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(Received for publication, February 19, 1997)
From the ABSTRACT Activation of the erythropoietin
receptor is essential for the survival, proliferation, and
differentiation of erythroid progenitors. To understand the role of
erythropoietin receptor (EpoR) activation in erythroid differentiation,
we infected primary erythroid progenitors with high-titer retrovirus
encoding the non-hematopoietic prolactin receptor. The infected
progenitors responded to prolactin in the absence of Epo by generating
fully differentiated erythroid colonies. Therefore, differentiation of
erythroid progenitors does not require an intracellular signal
generated uniquely by the EpoR; the EpoR does not have an instructive
role in erythroid differentiation. We also infected primary erythroid
progenitors with retrovirus encoding chimeric receptors containing the
extracellular domain of PrlR and the intracellular domain of either the
wild-type or truncated EpoRs. A chimeric receptor containing only
the membrane-proximal 136 amino acids of the EpoR cytoplasmic domain
efficiently supported prolactin-dependent
differentiation of erythroid progenitors. Substitution of the single
cytoplasmic domain tyrosine in this receptor with phenylalanine (Y343F)
eliminated its ability to support differentiation. The minimal EpoR
cytoplasmic domain required for erythroid differentiation is therefore
the same as that previously reported to be sufficient to support cell
proliferation (D'Andrea, A. D., Yoshimura, A., Youssoufian, H., Zon,
L. I., Koo, J. W., and Lodish, H. F. (1991) Mol. Cell.
Biol. 11, 1980-1987; Miura, O., D'Andrea, A. D., Kabat, D., and
Ihle, J. N. (1991) Mol. Cell. Biol. 11, 4895-4902; He,
T.-C., Jiang, N., Zhuang, H., Quelle, D. E., and Wojchowski, D. M. (1994) J. Biol. Chem. 269, 18291-18294).
INTRODUCTION The EpoR1 belongs to a large family of
cytokine receptors, many of which are required for the proliferation
and differentiation of hematopoietic as well as other cell types
(4-7). Throughout life, eight different hematopoietic lineages arise
from pluripotent stem cells in the bone marrow (8, 9). The exact role
of growth factors in this process is not clear and has been described broadly by two alternative hypotheses. The stochastic hypothesis suggests that commitment of a progenitor to a particular lineage is a
stochastic event, subsequent to which cell differentiation proceeds
along a predetermined program; growth factors are merely required to
ensure the survival and proliferation of committed progenitors
(10-14). The contrasting inductive, or instructive, hypothesis
attributes to growth factors a direct role in cell differentiation,
predicting that growth factor receptors transduce signals that uniquely
specify the differentiation outcome of a progenitor (15-17). A number
of hybrid hypotheses have also been proposed, where, for example,
committed progenitors arise stochastically, but their subsequent
differentiation and acquisition of the mature phenotype are uniquely
induced by lineage-specific growth factors (18).
Although Epo is essential for the production of red blood cells, it is
not thought to play a role in the generation of committed erythroid
progenitors from pluripotent progenitors: expression of recombinant
EpoR does not bias the lineage commitment of pluripotent hematopoietic
progenitors (19, 20), and normal numbers of committed erythroid BFU-e
and CFU-e progenitors are found in the fetal livers of
EpoR Some evidence for the capability of EpoR to promote the erythroid
phenotype comes from the ability of Epo to induce surface expression of
glycophorin (25) and transcription of the
We therefore examined whether the distal half of the EpoR cytoplasmic
domain is essential for differentiation of primary fetal liver
erythroid progenitors. We also examined whether the entirety of the
cytosolic domain of the EpoR can be replaced with the corresponding segment of a different receptor; we chose the prolactin receptor, which
plays no role in hematopoiesis, but belongs to the same subfamily of
cytokine receptors as EpoR, and shares many of its signaling
molecules.
EXPERIMENTAL PROCEDURES CHI338 was generated by ligating a
double-stranded oligomer containing a stop-codon in frame at position
339 of the EpoR between the BSP120I and HindIII sites of the
murine EpoR cDNA (31). CHI374 was made by inserting a stop-codon in
frame in the HindIII site of CHI. CHI374/Y343F was
constructed by ligating a polymerase chain reaction product containing
the point mutation into the BSP120I and HindIII sites of
CHI374. CHI442 was made by subcloning the BSP120I-EcoRI
fragment of pSFFV.tEpoR (32) into the BSP120I site of CHI. All
receptors were expressed in the retroviral expression vector MSCV
(33).
The 293 cell line expressing the
MuLv gag-pol proteins (34) was co-transfected with
penv.min encoding the mouse ecotropic MuLv envelope
glycoprotein, a gift of Dr. David A. Sanders (Purdue University), and
with pSV2neo. Fifty G418-resistant clones were selected; of
these, clone VE23 generated the highest titer of transducing
retroviruses following transient transfection with a reporter MSCV DNA
containing the lacZ gene. To generate transducing retroviruses, VE23 cells were transiently transfected using the calcium-phosphate method with MSCV retroviral constructs each encoding
the desired receptor. Culture supernatants were collected at 48 h
and either immediately frozen or used for infection.
Fetal livers from
BALB/c mouse embryos were harvested at days E13 to 15 and dissociated
by mechanical pipetting. The cells were subjected to a brief treatment
with distilled water to lyse non-nucleated cells, strained through a
70-micron cell filter, and washed in Iscove's modified Dulbecco's
medium (IMDM; purchased from Life Technologies Inc.) and 20% fetal
calf serum. The cells were resuspended either in virus-containing
culture supernatants containing 4 µg/ml polybrene or in control
culture medium with 4 µg/ml polybrene. After rocking for 3-4 h at
37 °C, the cells were recovered by centrifugation. For CFU-e
cultures, the cells were washed in IMDM and resuspended in serum-free
methylcellulose medium (1% methylcellulose in IMDM, 1% bovine serum
albumin, 100 ng/ml IGF-I, 7.7 µg/ml cholesterol, 5.6 µg/ml oleic
acid, 8 µg/ml L- A small aliquot of cells from each infection
was cultured in 20% fetal calf serum with 2 units/ml Epo and used at
36 h. The cells were washed three times in phosphate-buffered
saline containing 0.5% BSA, incubated at 4 °C with 50 µg/ml
monoclonal antibody M110 (gift of Dr. J. Dijane) in the presence of 200 µg/ml each of rabbit and goat IgG, followed by a
phycoerythrin-conjugated goat anti-mouse F(ab Five day 7 BFU-e bursts were individually
aspirated from each methylcellulose culture. Following cytospin, cells
were fixed in methanol, stained in diaminobenzidine, and counterstained
with Giemsa.
RESULTS AND DISCUSSION We used transient
transfection of the packaging line VE23 to generate high-titer
recombinant ecotropic retrovirus and infected day 14 fetal liver cells
in vitro with retroviruses encoding a series of chimeric
receptors. These contained the extracellular domain of the rabbit
prolactin receptor, which is not normally expressed by erythroid
progenitors, and the transmembrane and cytoplasmic domains of EpoR (CHI
(35)) or EpoRs truncated at amino acids 442 (CHI442), 374 (CHI374), or
338 (CHI338; Fig. 1). We then examined the ability of
the infected fetal liver cells to generate erythroid colonies when
cultured in semi-solid methylcellulose medium in the presence of
prolactin (Fig. 2). In parallel, we measured the level
of expression of the chimeric receptors by culturing a small aliquot of
the infected fetal liver cells in liquid medium in the presence of Epo.
After 36 h we incubated the cells with the monoclonal antibody
M110, specific for the prolactin receptor (36), and quantified the
fraction of M110-positive cells by FACS. The window we employed (M1,
Fig. 2A) provides a lower estimate of the fraction of fetal
liver cells infected by each of the chimeric receptors. Infection by
CHI varied from 10 to 25% (Figs. 2, A, C, and
D).
In each of five experiments, the ratio of
prolactin-dependent erythroid BFU-e and CFU-e colonies
formed by CHI-infected cultures to those formed by parallel uninfected
and erythropoietin-treated cultures was the same or higher than the
estimated rate of infection by CHI (Fig. 2). Specifically, Epo
supported the generation of 700-1000 CFU-e colonies (per 2 × 105 fetal liver cells; C); 10% of the fetal
liver cells expressed CHI, and this population generated 100 CFU-e colonies promoted by prolactin. Thus the chimeric receptor CHI,
containing the full-length cytoplasmic domain of EpoR, functions as
efficiently as the endogenous EpoR in supporting erythroid colony
development.
CHI442 and CHI374 were each able to support day 3 prolactin-dependent erythroid colonies in serum-poor medium
(CFU-e-derived colonies) and day 7 erythroid bursts (BFU-e-derived
bursts) to the same extent as CHI (Fig. 2, B and
C). CHI338 was not able to support erythroid colony
formation, although it was well expressed by the infected fetal liver
cells (Fig. 2, B and D). No significant quantitative differences were seen between CHI, CHI442, or CHI374 in
their response to prolactin (Fig. 2, B and C).
The reduction in numbers of colonies and bursts at high concentrations
of prolactin (Fig. 2, C and D) is expected,
assuming the PrlR, a member of the same family of cytokine receptors as
the Epo and growth hormone receptors, follows the same sequential
dimerization mechanism (37). Importantly, the colony and burst size,
morphology, and degree of hemoglobinization promoted by CHI, CHI442,
and CHI374 were similar to those promoted by Epo acting through the
endogenous EpoR (Fig. 3). Therefore, CHI374 contains a
minimal EpoR domain required for erythroid differentiation. Tyrosine
343 within this domain is essential for erythroid differentiation,
since its replacement in CHI374 with phenylalanine completely abolished
its ability to support CFU-e differentiation (CHI374/Y343F,
Fig. 2D). This same tyrosine is essential for mitogenesis
and is one of the four in the EpoR able to mediate STAT5 activation
(38-41). The minimal EpoR cytoplasmic domain required to support
erythroid differentiation is therefore the same as that previously
found to be sufficient to support cell proliferation (1-3).
The finding that CHI374 can fully support erythroid
differentiation suggests that there is no essential differentiation
signal emanating from the distal part of EpoR; if there is such a
differentiation signal, it must arise from the membrane-proximal part
of the receptor. To examine this point further, we infected progenitors
with retrovirus encoding the wild-type PrlR. Like the EpoR, activation
of the PrlR leads to Jak2 and STAT5 phosphorylation (35, 42, 43). In
hematopoietic cell lines such as Ba/F3, transfected PrlR supports Prl-mediated cell proliferation (35, 42). Figs. 2, C and
D, and 3 show that the PrlR is as efficient as CHI in
supporting formation of both CFU-e- and BFU-e-derived erythroid
colonies. CHI and PrlR were expressed at similar levels by the fetal
liver cells, and there was no significant difference between the
prolactin responsiveness of the CHI-infected and PrlR-infected cells
(Fig. 2, C and D). PrlR-supported colonies were
of the same size, morphology, and extent of hemoglobinization as those
that arose from prolactin-dependent CHI-infected
progenitors or control uninfected Epo-dependent
progenitors. Normal erythrocytes were present in cytospin preparations
of PrlR-supported colonies (Fig. 3). Thus, the cytosolic domains of the
Epo and Prl receptors were equally efficient in supporting erythroid
differentiation when the extracellular domain was activated by
dimerization by prolactin. There is therefore no requirement for an
EpoR-unique signal during the differentiation of erythroid
progenitors.
The unique phenotypic outcome of erythroid differentiation cannot
therefore be due to an essential, EpoR-generated instructive signal.
Erythroid differentiation from BFU-e and CFU-e progenitors apparently
proceeds along a predetermined program, supported by EpoR-activated
"generic" intracellular signals also common to other cytokine
receptors. The exact role of these EpoR-activated signal transduction
proteins is still to be determined. Our finding that the minimal domain
of EpoR able to support differentiation is the same as that required to
support cell proliferation raises the possibility that the role of the
EpoR-activated signals is only to support the survival and
proliferation of the differentiating progenitors, as proposed by the
stochastic hypothesis. It is possible, however, by analogy with studies
on the granulocyte-colony-stimulating factor or thrombopoietin
receptors (44, 45), that, to support erythroid gene expression,
additional signals are needed beyond those required for proliferation.
If this is the case, these signals must arise from the
membrane-proximal domain of the EpoR and cannot be unique to the EpoR.
The inability of tyrosine kinase receptors to fully replace EpoR
function (22, 23, 46) suggests that the signals required for
erythropoiesis, although not unique to the EpoR, are not shared by all
receptors.
Relatively little is known about how unique patterns of gene expression
are generated by different cytokine ligands, particularly since many of
the known signaling proteins are common to more than one receptor (47,
48). All of the signaling proteins known to be activated by the EpoR,
including JAK2, STAT5, SH-PTP1, Grb2, and Ras, are also activated by
other cytokine receptors (6, 7). Here we show that the unique outcome
of EpoR signaling is not due to any unique signals it may generate, but
instead is encoded in the cellular environment of erythroid
progenitors. The specificity of EpoR function in erythroid
differentiation is therefore a result of its unique expression by
erythroid progenitors.
FOOTNOTES ACKNOWLEDGEMENTS We thank D. Sjolly (Viagene, Inc.) for the
293 gag/pol line; David A. Saunders (Purdue University) for
penv.min; Jean Djiane for the M110 monoclonal antibody;
Glen Paradis for assistance with FACS analysis; and Dan Ory, Stefan
Constantinescu, Ralph Lin, Carlos Rodriguez, and Svetlana Bergelson for
helpful discussion.
REFERENCES
Volume 272, Number 22,
Issue of May 30, 1997
pp. 14009-14012
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
§,
**
Whitehead Institute for Biomedical Research,
Cambridge, Massachusetts 02142, the ¶ Laboratoire d'Oncologie
Cellulaire et Moleculaire, Hopital Cochin, 75014 Paris, France, and the
Department of Biology, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02138
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/ mutant mice (21). However, there is a unique
requirement for EpoR activation during the subsequent proliferation and
terminal differentiation of committed erythroid progenitors: the
EpoR/ CFU-e and BFU-e progenitors fail to give rise to
mature erythrocytes unless EpoR is expressed by retroviral infection
(21); and in vitro, other growth factors only partially
substitute for Epo (22-24). It is not known whether EpoR activation is
essential at this stage of erythroid differentiation because of an
EpoR-unique instructive signal or whether it is simply required for
functions that are not unique to EpoR, such as its proliferative and
anti-apoptotic effects.
-globin gene (26, 27) in
pre-B Ba/F3 cells expressing a transfected EpoR. However, the uncertain
lineage commitment of many cell lines and their incomplete
differentiation response makes them less suitable for the study of
signaling in differentiation. EpoR-mediated signaling for proliferation
can be studied in a number of interleukin-3-dependent cell
lines, where heterologous expression of EpoR allows Epo to support cell
proliferation (1, 2). Only the membrane proximal ~120 amino acids is
essential for this function (1-3). Similarly truncated mutants of
other cytokine receptors are also able to support mitogenic signaling
in such cells (17, 28-30). Since the greatest homology between
cytokine receptors is found in the Box 1 and Box 2 domains of their
membrane-proximal regions (30) (see Fig. 1), it might be expected that
this region would generate signals for functions common to all these
receptors such as cell survival and proliferation and that the
divergent membrane-distal regions would endow the specificity of
signaling presumed unique to each receptor.
Fig. 1.
Chimeric PrlR-EpoR receptors. PrlR
extracellular domain, EpoR transmembrane (TM), and
cytoplasmic domains. Shaded rectangles represent regions of
homology with the cytokine receptor superfamily (Box 1, Box 2). The
eight tyrosines in the cytoplasmic domain are marked with a
line.
[View Larger Version of this Image (23K GIF file)]
-phosphatidylcholine, 0.3 mg/ml
transferrin, 4 µl/100 ml monothioglycerol) and ovine-prolactin (the
National Hormone and Pituitary Program of the NIDDK, National
Institutes of Health, Bethesda, MD) as indicated. For BFU-e cultures,
they were resuspended in 30% fetal calf methylcellulose medium (Stem
Cell Technologies) with added spleen conditioned medium, 50 ng/ml rat
stem cell factor, and 500 ng/ml ovine prolactin. Control (uninfected)
cultures in each experiment had Epo added at 2 units/ml. Hemoglobinized
CFU-e colonies were scored on day 3 after staining with
diaminobenzidine. Hemoglobinized BFU-e colonies were scored on day
7.
)2. Cells
were scanned on a Becton-Dickinson cell scanner.
Fig. 2.
Truncated chimeric receptors and the PrlR
support erythropoiesis. A and B, a representative
experiment in which fetal liver cells were infected with chimeric
receptors and cultured for BFU-e burst formation. A, FACS
scan of fetal liver cells, cultured in 2 units/ml Epo for 36 h
postinfection and stained with the anti-PrlR monoclonal antibody M110.
The percent of cells with fluorescein isothiocyanate (FITC)
fluorescence within the range M1 is a lower estimate of the extent of
infection. B, corresponding BFU-e-derived bursts cultured in
methylcellulose in the presence of 500 ng/ml prolactin and no Epo.
Burst numbers represent mean ± S.E. of triplicate measurements,
scored on day 7 of culture. In parallel, and in the same experiment,
uninfected control methylcellulose cultures incubated with 2 units/ml
Epo generated 110 bursts per 2 × 105 cells.
C and D, prolactin dependence of CFU-e-derived
colonies in serum-poor methylcellulose medium. Two representative
experiments are shown out of four; each receptor was tested in at least
two experiments. Shown for each receptor is the fraction of infected fetal liver cells, as determined by culturing cells for 36 h
postinfection in 2 units/ml Epo and staining with fluorescent anti-PrlR
monoclonal antibody M110. In parallel, and in the same experiment,
uninfected control cultures incubated in serum-free methylcellulose
medium with 2 units/ml Epo generated between 700 and 1000 CFU-e
colonies/2 × 105 cells. E, PrlR is as
efficient as CHI in supporting BFU-e burst formation. Each
determination is the mean ± S.E. of three independent experiments. The mean fraction of M110-positive cells was 8% for the
PrlR and 9% for CHI. In parallel, uninfected control cultures incubated in serum-free medium with 2 units/ml Epo generated an average
of 90 bursts per 2 × 105 cells.
[View Larger Version of this Image (19K GIF file)]
Fig. 3.
Truncated chimeric receptors and the PrlR
give rise to erythroid colonies with normal morphology. A,
CFU-e and BFU-e colonies in methylcellulose. Cells were either not
infected and cultured in Epo or infected with the indicated receptor
and cultured in prolactin. Scale bar = 100 µm.
Panels a-d, day 3 CFU-e colonies in serum-poor medium.
Colonies were stained with diaminobenzidine. Panels e-h,
day 7 BFU-e bursts. B, cytospin preparations of individually aspirated BFU-e bursts showing normoblasts at different stages of
differentiation. Cells were incubated with diaminobenzidine, which
stains cytoplasmic hemoglobin brown, and counterstained with
Giemsa. Scale bar = 10 µm. Panel a,
uninfected cells cultured in Epo. Panels b and c,
PrlR-infected cells cultured in prolactin. r = reticulocyte; O = orthochromatophilic normoblast;
p = polychromatophilic normoblast;
Oe = orthochromatophilic normoblast in the process
of exonucleation.
[View Larger Version of this Image (85K GIF file)]
§
Howard Hughes Medical Institute Physician Postdoctoral Fellow.
**
To whom correspondence should be addressed. Tel.: 617-258-5216;
Fax: 617-258-6768; E-mail: lodish{at}wi.mit.edu.
1
The abbreviations used are: Epo, erythropoietin;
EpoR, erythropoietin receptor; Prl, prolactin; PrlR, prolactin
receptor; BFU-e, burst-forming unit erythroid; CFU-e, colony-forming
unit erythroid; IMDM, Iscove's modified Dulbecco's medium; IGF-I,
insulin-like growth factor 1; FACS, fluorescence-activated cell
sorting; MuLv, murine leukemia virus.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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R. Stoffel, S. Ziegler, N. Ghilardi, B. Ledermann, F. J. de Sauvage, and R. C. Skoda Permissive role of thrombopoietin and granulocyte colony-stimulating factor receptors in hematopoietic cell fate decisions in vivo PNAS, January 19, 1999; 96(2): 698 - 702. [Abstract] [Full Text] [PDF]
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M. Socolovsky, A. E.J. Fallon, and H. F. Lodish The Prolactin Receptor Rescues EpoR-/- Erythroid Progenitors and Replaces EpoR in a Synergistic Interaction With c-kit Blood, September 1, 1998; 92(5): 1491 - 1496. [Abstract] [Full Text] [PDF]
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R. C. Gregory, N. Jiang, K. Todokoro, J. Crouse, R. E. Pacifici, and D. M. Wojchowski Erythropoietin Receptor and STAT5-Specific Pathways Promote SKT6 Cell Hemoglobinization Blood, August 15, 1998; 92(4): 1104 - 1118. [Abstract] [Full Text] [PDF]
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K. W. Harris, X.-J. Hu, S. Schultz, M. O. Arcasoy, B. G. Forget, and N. Clare The Distal Cytoplasmic Domain of the Erythropoietin Receptor Induces Granulocytic Differentiation in 32D Cells Blood, August 15, 1998; 92(4): 1219 - 1224. [Abstract] [Full Text] [PDF]
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J. Jacob, J. S. Haug, S. Raptis, and D. C. Link Specific Signals Generated by the Cytoplasmic Domain of the Granulocyte Colony-Stimulating Factor (G-CSF) Receptor Are Not Required for G-CSF-Dependent Granulocytic Differentiation Blood, July 15, 1998; 92(2): 353 - 361. [Abstract] [Full Text] [PDF]
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M. Socolovsky, H. F. Lodish, and G. Q. Daley Control of hematopoietic differentiation: Lack of specificity in signaling by cytokine receptors PNAS, June 9, 1998; 95(12): 6573 - 6575. [Full Text] [PDF]
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M. A. Goldsmith, A. Mikami, Y. You, K. D. Liu, L. Thomas, P. Pharr, and G. D. Longmore Absence of cytokine receptor-dependent specificity in red blood cell differentiation in vivo PNAS, June 9, 1998; 95(12): 7006 - 7011. [Abstract] [Full Text] [PDF]
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S. N. Constantinescu, H. Wu, X. Liu, W. Beyer, A. Fallon, and H. F. Lodish The Anemic Friend Virus gp55 Envelope Protein Induces Erythroid Differentiation in Fetal Liver Colony-Forming Units-Erythroid Blood, February 15, 1998; 91(4): 1163 - 1172. [Abstract] [Full Text] [PDF]
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S. Bergelson, U. Klingmuller, M. Socolovsky, J. G. Hsiao, and H. F. Lodish Tyrosine Residues within the Intracellular Domain of the Erythropoietin Receptor Mediate Activation of AP-1 Transcription Factors J. Biol. Chem., January 23, 1998; 273(4): 2396 - 2401. [Abstract] [Full Text] [PDF]
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M. Ogilvie, X. Yu, V. Nicolas-Metral, S. M. Pulido, C. Liu, U. T. Ruegg, and C. T. Noguchi Erythropoietin Stimulates Proliferation and Interferes with Differentiation of Myoblasts J. Biol. Chem., December 8, 2000; 275(50): 39754 - 39761. [Abstract] [Full Text] [PDF]
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T. J. Pircher, J. N. Geiger, D. Zhang, C. P. Miller, P. Gaines, and D. M. Wojchowski Integrative Signaling by Minimal Erythropoietin Receptor Forms and c-Kit J. Biol. Chem., March 16, 2001; 276(12): 8995 - 9002. [Abstract] [Full Text] [PDF]
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