolyzes GTP, and 
subunits that form a functional monomer. Receptor activation induces
the exchange of GDP for GTP on the G
subunit. Once GTP
is bound, the
subunit dissociates both from the receptor and from

. The free
and 
subunits each activate target
effectors. However, a number of single-spanning transmembrane receptors
such as receptors for EGF (21), insulin and insulin-like growth factor (IGF)-I and IGF-II (22-25), fibroblast growth factor (26), and T
lymphocyte receptors (27, 28) have been reported to activate G
proteins. In some cases a physical association, in addition to a
functional coupling, has also been demonstrated between a single-spanning membrane receptor and G proteins (29-32).
Heterotrimeric G proteins could be important intermediates in the
signal transduction of hematopoietic cytokines. Changes in the
expression level and GTPase activity of G
16, a member of
the Gq family of G proteins uniquely expressed in
hematopoietic cells, may modulate cellular proliferation or
differentiation in T lymphocytes and in MB-02 erythroleukemia cells
(33, 34). Pertussis toxin (PT) modifies the response to several
hematopoietic growth factors. PT catalyzes the ADP-ribosylation of the
Gi family of G proteins and uncouples G proteins from
surface receptors. PT inhibits the signal transduction and/or
proliferation induced by interleukin (IL)-1, IL-3,
granulocyte-macrophage colony-stimulating factor (GM-CSF) and
colony-stimulating factor-1 (CSF-1) (35-38) in hematopoietic cells.
Expression of a dominant negative mutant of G
i2 also
inhibits cell proliferation in response to CSF-1 in BAC 1.2F5
macrophage cell line (39). In erythroid precursor cells a pertussis
toxin-sensitive G protein identified as G
i2 is required
for the regulation of voltage-independent calcium channels by Epo (40,
41). The increase in [Ca2+]i appears to be a
stage of differentiation specific and restricted to differentiating
erythroblasts (42).
In the present study we demonstrate the constitutive association of
Gi with the EpoR in hematopoietic cell lines as well as in
erythroid progenitors. We show that the C-terminal region of EpoR is
required for Gi protein binding. In addition, Epo activates G protein in cell membranes and induces the release of Gi
bound to the EpoR in hematopoietic cells. Thus, EpoR appears to be
physically and functionally coupled to G proteins.
 |
EXPERIMENTAL PROCEDURES |
Antibodies and Reagents--
Anti-phosphotyrosine (anti-Tyr(P))
monoclonal antibodies 4G10 and PY72 were produced from hybridoma cell
lines kindly provided by B. Drucker (Portland, OR) and B. Sefton (La
Jolla, CA), and were affinity purified by chromatography on
phosphotyramine. Anti-EpoR antiserum used for immunoprecipitation was
produced against a fusion protein between glutathione
S-transferase and the cytoplasmic portion of human EpoR.
Peroxydase-conjugated anti-rabbit antibodies were purchased from
Amersham Pharmacia Biotech. Antibodies specific for human EpoR used for
immunoblotting were purchased from Santa Cruz (sc-695) and anti-JAK2
antiserum from Upstate Biotechnology Inc. (catalog number 06-255). The
fusion protein between the maltose-binding protein (MalE) and
the cytoplasmic region of EpoR was described previously (43). Purified
recombinant human Epo (specific activity, 120,000 units/mg) was a gift
of Dr. M. Brandt (Roche Molecular Biochemicals). Pertussis toxin was
purchased from Alexis, ATP was from Amersham Pharmacia Biotech, and GTP
and GTP
S were from Sigma.
DNA Constructs and Expression Vectors--
The murine EpoR
mutant F1-Y58 that contains a deletion between Glu377 and
Tyr431 was described previously (9). A panel of EpoR
deletion mutants was produced from polymerase chain reaction-amplified
fragments. In mutants
41,
24,
20, and
5, stop codons were
inserted just after codons 442, 459, 463, and 478, respectively (see
Fig. 6). All receptor constructs were subcloned into a modified
pCDNA3 expression vector where the cytomegalovirus promotor was
changed to Rous sarcoma virus and the neomycin resistance gene was
replaced by puromycin. The fidelity of all constructs was confirmed by sequencing.
Cell Lines and Stimulation--
The human leukemic cell line UT7
(44) was maintained in
-minimum essential medium supplemented with
5% fetal calf serum, penicillin, streptomycin, 2 mM
L-glutamine, and 2 units/ml Epo. TF-1 (45) and MO7E cell
lines (46) were cultured in
-minimum essential medium supplemented
with 10% fetal calf serum and 2.5 ng/ml GM-CSF. TF-1-ER (47) and
MO7E-ER (8) cell lines were obtained after infection of TF-1 and MO7E
with an amphotropic virus encoding a murine EpoR and were cultured with
Epo. FDCP-1 myeloid cells were grown in
-minimum essential medium
supplemented with 5% fetal calf serum and 3% WEHI conditioned medium
as a source of IL-3. After transfection with EpoR expression vectors
and selection in Epo, cells were grown in the presence of Epo. 32D
myeloid cells expressing wild-type EpoR and W282R mutant (48) were
generously provided by G. D. Longmore (St. Louis, MO). Erythroid
progenitors were purified from human umbilical cord blood cells as
described previously (49). Briefly CD34+ cells were cultured for 7 days in serum-free conditions in the presence of interleukin-3,
interleukin-6, and stem cell factor. CD36+ cells were then purified and
expanded for 2 additional days in the presence of the same cytokines
plus 2 units/ml Epo. CHO-ER cell line, a kind gift of E. Goldwasseur (Chicago, IL), was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.
For Epo triggering experiments, exponentially growing UT7 or TF-1 cells
were washed and incubated for 16-18 h in Iscove's modified
Dulbecco's medium supplemented with 0.4% bovine serum albumin and 20 µg/ml iron saturated transferrin, in serum-free conditions. Normal
erythroid progenitors were washed and incubated for 4 h in
serum-free Iscove's modified Dulbecco's medium in the presence of 5%
bovine serum albumin, 50 µg/ml insulin, and 1 mg/ml transferrin.
FDCP-1 cells expressing EpoR and MO7E-ER cells were Epo starved by
replacing Epo, respectively, with WEHI conditioned medium and GM-CSF
one to 2 days prior to stimulation. The cells (1 × 107/ml) were stimulated with 10 units/ml Epo at 37 °C as
described in the text.
Immunoaffinity Chromatography--
Cells (1 × 109) were lysed on ice with buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet
P-40, 1 mM orthovanadate, 2 mM EGTA, 30 mM disodium pyrophosphate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptine, 10 µg/ml
aprotinine, 10 µg/ml EG4, and 1 µg/ml pepstatine. Anti-Tyr(P)
columns were prepared by covalently cross-linking affinity purified
PY72 monoclonal antibody to Sepharose beads (Hi TrapTM
NHS-activated Sepharose from Amersham Pharmacia Biotech). Solubilized proteins were applied to the anti-Tyr(P) column (1 ml) using a fast
protein liquid chromatography system. The column was washed successively with 10 column volumes of lysis buffer and 30 volumes of
washing buffer (same as lysis buffer with 0.1% Nonidet P-40 instead of
1%). Then elution was done with the washing buffer supplemented with
40 mM phenyl phosphate. Fractions were collected and
analyzed for protein content by silver staining and for Tyr(P) content
by immunoblotting. The peak of tyrosine phosphorylated proteins,
which coincided with the peak of eluted proteins, was pooled. Protein
quantification was carried out with the micro BCA assay (Pierce).
Rabbit Immunization and Antisera Analysis--
Anti-Tyr(P)
reactive proteins (1 × 1010 cell equivalents) in
complete Freund's adjuvant were used to immunize two New Zealand White
rabbits as follows. Two intradermic injections (50 µg) were followed
by footpad injections with 25 µg of proteins. Bleeds were collected
15 days after each boost and screened for the production of antibodies.
Because expression cloning relies on the detection of denatured
antigens, immune antisera were tested by immunoblotting on UT7 cell
lysates. The rabbit antiserum from the rabbit with the highest antibody
titer and the lowest reactivity of the preimmune serum was selected for
further screening of the cDNA library. This serum was also tested
for its ability to precipitate proteins known to be tyrosine
phosphorylated following Epo stimulation and was shown to contain
anti-Shc antibodies (data not shown). IgG were purified and antibodies
reacting with bacterial proteins were removed by absorption on column
of bacterial proteins cross-linked to Sepharose beads (Hi TrapTM
NHS-activated Sepharose from Amersham Pharmacia Biotech).
EXlox Library Screening--
Absorbed IgG were used to screen
a commercial (Novagen RD Systems Europe) 16-day-old murine embryo
cDNA library in
EXlox vector, according to the
manufacturer's instructions. Screening conditions were chosen to get
an optimal signal-to-noise ratio and no reactivity with preimmune serum
in immunoblot. This was obtained by initially testing the reactivity of
the selected serum with a Shc clone previously isolated from the same
library, a kind gift of J. Finidori (Paris, France). Briefly 2 × 106 recombinant plaques were initially screened with IgG (3 µg/ml) in 25 mM Tris-HCl, pH 7.5, 0.1% Tween, 140 mM NaCl, 3 mM KCl containing 5% (w/v) low fat
powder milk. Colorimetric detection was carried out with anti-rabbit
IgG conjugated to alkaline phosphatase and the bromochloroindolyl
phosphate/nitro blue tetrazolium substrate (Promega). A second screen
confirmed the reactivity of positive phages. Selected phages were
plaque purified by conventional methods, and autosubcloning in plasmid
vector was generated with the loxP-cre system. The insert
cDNA sequence was determined using PerkinElmer Life Sciences
automatic sequencing. Nucleotidic and protein data bases were screened
with the BLAST program.
Immunoprecipitation, MalE Precipitations, and
Immunoblotting--
Immunoprecipitations, MalE precipitations, and
immunoblots were performed as described previously (4, 8). Proteins
were immunoblotted with a mixture of 4G10 (0.3 µg/ml) and PY72 (1 µg/ml) to detect tyrosine phosphorylated proteins, with anti-G
antibodies (1 µg/ml), anti-EpoR antibodies (1 µg/ml), or anti-JAK2
antiserum (1:1000). Bound antibodies were detected by incubation with
horseradish peroxidase-coupled secondary antibodies and the enhanced
chemiluminescence system (Amersham Pharmacia Biotech).
Preparation of Cell Membranes--
After washing UT7 cells in
phosphate-buffered saline, the cells were suspended in hypotonic buffer
(10 mM Tris-HCl, pH 7.4, 10 mM KCl, 2 mM EDTA) in the presence of protease inhibitors and homogenized with a Dounce pestle. After addition of 0.25 M
sucrose, nuclei and unbroken cells were removed by centrifugation at
375 × g, and a membrane enriched fraction was obtained
by centrifuging the supernatant at 150,000 × g for 45 min. The resulting membrane pellet was stored at
80 °C in 25 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM
EGTA, 10% glycerol or resuspended in appropriate buffer and used
immediately for [35S]GTP
S binding. CHO-ER membranes
were prepared as described previously (50).
ADP-ribosylation Assay--
ADP-ribosyltransferase activity was
measured by following the incorporation of
[32P]ADP-ribose (51). To assess the activity present in
immunoprecipitates, immune complexes bound to protein G-Sepharose were
washed in 50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA,
1 mM ATP, and 0.1 mM GTP. Immunoprecipitated proteins were suspended in 50 µl of the same buffer containing 2 µCi of adenylate [32P]NAD (PerkinElmer Life Sciences;
800 Ci/mmol) and 7 µg/ml activated pertussis toxin. The toxin (17 µg/ml) was preactivated immediately before use for 1 h at room
temperature in 50 mM Tris-HCl, pH 7.5, containing 62.5 mM dithiothreitol. ADP-ribosylation was carried out at
37 °C for 1 h, and the reaction was stopped by adding
SDS sample buffer. Samples were boiled for 5 min, and the
proteins were separated on a 10% SDS-polyacrylamide gel followed by
transfer to nitrocellulose and autoradiography.
To assess the effect of Epo on pertussis toxin-induced ADP-ribosylation
in cell membranes, membranes (50 µg) were initially incubated in 35 µl of buffer A (25 mM Hepes, pH 7.5, 150 mM
NaCl, 1 mM EDTA, 100 µM GTP, 10% glycerol)
with or without Epo for 10 min at 37 °C. Other additions or
deletions are as noted under "Results" and in the figure legends.
Then membranes were combined with 35 µl of 2× buffer B (100 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM ATP, 0.2 mM GTP). After addition of
adenylate [32P]NAD and activated pertussis toxin in 1×
buffer B, ADP-ribosylation was performed as described above.
GTP
S Binding Assay--
The [35S]GTP
S
binding was measured as described (52) with slight modifications.
Membranes (~30 µg) were suspended in an assay volume of 50 µl of
buffer containing 20 mM Hepes, pH 7.4, 10 mM MgCl2, 100 mM NaCl, 10 µM GDP.
Following a preincubation of membranes in the presence or absence of
Epo for 15 min at 30 °C, the assay was initiated by adding
[35S]GTP
S (New England Nuclear, 1250 Ci/mmol) to yield
a final concentration of 0.3 nM. Nonspecific binding was
measured in the presence of 100 µM unlabeled GTP. After
60 min at 30 °C, 0.5 ml of ice-cold buffer containing 20 mM Hepes, pH 7.4, 20 mM MgCl2, 100 mM NaCl, 100 µM GTP was added. Bound and free
[35S]GTP
S were separated by filtration over glass
fiber filters and three washings in the same buffer without GTP.
 |
RESULTS |
Screening of a Bacterial Expression Library with Antibodies
Specific for Anti-Tyr(P) Reactive Fractions of Epo-stimulated
Cells--
To purify proteins that become tyrosine phosphorylated upon
Epo stimulation and their associated proteins, our initial concern was
to get a high level of tyrosine phosphorylation. UT7 cells were
selected for their high surface EpoR expression (~7000
receptors/cell) and their ability to proliferate in response to Epo. An
optimal system was obtained by stimulating the cells with 10 units/ml Epo for 10 min at 37 °C and adding pervanadate (100 µM
vanadate and 50 µM H2O2) for the last 2 min
of stimulation. The low pervanadate concentration used did not modify
the basal level of tyrosine phosphorylated proteins in the absence of
Epo but allowed a significant increase of Epo-induced tyrosine
phosphorylation (data not shown). Epo-stimulated cells were
solubilized, the soluble fraction was loaded on anti-Tyr(P) column, and
anti-Tyr(P)-reactive fractions were eluted with phenyl phosphate. As
shown in Fig. 1A, most of tyrosine phosphorylated proteins present in Epo stimulated cells (lane 1) were purified on the anti-Tyr(P) column (lane
4). Comparison of the silver-stained protein profiles of the
anti-Tyr(P) reactive fractions with the anti-Tyr(P) Western blotting
profiles of the same fractions revealed that the purified proteins
contained several proteins that were not tyrosine phosphorylated (data
not shown). Proteins recovered from the column represented about 0.06%
of solubilized proteins, a value in good agreement with EGF receptor substrates previously isolated with a similar procedure (20). Polyclonal antisera were generated using the entire pool of anti-Tyr(P) reactive proteins as immunogen (see "Experimental Procedures"). We
next used these antibodies for immunological screening of a murine
embryo cDNA library in
EXlox vector and identified
several positive plaques. One of the cDNA encoded the
G
2 subunit of heterotrimeric G proteins and is the
subject of the present study. It contained the entire coding sequence
of the G
2 subunit of heterotrimeric G proteins and the
5' noncoding sequence (53). We first wanted to exclude the possibility
that the G
2 clone was isolated because of the
cross-reactivity of the antibodies used for expression cloning with a
protein present in the immunogen but different from G
2.
As shown in Fig. 1B the G
protein was detected by
immunoblotting with anti-G
antibodies in UT7 cell lysates as well as
in anti-Tyr(P) reactive proteins purified from the soluble fraction of
Epo-stimulated cells. As expected G
was not present in a preparation
of purified proteins isolated from nonstimulated cells. Thus, the
isolation of the G
cDNA with the expression cloning strategy
suggests that an heterotrimeric G protein could potentially be tyrosine
phosphorylated or associated with a tyrosine-phosphorylated protein
upon Epo activation.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
G detection in
anti-Tyr(P) affinity purified proteins from Epo-stimulated cells.
A, immunoaffinity chromatography. Epo- and serum-starved UT7
cells were stimulated with 10 units/ml Epo for 10 min at 37 °C. Cell
lysates were loaded on anti-phosphotyrosine (pTyr) column as
described under "Experimental Procedures." Proteins bound to the
column were eluted with 40 mM phenyl phosphate. Proteins
before purification (lane 1) in the column flow-through
(lane 2) and in phenyl phosphate eluted proteins
(lanes 3-5) were separated by SDS-PAGE and transferred to
nitrocellulose membrane. Tyrosine phosphorylated proteins extracted
from 2 × 105 cells were detected by immunoblotting
with anti-Tyr(P) antibodies. B, G detection in
anti-Tyr(P) reactive fractions. Starved UT7 cells were stimulated (+)
or not ( ) with 10 units/ml Epo for 10 min at 37 °C. Cell lysates
were loaded on anti-phosphotyrosine column and anti-Tyr(P) reactive
fractions were pooled. Purified proteins (PP, 5 × 106 cell equivalents), or total cell lysates
(Lysate, 2 × 105 cell equivalents) were
analyzed by SDS-PAGE and immunoblotting with anti-G
antibodies.
|
|
G
Subunit Associates with EpoR--
When G
was
immunoprecipitated from Epo-stimulated UT7 cells, we never detected a
tyrosine phosphorylated form of the protein by immunoblotting with
anti-Tyr(P) antibodies. This suggests that G
is not phosphorylated
upon Epo activation. Because G
was present in anti-Tyr(P) reactive
fractions (Fig. 1B), we hypothesized that G
may have been
copurified because of its association with another tyrosine-phosphorylated protein upon Epo stimulation. We then wanted to
determine whether G
was associated with the EpoR, one of the highly
phosphorylated proteins upon Epo activation (4). UT7 cells were
incubated or not with Epo at 37 °C for various times, and after cell
solubilization, the EpoR was precipitated. As shown in Fig.
2A, immunoblotting with
anti-G
antibodies revealed the presence of G
when cell lysates
were precipitated with anti-EpoR antiserum but not with preimmune
serum. The amount of G
coprecipitated with EpoR decreased following
Epo activation, suggesting that G
is constitutively associated with
EpoR and dissociates from the activated receptor. We extended our
analysis to determine whether the interaction between G
and EpoR
could also be evidenced in vitro. Proteins binding to the
cytoplasmic region of the EpoR were isolated from UT7 cell lysates,
using a recombinant fusion protein between MalE and the cytoplasmic
region of EpoR or a control MalE protein bound to amylose resin (Fig.
2B). When bound proteins were analyzed by immunoblotting
with anti-G
antibodies, G
was detected only when MalE-EpoR fusion
protein was used. We conclude that G
binds to EpoR both in
vivo and in vitro and that the interaction between G
and EpoR occurs through the intracellular region of EpoR.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
G association with
erythropoietin receptor in UT7 cells. A,
immunoprecipitation of G with anti-EpoR antibodies. Epo-starved UT7
cells were incubated with 10 units/ml Epo for the indicated time at
37 °C and then lysed. Cell lysates were immunoprecipitated with
anti-EpoR antiserum (EpoR) or preimmune serum (C). Precipitated
proteins (2 × 107 cell equivalents), and whole
lysates (5 × 105 cell equivalents) were analyzed by
SDS-PAGE and anti-G immunoblotting. B, in
vitro association of EpoR intracellular domain with G . Lysates
from UT7 cells (1.5 × 107) were incubated with
immobilized MalE or MalE-EpoR fusion proteins. Bound proteins were
resolved by SDS-PAGE and analyzed by anti-G
immunoblotting.
|
|
G
i Subunit Associates with EpoR--
G proteins are
associated to seven transmembrane receptors in an heterotrimeric form
where the
subunit is associated to
/
. Because EpoR is not a
"classical" G protein-coupled receptor, we investigated whether
only G
binds to the EpoR or whether the
chain of G proteins was
also associated to the EpoR. Several forms of G
were identified in
UT7 cells by immunoblotting including Gi, Gs,
and Gq. Our initial attempt to detect G
coprecipitated with the EpoR by immmunoblotting was unsuccessful because of the high
nonspecific background in the region of G
migration. We used another
method to investigate whether G
was bound to the EpoR, assuming that
Gi/o would be a good candidate because a pertussis
toxin-sensitive G protein has been shown to be required for
Epo-dependent calcium activation in erythroid precursors
(40). The
subunit was detected by following the in vitro
incorporation of [32P]ADP-ribose in the presence of
preactivated pertussis toxin. This toxin catalyzes the ADP-ribosylation
of
i/o/t subunits of G proteins.
t
expression is restricted to the nervous system, and
o is
not expressed in erythroid precursors or erythroleukemia cells (40,
54). This was confirmed in UT7 cells. Indeed we did not detect
o protein by immunoblotting with anti-G
o
antibodies. In initial experiments we observed that a 41-kDa band was
ADP-ribosylated in vitro in UT7 membranes. Labeling was
linear for up to 1 h and pertussis toxin-dependent
(data not shown). Therefore, this band is presumptively referred to as
G
i, the
subunit of the inhibitory guanine nucleotide
regulatory protein.
The EpoR was immunoprecipitated from the soluble fraction of UT7 cells,
and the ADP-ribosyltransferase activity present in the precipitates was
measured. Fig. 3 illustrates that
G
i coprecipitated with the EpoR in resting UT7 cells,
and cell stimulation with Epo decreased the amount of ADP-ribosylated
i associated with the EpoR. On the contrary, the amount
of EpoR remained constant. The G protein-EpoR complex was recovered in
the 150,000 × g supernatant of solubilized cells and
thus corresponded to solubilized complexes and not to membrane
fragments (data not shown). These data show that G
i is
constitutively bound to the EpoR, more likely as an
/
/
heterotrimer because G
i is a better substrate for PT in its heterotrimeric form (51, 55). They also suggest that the G protein
dissociates from the EpoR upon activation. To exclude the possibility
that the G protein was precipitated by anti-EpoR antiserum because the
EpoR and the G protein share a common epitope, the association was also
studied both in cells that express an exogeneous EpoR and in cells that
do not express EpoR. In MO7E cells expressing a murine EpoR,
ADP-ribosylated
i was coprecipitated with EpoR but no
41-kDa band was detected in cells that do not express EpoR (Fig.
4A).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3.
Coprecipitation of
G i with EpoR in UT7 cells.
Lysates from Epo-starved UT7 cells (1 × 107 cell
equivalents), stimulated (+) or not ( ) for 10 min with 10 units/ml
Epo, were immunoprecipitated (IP) with anti-EpoR antiserum
(EpoR) or preimmune serum (C). Immunoprecipitates
were subjected to in vitro ADP-ribosylation as described
under "Experimental Procedures." The proteins were separated by
SDS-PAGE and transferred to nitrocellulose membrane, and
G i was visualized by autoradiography. EpoR protein was
detected by immunoblotting.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
G i
association with EpoR in EpoR transfected cells and in erythroid
progenitors. A, MO7E cells expressing murine EpoR
(MO7E-ER) or not (MO7E) were stimulated with 10 units/ml Epo for 30 min. Cell lysates (1 × 107 cell
equivalents) were immunoprecipitated with anti-EpoR antiserum.
B, erythroid progenitors were deprived of growth factor and
stimulated (+) or not ( ) with Epo for the indicated time. After cell
solubilization the EpoR was precipitated, and G i was
detected in the precipitates by in vitro ADP-ribosylation
followed by autoradiography.
|
|
Having established an association between G protein and EpoR in cell
lines that express endogenous or exogenous receptor, we wanted to
determine whether G protein binding to EpoR holds true in normal human
erythroid progenitors. CD36+ red cell progenitors were isolated after
culture of CD34+ cells from umbilical cord blood (49) and then deprived
of growth factors and stimulated or not with Epo. The data shown in
Fig. 4B demonstrate that an association between
i and EpoR also exists in normal erythroid progenitors
and that Epo significantly reduces the amount of Gi associated with the EpoR.
Gi Binds to the C-terminal End of EpoR Cytoplasmic
Region--
To identify the region of the EpoR involved in G protein
binding, we first examined Gi association to EpoR in TF-1
cells (Fig. 5). This human
erythroleukemia cell line was previously shown to overexpress an
abnormal EpoR caused by a deletion of the 96 C-terminal amino acids,
together with a minor expression of full-length EpoR (47, 56). TF-1
cells were solubilized, and the G
i protein coprecipitated with EpoR was detected by in vitro
ADP-ribosylation in the presence of pertussis toxin. Very little
G
i was detected in anti-EpoR immunoprecipitates either
before or after Epo stimulation, suggesting that these cells have a
defect in G
i binding to EpoR. When the cells were
infected with a virus encoding a normal murine EpoR (TF-1-ER),
G
i binding to the EpoR was restored. This suggests that
the C-terminal end of EpoR is necessary for G
i binding
to EpoR.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 5.
Defect in Gi association to EpoR
in TF-1 cells. TF-1 cells or TF-1 expressing murine EpoR (1 × 107 cell equivalents) were deprived of growth factor and
stimulated (+) or not ( ) with Epo for 15 min. Cell lysates were
immunoprecipitated (IP) with anti-EpoR antiserum (EpoR) or
preimmune serum (C) and G i coprecipitated
with EpoR was detected by in vitro ADP-ribosylation.
|
|
To examine this possibility in greater detail we next studied the
association of G
i with wild-type murine EpoR or
C-terminal deletion mutants (Fig.
6A) expressed in FDCP-1 cells.
In FDCP-1 cells transfected with the murine EpoR deletion mutants
41
and
24, no G
i was found associated with EpoR, and a
weak binding was detected in mutant
20 (Fig. 6B). The
absence of binding was not due to a decrease in EpoR surface expression
because FDCP-1 cells transfected with wild-type EpoR,
41,
24, and
20 EpoR C-terminal deletion mutants expressed, respectively, 695, 580, 685, and 1820 125I-Epo binding sites on the cell
surface. G
i binding to the
5 EpoR deletion mutant and
to EpoR mutant F1-Y58 deleted between amino acids 377 and 431 was
similar to the binding to the wild-type receptor. We controlled that a
similar fraction of EpoR bound 125I-Epo was precipitated
with anti-EpoR antibodies for the different EpoR mutants. In addition G
protein expression, as detected by anti-G
immunoblotting was similar
in FDCP-1 cells transfected with either wild-type EpoR or EpoR deletion
mutants (data not shown). These results show that the region between
amino acids 459 and 479 in the C-terminal end of EpoR is necessary for
heterotrimeric G protein binding to the EpoR. It cannot be excluded
that amino acids 432-458, also present in F1-Y58 mutant, contribute to
the binding.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 6.
Gi protein binding to
the C-terminal end of EpoR intracytoplasmic region. FDCP-1 cells
or FDCP-1 cells expressing murine EpoR or deletion mutants of EpoR
(1 × 107 cell equivalents) were starved of Epo and
stimulated for 30 min with Epo. A, schematic representation
of the different EpoR mutants. B, G i
coprecipitated with EpoR as performed in Fig. 3. WT, wild
type.
|
|
Epo Induces the Release of G Protein from the EpoR--
A decrease
in G protein association with EpoR following Epo addition was
constantly observed in the different hematopoietic cells studied. The
kinetic of G protein binding to EpoR was analyzed in UT7 cells
stimulated or not with Epo for various times at 37 °C. The amount of
Gi coprecipitated with the EpoR in the soluble fraction was
determined by measuring the 32P incorporated in the 41-kDa
protein following in vitro ADP-ribosylation (Fig.
7). Epo induced a rapid decrease in the
amount of G
i bound to the EpoR. About 50% of the
binding was lost after 10 min of incubation, and then the amount of
binding decreased more slowly. We conclude that heterotrimeric
Gi is constitutively associated with EpoR and Epo induces
the dissociation of EpoR-bound Gi. To determine whether
Epo-induced JAK2 tyrosine kinase activation is required for
Gi release from the EpoR, we investigated whether the
decrease in EpoR-bound Gi was observed in 32D cells
expressing EpoR W282R mutant. This mutant has lost the ability to bind
JAK2 and activate the kinase in response to Epo (Refs. 5 and 48 and
data not shown). As shown in Fig. 8 Epo
activation induced Gi release in cells expressing the
normal as well as the mutant receptor. Thus JAK2 activation is not
required for the release of EpoR-bound Gi.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 7.
Decrease of Gi coprecipitated
with EpoR after Epo stimulation. UT7 cells were deprived of growth
factor and stimulated with 10 units/ml Epo for the indicated time. Cell
lysates were immunoprecipitated with anti-EpoR antibodies. The
immunoprecipitates were subjected to in vitro
ADP-ribosylation. The proteins were separated by SDS-PAGE and
transferred to nitrocellulose membrane. G i was
visualized by autoradiography and the amount of 32P present
in the bands was quantified with a PhosphorImager. Data are the
means ± S.E. of three individual experiments.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 8.
Decrease of Gi coprecipitated
with EpoR in 32D cells expressing EpoR mutant defective in Jak2
activation. 32D cells expressing murine EpoR or EpoR mutant W282R
(1 × 107 cell equivalents) were starved of Epo and
stimulated for 30 min with 10 units/ml Epo. Analysis of
G i coprecipitated with EpoR was followed by in
vitro ADP-ribosylation. WT, wild type.
|
|
G Protein Activation by Epo--
G protein-coupled receptors
activate G proteins resulting in GDP exchange for GTP and heterotrimer
dissociation in
and 
subunits. To investigate whether the G
proteins associated with EpoR are activated upon receptor stimulation
with Epo, we first monitored alterations of pertussis toxin-catalyzed
ADP-ribosylation in isolated cell membranes. This assay was used to
assess G
i protein subunit conformational changes,
because the
subunit serves as a good substrate for the toxin only
when G proteins are in the trimeric form (51, 55). Results showed that
preincubation of UT7 cell membranes with Epo inhibited subsequent
ADP-ribosylation of the 40-kDa substrate (Fig.
9A, lanes 4 and
5). The action of Epo is dependant on the Mg2+
concentration (Fig. 9A, lanes 1-4), which is
also essential for Gi protein trimer dissociation (57).
GTP
S, the nonhydrolyzable GTP analog that causes 

subunit
dissociation and irreversibly activates Gi, inhibited
pertussis toxin-catalyzed ADP-ribosylation independently of Epo. These
data show that Epo induces an alteration in G
i and
suggest that Epo activation leads to the heterotrimer dissociation.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of Epo on pertussis toxin-induced
ADP-ribosylation and on
[35S]GTP S binding to
membranes. A, inhibition of pertussis toxin-induced
ADP-ribosylation by Epo. UT7 cell membranes were preincubated with 10 units/ml Epo or 100 µM GTP S for 10 min in the presence
of increasing concentrations of Mg2+ using
Mg2+-EDTA buffer before ADP-ribosylation by pertussis
toxin. The proteins were separated by SDS-PAGE, and G i
was visualized by autoradiography. B, increase in
[35S]GTP S binding by Epo. UT7 or CHO-ER membranes were
pretreated or not with Epo for 15 min, and [35S]GTP S
binding was determined as described under "Experimental
Procedures." Data are the means ± S.E. of duplicate samples and
are expressed as percentages of values from untreated cell
membranes.
|
|
GPCR-catalyzed activation of G proteins is associated with an
enhancement of their GTP binding. We have therefore examined the effect
of Epo on the nonhydrolyzable GTP analog GTP
S binding to cell
membranes. In the presence of Epo, the amount of nucleotide that
specifically bound was increased by 18% in UT7 cell membranes and by
56% in CHO-ER cell membranes (Fig. 9B). The increase in [35S]GTP
S binding observed in Epo-activated CHO-ER
cells is similar to the increase detected in HEK293 cells expressing a
GPCR, the Mel1a melatonin receptor, following activation with melatonin (Ref. 52 and data not shown).
 |
DISCUSSION |
In the present investigation, we provide the first demonstration
of the physical association between the heterotrimeric G protein of the
Gi family and the erythropoietin receptor, both in
hematopoietic cell lines and in human normal erythroid progenitor cells. The Gi protein associated with the EpoR is more
likely in an heterotrimeric conformation. Indeed both G
and
G
i, identified as a 41-kDa ADP-ribosylated band,
coprecipitated with the EpoR. By utilizing natural and engineered
truncated EpoR mutants, we showed that the intracellular C-terminal end
of the EpoR is required for G protein association. The association
between the G protein and EpoR is constitutive. Classical G
protein-coupled receptors are believed to associate with G proteins
following ligand activation (58), although they may in some cases be
preassociated. Actually, the association between a Gi
protein and a single-spanning transmembrane receptor has previously
been reported. A peptide corresponding to the intracytoplasmic sequence
of
-1,4-galactosyltransferase, the sperm receptor for the mouse egg,
bound a heterotrimeric G protein that contained the G
i
subunit (30). G
i was found to copurify with the
chain of insulin receptor isolated from adipocyte plasma membranes
(31). A transient association between G
i and the EGF
receptor also occurred in rat hepatocytes after ligand activation (29,
59), and G
i and G
were recently shown to
constitutively bind the IGF-I receptor (60). A region of 18 amino acids
in the C terminus of EpoR is necessary for Gi binding to
EpoR. A 14-residue sequence in the IGF-II receptor, with several basic
residues, presents structural similarity with the terminal portion of
the third cytoplasmic loops of most G-coupled receptors. This sequence
activates and directly interacts with Gi proteins (61, 62),
but the EpoR cytoplasmic domain does not contain such a G
protein-binding motif. Another possibility is that the interaction
between Gi and EpoR occurs through an adaptor protein bound
to the EpoR. The tyrosine kinase Jak2 and the docking protein IRS2
constitutively bind to the EpoR membrane proximal region (5, 15).
Because G protein binding to EpoR involves the C-terminal end of the
receptor, these proteins are unlikely to play a role of adaptor between
G protein and EpoR, and another component whose nature is unknown could
be required. The ability of the EpoR mutant W282R to bind
Gi in the absence of Jak2 binding also excludes Jak2 as a
potential adaptor.
Epo induces the release of Gi from the EpoR. Hallak and
co-workers (60) have recently reported that heterotrimeric
Gi is constitutively associated with IGF-I receptor and
that IGF-I also induces the release of the G
subunit from the
IGF-I receptor. Jak2 tyrosine kinase is probably not required for
Gi protein release from the EpoR because this process still
occurs in 32D cells that express the EpoR mutant W282R defective in
Jak2 activation. It cannot be excluded that other tyrosine kinases
involved in Epo activation such as Lyn, Syk, or c-Fes (6-8) may be
required for Gi release. Although Jak2 plays a pivotal role
in EpoR signaling (63, 64), Gi protein release from EpoR
could participate in a new Jak2-independent pathway.
Following activation of heterotrimeric G proteins by classical GPCR,
both the
and the 
subunits dissociate from the receptor and
activate target effectors (58, 65). Epo inhibited PT-catalyzed ADP-ribosylation of G
i in UT7 cell membranes in a
Mg2+-dependant manner, suggesting that the
subunit also
dissociates from the 
subunit complex of the heterotrimeric G
protein after Epo activation. Similar data were obtained in membranes
activated with other single-spanning transmembrane receptors, such as
insulin and IL-1 (66, 67), suggesting that other receptors that GPCR can modify G protein conformation and potentially induce trimer dissociation.
In addition to demonstrating the physical association of Gi
with the EpoR, our data show that Epo increases GTP
S binding in cell
membranes, providing evidence that EpoR can activate G proteins. Some
studies have suggested that Epo may increase adenylate cyclase activity
and cAMP levels (40, 68), whereas others did not report any
modification of cAMP (69). However, to our knowledge, no studies have
reported a decrease in cAMP levels in response to Epo stimulation. In
erythroid precursors a PT-sensitive G
i2 has been
demonstrated to regulate an Epo-modulated Ca2+ channel (40,
41), and G protein activation by GTP
S mimics the rise in
[Ca2+]i (41). These data support an important
role for heterotrimeric G proteins in the signaling pathways of Epo.
Epo-stimulated changes in [Ca2+]i were not
detected in nonhemoglobinized or poorly hemoglobinized early
erythroblasts with a large proliferative capacity. In contrast, in
hemoglobinized late erythroblasts with a reduced proliferative capacity, Epo increased [Ca2+]i (42), suggesting
that regulation of [Ca2+]i may be restricted to
rather mature precursors. Recently, tyrosine 460 in the
intracytoplasmic domain of EpoR was shown to be critical for
Epo-stimulated Ca2+ influx, in CHO cells as well as in
Ba/F3 hematopoietic cells transfected with EpoR mutants (70).
Interestingly the C-terminal end of EpoR required for Gi
binding to EpoR includes the tyrosine 460 (Fig. 6). Although in
erythroid precursors the role of Gi protein in Epo-induced
Ca2+ activation has been clearly established, transducers
that bind to Tyr460 have not yet been identified (70).
Future investigations should allow the identification of the components
that participate in G protein and calcium activation by Epo.
Heterotrimeric Gi proteins could be important intermediates
in cell proliferation mediated by hematopoietic growth factors. Indeed
PT inhibits cell growth induced by IL-1 (38), IL-3 (35), CSF-1 (35),
and GM-CSF (36). Expression of a dominant negative G
i2
inhibits cell proliferation mediated by CSF-1 receptor in macrophages
(39) and cell proliferation and transformation in NIH3T3 cells
transfected with the oncogenic form of the CSF-1 receptor v-fms (71).
PT inhibits Epo-stimulated erythroid colonies formation in rat
erythroid progenitor cells from fetal liver (72). This suggests that
Gi proteins could be involved in Epo-dependent cell growth. Further studies will be required to elucidate the functional significance of G protein coupling to EpoR.
In conclusion, the results presented here show that Gi protein
is physically associated with EpoR. Epo activates G protein in cell
membranes and induces the release of Gi bound to the
receptor in hematopoietic cells. Our data strongly suggest that
Gi proteins are important components in Epo signaling.