From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
REVIEW ARTICLE
Structure, Function, and Activation of the Erythropoietin Receptor
By Hagop Youssoufian, Gregory Longmore, Drorit Neumann, Akihiko Yoshimura, and Harvey F. Lodish
E
RYTHROPOIETIN (EPO) is the principal growth factor
that promotes the viability, proliferation, and differentiation of mammalian erythroid progenitor cells, functions
that are transduced by the specific cell surface EPO receptor
(EPO-R). In contrast to many other hematopoietic growth
factors, EPO primarily affects relatively mature erythroid cells,
although it may also influence the growth and behavior of
other cells. The recent cloning of the EPO-R gene has greatly
facilitated the molecular analysis of the structure and function
of this receptor, and provided a fundamental link to earlier
biochemical and cellular studies of erythropoiesis. Here we
describe recent studies on the structure, function, and activation of this receptor. One focus will be on mechanisms by
which the activated receptor can induce cellular proliferation
in the absence of EPO. This can be achieved by binding of
a retrovirus envelope glycoprotein to the EPO-R or by a specific point mutation in the exoplasmic domain of the EPOR. EPO-independent activation of the EPO-R can contribute
to the development of erythroleukemia in animal model systems. A second focus will be on the structure and control of
expression of the EPO-R gene.
MAMMALIAN ERYTHROPOIESIS
AND ERYTHROID CELL LINES
Within the fetal liver and adult bone marrow (BM),
pluripotential hematopoietic stem cells generate erythroid
progenitors. The earliest cells identified ex vivo as erythroid
progenitors are the slowly proliferating burst-forming uniterythroid (BFU-E), which are not responsive to EPO and do
not express the EPO-R. After 48 to 72 hours of growth in
the presence of interleukin-3 (IL-3),132granulocyte-macrophage colony-stimulating factor (GM-CSF),'.* or Steel factor
(SF),3"mature" BFU-E develop that express the EPO-R and
are weakly responsive to EP0.4 After another 4 to 5 days in
culture, these cells give rise to smaller colony-forming unitserythroid (CFU-E). CFU-Es are highly responsive to EPO
and generate erythroblast colonies in 7 days (in humans) or
2 days (in m i ~ e ) .The
~ , ~sensitivity of these erythroid precursor
cells to EPO is transient; it declines gradually with increasing
maturation such that cells beyond the erythroblast stage are
no longer dependent on EPO, and concomitantly display
fewer EPO-Rs per cell.'
Human and mouse CFU-E cells and mouse fetal liver cells
proliferate and differentiate in response to EPO, as do splenic
erythroid precursors derived from mice infected with an anemic strain of Friend spleen focus-forming virus (SFFV-A; see
below). In culture such cells express approximately 300 to
1,000 EPO-binding sites on their surfaces: 20% are high affinity (kd = 100 pmol/L), and 80% are considered low affinity
(kd = 600 pmol/L).*-" In contrast, EPO-Rs of only a single
affinity (kd = 100 to 200 pmol/L) were found during differentiation of human BFU-Es to reticulocytes.'* Many features
of mammalian erythropoiesis are recapitulated by immortalized cell lines, including EPO-independent proerythroblasts
isolated from the spleens of mice infected with a polycythemic
Blood, Vol81, No 9 (May l ) , 1993:pp 2223-2236
strain of Friend virus (SFFV-P). These cells, known collectively as murine erythroleukemia (MEL) cells, generally express less than 1,000 surface EPO-binding sites per cell, but,
unlike CFU-Es, they express receptors of only a single affinity
(240 to 1,000 pmol/L).'," However, they differ from normal
progenitors in at least two respects: they do not require EPO
for proliferation nor do they differentiate in its presence, although a number of chemicals, such as dimethylsulfoxide
and hexamethylene-bis-acetamide, successfully induce erythroid differentiation. In cells expressing both high- and lowaffinity receptors, only the high-affinity form might be responsible for the biologic effects of EPO, as few low-affinity
receptors are expected to be occupied at physiologic concentrations of EPO.'.''
FUNCTIONAL DOMAINS OF THE EPO-R
The cDNA of the murine EPO-R encodes a protein of 507
amino acids that contains a single hydrophobic membrane
spanning domain (Fig 1). The amino acid sequence of the
human EPO-R13 is 82%, identical to that of the mouse prot e i ~ Following
'~
the cloning of the cDNA for the EPO-R,
cDNAs encoding other hematopoietic growth factor receptors
were isolated in rapid succession. Sequence analysis identified
a new family of receptors that share two distinctive features
in their exoplasmic domains: a set of four conserved Cys
residues, and a five-residue motif located close to the transmembrane domain, Trp-Ser-X-Trp-Ser (WSXWS), where X
represents any amino acid.l5-'' Members of this family include
receptors for EPO, IL-2, 1L-3, IL-4, IL-5, IL-6, IL-7, GMCSF, G-CSF, leukemia inhibitory factor (LIF), growth hormone (GH), prolactin, neurociliary trophic factor (CNTF),
and the env gene of mouse myeloproliferative leukemia virus,
v-mpl, along with its cellular homolog, c-mpl.15-23
Expression of the EPO-R in normally IL-3-dependent cell
lines such as Ba/F3 or 32D allows cell proliferation in the
presence of either EPO or IL-3, indicating that the cloned
EPO-R is capable of generating a proliferative signal in these
cell^.^^.'^ In an effort to delineate the domains essential for
signal transduction, we and others26-28
have introduced several
deletions in the cytoplasmic tail of the EPO-R. Two regions
From the Department of Medicine, Hematology-OncologyDivision,
Brigham and WomenS Hospital, Boston, MA; Whitehead Institute
for Biomedical Research, Cambridge, MA: and the Department of
Biology, Massachusetts Institute of Technology, Cambridge, MA.
Submitted October 27, 1992: accepted December 21, 1992.
Supported in part by National Institutes of Health (NIH) PhysicianScientist Awards HL02277 (H.Y.) and AGO0294 (G.L.). Henry M .
and Lillian Stratton Foundation Award (H. Y.), Weizman Fellowship
(D.N.), and NIH Grant HL32253 (H.F.L.).
Address reprint requests to Hagop Youssoufian,MD, HematologyOncology Division, Brigham and Women'sHospital, 221 Longwood
Ave, LMRC 620, Boston, M A 02115.
0 1993 by The American Society of Hematology.
0006-4971/93/8109-0030$3.00/0
2223
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2224
extracellular
YOUSSOUFIAN ET AL
nH c=
a (1,
I 3 1 2 9 4 ACTIVATING MUTATION
/-q
WSXWS
transmembrane
SERINE-RICH SIGNAL
TRANSDUCTION DOMAIN
F
NEGATIVE REGULATORY DOMAIN
COOH
Fig 1. Functional organization of the EPO-R molecule. The schematic diagram shows the relative position of the four conserved
exoplasmic Cys residues (black bars; C), a fifth exoplasmic Cys (C
in parenthesis)which is not present in all members of the cytokine
receptor superfamily. Also shown are the R129C activating mutation, the WSXWS motif, and the cytosolic-facing negative regulatory and signal transduction domains.
of opposing function were defined: a membrane-proximal
region of about 100 amino acids that is sufficient for transduction of proliferative signals, and a distal region that
downmodulates such signals. The proximal region is homologous to the corresponding segment of the IL-2-R pchain, which is also sufficient for signal transd~ction.’~
Deletion of the carboxy-terminal 40 to 90 amino acids of
the EPO-R allows Ba/F3 cells to proliferate in as little as 1
pmol/L EPO, one tenth that required for proliferation of
cells expressing the wild-type EPO-R. Because the deletion
had no effect on the number or affinity of cell-surface EPORs, occupancy of fewer cell-surface receptors by EPO than
in cells expressing the wild-type EPO-R was sufficient to generate the growth-promoting intracellular signal. Thus, the
carboxy-terminal region of the cytoplasmic domain is thought
to downmodulate the intracellular growth signal (see
Interestingly, this inhibitory region also appears to downmodulate the proliferative action of GM-CSF in myeloid
progenitor FDC-PI cells transfected with the EPO-R CDNA?’
suggesting the possibility of “cross-talk” between this family
of receptors. The carboxy-terminal region includes putative
phosphorylation sites or binding sites for a Tyr kinase (Fig
The precise residues involved and the mechanism for
downmodulation of the EPO-R and GM-CSF-R remain to
be established.
The conserved WSXWS motif was predicted to be an essential component of the ligand-binding site of cytokine receptor~.’~,’~
The motif is also found in complement precursor
proteins (C7, CS, and C9), thrombospondin, and properdin,
and is thought to mediate protein-protein interactions.”
Lymphoid Ba/F3 cells expressing mutant EPO-Rs lacking
part or all of this motif, or containing a single Gly insertion
between the two WS residues, express few, if any, EPO-Rs
on the cell surface and do not grow in the presence of EP0.31
Biochemical and immunohistochemical analysis show that
the mutant receptors are retained in the endoplasmic reticulum and are unable to bind EPO. In the case of the IL-2R, mutation of either or both Ser residues in the WSXWS
motif of the IL-2-R @-chaindoes not affect binding of IL-2
or its ability to trigger cell proliferation, whereas substitution
of either of the two Trp residues to Gly or Ser destroys IL-2
binding.32Thus, the Trp residues as well as the spacing between them seem to be critical features of receptor structure
and function.
The three-dimensional structure of the exoplasmic domain
of the related GH receptor consists of two sub-domains, each
of which contains seven p-sheets (Fig 2).33The four conserved
Cys residues form two disulfide bonds that stabilize the structure of the membrane-distal sub-domain. The sequence homologous to WSXWS is not present in the ligand-binding
region. Rather, it is an integral component ofthe membraneproximal subdomain, consistent with the finding that EPOR molecules mutant in this region are unable to fold into
structures that either bind EPO or exit the endoplasmic reticulum.
OLIGOMERIC ORGANIZATION AND METABOLISM
OF THE EPO-R
For several members of the cytokine receptor superfamily,
generation of high-affinity receptors requires the formation
of hetero-oligomers. Examples include receptors for IL-2, IL3, IL-5, IL-6, GM-CSF, and CNTF.34-42
The high-affinity receptors for human GM-CSF, IL-3, and IL-5 share a common
subunit (called @ - s ~ b u n i t )as
, ~do
~ ,receptors
~~
for LIF, IL-6,
and CNTF.4’,42While the oligomeric structure of the EPOR on the cell surface is not yet unequivocally established,
various lines of evidence indicate that the EPO-R exists as a
multimeric complex.
Cross-linking studies of 1251-EP0to most EPO-responsive
cell lines, regardless of ligand affinity, have identified two
nonglycosylated or poorly glycosylated polypeptides of molecular weights 100 to 105 Kd and 85 Kd.43Peptide mapping
of these proteins shows that they are similar or identical in
structure,44 suggesting that they are derived either by alternative splicing from the same mRNA or by differential proteolytic processing. In COS cells transfected with the EPOR cDNA, radiolabeled EPO bound to the cell surface can be
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
STRUCTURE, FUNCTION, AND ACTIVATION OF EPO-R
cross-linked to polypeptides of apparent molecular weights
65 Kd and -105 Kd.I4 The 65-Kd polypeptide is almost
certainly the product of the cloned EPO-R cDNA: it is immunoprecipitated by antisera specific to the cloned EPO-R,
and is immunologically distinct from the 85- and 100-Kd
proteins!’ Whiie the relationship between these polypeptides
I
P-
\
I
2225
is not clear, the overall data are consistent with the notion
that the 65-Kd EPO-R exists as a multimeric complex with
an 100- to 105-Kd polypeptide.
What is the structure of the high-affinity receptor, and what
roles, if any, do the 85- and 100- to 105-Kd polypeptides
play in generating high-affinity EPO binding? Correlations
between the polypeptides cross-linked to EPO and ligand affinities have produced somewhat disparate results. Initially,
DAndrea et all4 reported that expression of the murine EPOR cDNA in COS cells generates both high-affinity and lowaffinity receptors, even though the cDNA was derived from
a cell line (MEL clone 745, derived from a mouse infected
with SFFV-P) that contains only low-affinity receptors. Others
have observed only low-affinity receptors on the surface of
transfected COS cells (D. Hilton and S. Watowich, unpublished observations, October 1992). Mutation of Arg- 129 to
Cys in the exoplasmic domain of the EPO-R (R129C; see
below) causes formation of disulfide-linked homodimers,
many of which are found on the cell surface, and results in
low-affinity receptors that generate an intracellular growth
signal in the presence or absence of EPO.*
By analogy with two related receptors, additional inferences
may be drawn about the oligomeric structure of the EPO-R.
The possibility that EPO itself causes formation of noncovalent EPO-R homodimers on the cell surface is supported by
analysis of the crystal structure of GH-R.33Like EPO, GH
is monomeric, and a soluble form of its receptor forms noncovalent homodimers in the presence of a single GH ligand
(Fig 2). Is the homodimer sufficient to generate a high-affinity
binding site? The G-CSF-R, another related receptor, is able
to form a homodimer that is sufficient to generate a highaffinity receptor!’
The EPO-R appears to have features of
both receptors; while the 65-Kd polypeptide can most likely
form a homodimer, it is probably not sufficient to generate
a high-affinity receptor, as illustrated by the R 129C mutation,
which generates only a low-affinity receptor complex, at least
when expressed in Ba/F3 cells!6
Ligand-occupied receptors are internalized by endocytosis,
and the bound EPO is degraded in a lysosomal compart-
-
I
-P
PROTEIN
KINASES
3
software; photocourtesy of A.M. deVos, Genentech, Inc.) Adapted
with p e r m i ~ s i o nCopyright
.~~
1992 by the AAAS. (B)Hypothetical
model of the EPO-R complex. By analogy to the GH-R complex, a
single moleculeof EPO is shown to bind to the extracellulardomains
of an EPO-R dimer. The EPO bindingsite is distinctfromthe WSXWS
motif. Additional protein-protein interactions occur within the
transmembranedomain of EPO-R (eg, with gp55) and betweenputative protein kinases and the EPO-R cytoplasmic domain. Phos-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2226
YOUSSOUFIAN ET AL
ment." The intracellular fate of the receptor is only partly
understood. Antipeptide antisera generated against the predicted amino- or carboxy-terminal sequences of the mouse
EPO-R immunoprecipitate two forms of the EPO-R from
pulse-labeled, EPO-R-expressing heterologous cells
(NIH3T3, Ba/F3, HCD57), a nonglycosylated species of molecular weight 62 Kd, and a 64-Kd form that is presumably
localized to the endoplasmic reticulum or another premedial
Golgi compartment, displaying endoglycosidaseH (endoH)sensitive oligosaccharides.30~31~48
Approximately half of the
newly made EPO-R is degraded rapidly (tl,2 -40 to 60 minutes) without acquiring endoH-resistance (presumably being
degraded in the endoplasmic reticulum), while the rest becomes 66 Kd because of the acquisition of an endoH-resistant
oligosaccharide in the Golgi. The 66-Kd species also has a
half-life of 40 to 60 minutes, and less than 5% is found on
the cell surface. Presumably, the balance of the polypeptide
is in vesicles en route from the Golgi to the plasma membrane,
lysosomes, or postendocytotic vesicles derived from the
plasma membrane. The 66-Kd species is apparently degraded
in lysosomes, because inhibitors of lysosome degradation
cause accumulation of both intact 66-Kd EPO-R and an endoproteolytic fragment in lysosomes (D. Neumann and H.F.
Lodish, unpublished observations, October 1992).These calculations are derived primarily from transfected Ba/F3 cells
expressing newly synthesized EPO-R polypeptides, and it is
possible that the metabolic fate of the EPO-R may be significantly different in erythroid cells in which putative accessory
molecules are expressed. Thus, by analogy to the T-cell receptor
the stability of the EPO-R in erythroid
cells and its surface expression may be dictated by the presence
of accessory subunits. The pp130 is one such l and id ate.^'
TRANSMEMBRANE SIGNALING BY THE EPO-R
Given the overall functional similarities among members
of the cytokine receptor superfamily, any molecular insight
into the mechanism of signaling by one member may be
applicable to others. Sequence analysis of the cytoplasmic
domains from these receptors has provided no clear understanding of the signal transduction system(s) to which they
are coupled. The cytoplasmic domains not only lack homologies to known protein kinases, they also differ greatly
in length and show no clear homologies among different
members, save for moderate regions of homology between
segments ofthe EPO-R and IL-2-R
Thus, despite
the availability of the cloned receptor, growth-regulatory signals transduced by the EPO-R remain poorly understood.
Before cloning of the receptor cDNA, it was shown that EPO
induces a rapid Ca2+influx in erythroid cells infected with
Friend virussoand in immature erythroblast^.^^.^' By contrast,
mouse CFU-Es did not show a similar increase in intracellular
ca2+
.53 Intracellular cyclic adenosine monophosphate
(CAMP)levels increased during EPO-induced signaling as a
result of adenylate cyclase a c t i v a t i ~ n .Partially
~ ~ . ~ ~ conflicting
observations were reported in two EPO-responsive cell lines:
while EPO induced a rapid increase in cAMP levels and erythroid differentiation in SKT6 cells,56it had no effect on
cAMP levels in TSAS cells, even though cAMP potentiated
the differentiation of these cells in combination with EP0.57
The signal transduction pathway of the EPO-R may include
activation of phospholipases A2 and C, resulting in the liberation of diacylglycerol and arachidonate and subsequent
formation of lipoxygenasemetabolites of a r a ~ h i d o n a t eEPO
.~~
had no effect on phosphoinositide turnover.56 It is possible
that EPO signaling is mediated by more than one intracellular
pathway.
A major difficulty encountered in the analysis of EPO-Rmediated signal transduction has been the paucity of receptors
on the cell surfaces9and lack of cell lines that depend exclusively on EPO for proliferation and differentiation. The latter
problem has been partly overcome by the generation of heterologous cell lines expressing the EPO-R. An instructive set
of results has emerged from comparison of hematopoietic
and nonhematopoietic cells transfected with the EPO-R. Stable expression of the EPO-R in IL-3-dependent murine hematopoietic cells, such as lymphoid Ba/F3 cells24and myeloid/erythroid progenitor FDC-P1 cells28,60and DA-3,2536'
allows them to grow in the presence of either EPO or IL-3.
Similarly, expression of the receptors for IL-2,29IL-6,35GMCSF,38 and G-CSp2 in Ba/F3 cells also allows the corresponding hormones to replace IL-3 in supporting growth,
and expression of the human IL-4-R confers IL-4 dependence
on CTLL cells that are normally IL-2-de~endent.~~
However,
surface expression of the EPO-R in NIH-3T3 fibroblasts is
not accompanied by EPO-induced proliferation,48s59
indicating that some components of the EPO-R signal transduction
mechanism may be unique to hematopoietic cells. The ability
of various members of the cytokine receptor family to substitute for each other suggests conservation in the mechanism
of signaling by these receptors. Nevertheless, these effects must
be interpreted with caution, since IL-3 autonomy may be
conferred by other classes of receptors to such factor-dependent cells. For example, even though the epidermal growth
factor (EGF) receptor is not a member of the cytokine receptor superfamily, it confers EGF responsiveness on 1L-3dependent cells.64
Expression of the constitutively active EPO-R (R129C) in
primary cultures of mou.se fetal liver cells completely abrogates the EPO requirement for CFU-E to form red blood
cells (RBCs). In the absence of exogenous growth factors, the
R 129C EPO-R does not enhance development of BFU-E or
render other types of progenitor cells independent of IL-3 or
GM-CSF, which is in contrast to the situation with the IL3-dependent cell lines described earlier. However, when fetal
liver progenitors are cultured in the presence of SF (but not
in the presence of IL-3 or GM-CSF), EPO-R(R129C) augments the growth and differentiation of erythroid bursts,
mixed erythroid/myeloid, and GM colonies. Thus, aberrantly
expressed and constitutively activated EPO-Rs can stimulate
proliferation of some GM progenitor^.^^
Nevertheless, some clues have emerged from closer sequence comparisons of the cloned receptors and mutagenesis
experiments. A subset of the family, including the receptors
for EPO, IL-2, IL-3, and IL-4, contains a high percentage of
Ser and Pro residues in the cytoplasmic region, which suggests
that this subset may share similarities in signaling. In the case
of receptors with Tyr-kinase domains, such as those for colony-stimulating factor- 1 (CSF-1) and EGF, the carboxy-ter-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
STRUCTURE, FUNCTION, AND ACTIVATION
OF EPO-R
minal regions contain Tyr residues that are subject to
auto-phosphorylation, thereby downmodulating receptor
signaling.66A similar situation may exist for the EPO-R.
Comparison of the phosphorylation profiles of the wild-type
EPO-R with EPO-R mutants having deletions of 30 or 40
residues at the carboxy-terminus shows weak autophosphorylation of the mutant receptors in vitro. As described earlier,
the carboxy-terminal region downmodulates proliferation in
response to EP0.26-28
Phosphorylation of Tyr residues in this
region of the EPO-R could negatively regulate intracellular
signaling. Alternatively, the carboxy-terminal region could
act as a recognition site for a protein kinase that phosphorylates another part of the EPO-R (Figs 1 and 2), as suggested
recently for the 6-chain of the IL-2-R.67This region of the
EPO-R may also interact directly or indirectly with neighboring receptors on hematopoietic cells, such as the GMCSF-R complex, and may be regulated by the same molecule,
possibly a novel protein kinase. The auto-phosphorylation
sites of Tyr-kinase receptors bind to proteins that contain src
homology 2 (SH2) domains. These sequence motifs are found
in src protein, other proto-oncogenes, noncatalytic regions
of cytoplasmic Tyr-kinases, GTPase activating protein (GAP),
phosphoinositide (PI)-kinase, and phospholipase C (PLC)y.66In several cases, SH2 domains have been shown to bind
to Tyr-phosphorylated proteins, including the Tyr-phosphorylated receptor for platelet-derived growth factor
(PDGF). We do not know whether the phosphorylated carboxy-terminal region of the EPO-R also binds to an SH2containing protein.
EPO generates signals for both differentiation and proliferation of erythroid progenitors. This dual action can be dissected by treatment with herbimycin, which selectively inhibits the proliferative activity of EP0.6BUnfortunately, EPOinduced differentiation has been difficult to analyze at the
molecular level, because Ba/F3 and FDCP-1 cells that express
transfected EPO-Rs fail to differentiate in response to EPO,
even though they become dependent on it for proliferation.
Thus, it has not been possible to reconstitute the differentiation response with the cloned EPO-R. The intriguing possibility that additional molecules coupled to the EPO-R may
be involved in transducing differentiation signals is suggested
by the cross-linking of '251-EP0 to EPO-responsive cells,
which shows a unique 119-Kd p ~ l y p e p t i d ethat
~ ~ is not seen
in the transfected cells.
PHOSPHORYLATION AND EPO-INDUCED SIGNALING
Protein phosphorylation is an important early step in
signal transduction by growth factor receptors, many of
which contain intrinsic Tyr kinase domains and exhibit
ligand-dependent Tyr phosphorylation. Examples of such
receptors include those for PDGF, EGF, CSF-1, and insulin.66Several observations suggest that phosphorylation
also plays an important role in EPO-R signaling and
erythropoiesis. Tyr-kinase inhibitors block the growth of
EPO-dependent erythroleukemia cells and induce erythroid differentiation.68370Murine retroviruses encoding
Tyr-kinases such as the ubi7' and src7' oncogenes efficiently
transform erythroid cells. In EPO-dependent cell lines both
EPO and IL-3 induce rapid phosphorylation of Tyr residues
2227
in several common and several unique cellular prot e i n ~ . ~These
~ , ' ~ data suggest the involvement of a protein
Tyr-kinase in EPO-mediated signal transduction. However, Ser/Thr phosphorylation has also been implicated in
EPO action: inhibitors of protein kinase C block transcriptional induction of c-myc by EP0,74an effect presumably mediated by the EPO-R. Inhibitors of Tyr-protein
kinases as well as of Ser/Thr protein kinases inhibit the
EPO-induced transition of HCD57 cells from the Go to
the S phase of the cell cycle. A regulatory role for Tyr
phosphatases was also ~uggested.~'
EPO also induced the
activity of nuclear protein kinase C in erythroid progenitor
cells.76
As there is no obvious kinase motif in the cytoplasmic
domain of cytokine receptors, attention has been directed
toward the identification of cytosolic proteins that may couple
ligand-activated receptors to downstream signals. One such
protein may be the c-raf proto-oncogene, which encodes Raf1. a ubiquitously expressed 74-Kd cytosolic Ser/Thr protein
kinase.77EPO induces rapid phosphorylation of serine and
tyrosine residues on Raf- 1 in the murine erythroid cell line
HCD-57 and in EPO-R-expressing FDC-P1 cells, an effect
similar to that seen after stimulation with IL-3, GM-CSF,6°378
or IL-2.79This is in contrast to the phosphorylation of Raf1 induced by other classes of mitogens (eg, T-cell activators,
PDGF, EGF, insulin, and CSF-l), which occurs predominantly on Ser residues. Finally, antisense oligodeoxyribonucleotides that specifically reduce the intracellular concentration of Raf- I also inhibit both IL-3- and EPO-stimulated
DNA synthesis.60
As noted earlier, the EPO-R itself appears to undergo
phosphorylation. After EPO stimulation of EPO-R-expressing Ba/F3, UT-7, or DA-3 cells most of the receptor remains
as a 64- to 66-Kd nonphosphorylated species, while a small
fraction is converted to a phosphorylated 72-Kd species.25330380
The 72-Kd form of the EPO-R is concentrated on the cell
surface.30By contrast, this species is virtually undetectable
in either NIH-3T3 fibroblast cells expressing the wild-type
EPO-R (A. Yoshimura and H.F. Lodish, unpublished observations, July 1992) or in Ba/F3 cells expressing a mutant
form of the EPO-R that lacks part of the cytoplasmic domain
essential for signal t r a n s d u ~ t i o n Thus,
. ~ ~ ~ formation
~~
of the
72-Kd phosphorylated EPO-R species may be closely related
to the proliferative action of the receptor. As it might be
predicted for a signal-competent molecule, the level of phosphorylated EPO-R decreases to baseline within a few minutes
of EPO stimulation. In addition to the 72-Kd species, in vitro
phosphorylation of partially purified complexes of EPO and
cell-surface EPO-R show an associated 130-Kd protein
(ppl30), which may be a protein kinase that is copurified
with the EPO-R complex or a substrate of the receptor-associated kinase(s) (Fig 2). Anti-phospho-Tyr antibodies precipitate both the 72-Kd EPO-R and the associated ppl30,
indicating that both molecules contain p h ~ s p h o - T y r . ~ ~
In summary, the cumulative data suggest that the EPO-R
undergoes phosphorylation in response to EPO, which in turn
induces the phosphorylation of other cytoplasmic or membrane proteins. The precise sequence of events that eventually
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2228
YOUSSOUFIAN ET AL
culminate in a growth signal, as well as the counter-signals
that turn off the receptor, remain to be elucidated.
EXPRESSION OF THE EPO-R IN NONERYTHROID CELLS
Murine megakaryocytes and their precursors respond to
EPO in serum-free culture”; these cells exhibit cell-surface
EPO receptors of a single affinity (kd -300 pmol/L).82 In
addition, EPO administration to rodent^*^.*^ or humans” results in a significant increase in the platelet count and marrow
CFU-megakaryocytes (CFU-MK), although the effects are
generally more impressive in rodents. These observations
strongly suggest the presence of functional EPO-Rs on the
surface of megakaryocytes.
In nonhematopoietic cells, binding studies have shown receptors of a single affinity (kd = 1,000 pmol/L) in rat and
mouse placenta,*6as well as in embryonic hamster yolk sac”
and in endothelial cells of human umbilical vein and bovine
adrenal capillary.** Northern blot analysis of RNA from
mouse placenta confirms the presence of EPO-R transcripts
(D. Neumann and H.F. Lodish, unpublished observations,
October 1992). What is the function, if any, of EPO and
EPO-R on these cells? The observation that radiolabeled EPO
can be transferred to the fetus from the maternal circulationg9
suggests that the EPO-R may be involved in transcellular
transport of EPO, similar to the function of the IgA receptor
in transepithelial transport of Igs.” EPO also enhances the
migration of endothelial cells.**Therefore, quite apart from
its function in erythroid cells, the EPO-R may play roles in
nonhematopoietic cells that are hitherto unappreciated.
ACTIVATION OF THE EPO-R: MUTATIONS AND
INTERACTION WITH A VIRAL PROTEIN
At least three different mechanisms are currently known
to trigger the proliferative functions of the EPO-R: (1) binding
of its natural ligand, EPO; (2) binding of the env gene product
of a murine erythroleukemia retrovirus; and (3) the R129C
mutation in the extracellular domain ofthe EPO-R. The latter
two mechanisms, while so far unique to the EPO-R, suggest
that leukemogenic viruses or mutations in cytokine receptors
play roles in the pathogenesis of many types of leukemia.
Friend virus is a complex of a replication-competent murine leukemia virus (F-MuLV) and a replication-defective
spleen focus-forming virus (SFFV). Although this complex
is acutely transforming and produces erythroleukemia when
injected into adult mice, neither virus in the complex contains
oncogenes. The two known strains of SFFV (the anemic and
polycythemic strains) differ in the early manifestations of
disease, although both ultimately give rise to erythroleukemia.” Genetic studies have shown that the virus responsible
for induction of erythroleukemia is the defective SFFV, specifically the transmembrane protein product of its env gene,
called gp55.92*93
The gp55 encoded by SFFV-P binds to the
EPO-R and activates it for proliferation, resulting in growth
of the host cells irrespective of the presence of EP0.24,94
While
a large portion of the gp55-EPO-R complex accumulates in
the endoplasmic reticulum, some of it is expressed on the
cell surface; this fraction is thought to be essential for the
transforming ability of SFFV.95The binding of gp55 does
not interfere with the binding of EPO to the EPO-R,26,96
im-
plying that these “ligands” bind to different sites on the exoplasmic domain of the EPO-R. It is principally the transmembrane domain of gp55 that confers EPO-independent
cell growth.97When chimeras of the EPO-R and the IL-3-R
polypeptides were expressed in Ba/F3 cells, only those molecules that contained the transmembrane domain of the EPOR could be activated for proliferation by gp55, thereby
emphasizing the interaction of gp55 and EPO-R via their
transmembrane domains.’*
Considerably less is known about the mechanism by which
the SFFV-A strain of Friend virus causes leukemia. Leukemic
cell lines isolated from animals infected with SFFV-A remain
dependent on EPO for growth, and despite much effort it has
not been possible to demonstrate functional or physical interactions between the EPO-R and the env gene product of
SFFV-A. The nucleotide sequences of env genes from the
“A” strains have been compared with their SFFV-P counterparts to identify potentially important difference^.^^ Typically, env genes of SFFV are the products of recombinations
and deletions of their counterpart genes from other retroviruses: the amino termini of gp55 molecules are derived from
the env genes of endogenous mink cell focus-inducing (MCF)
viruses, and the carboxy termini from ecotropic F-MuLV
env-like sequences. The greatest sequence disparity between
the env genes of SFFV-P and SFFV-A resides in and around
the transmembrane domain. Chimeras of the env genes of
the SFFV-A and SFFV-P viruses indicate that the -40 amino
acids, including the transmembrane region and carboxy-terminus, determine the ability of the env gene to stimulate
EPO-independent pr~liferation.~’
These observations suggest
that the transmembrane region of SFFV is important for activation of the EPO-R. The env protein of the MCF virus can
also bind to and activate the EPO-R, and possibly the IL-2pR,’OOthrough interaction of its amino-terminal domain with
the EPO-R. Lastly, the replication-competent helper virus FMuLV can also produce leukemias of multiple lineages in
newborn mice, but not in adults, suggesting that erythroleukemias induced by F-MuLV result from the generation of
MCF-like env genes in adult mice.’OO
Two classes of mutations that enhance the activity of the
EPO-R have been identified by a genetic selection strategy
that takes advantage of the inherent mutagenic potential of
retroviral transduction systems. The first class (tEPO-R) includes the carboxy-terminal deletions noted earlier. The
cDNA of tEPO-R appears to have sustained a deletion spanning the 3‘ coding and noncoding region, resulting in the
replacement of the terminal 42 amino acids (from Gly 1424
to Gly 16 16) of the wild-type EPO-R with two amino acids,
Ala and Leu.26This mutation renders the receptor hypersensitive to bound EPO allowing cells expressing tEPO-R to grow
in 1 pmol/L EPO, one tenth the physiologic concentration,
but not in its absence.26Otherwise, as stated earlier, Ba/F3
cells expressing tEPO-R and wild-type EPO-R have similar
numbers of cell-surfaceEPO-Rs, similar binding affinities for
EPO, and similar characteristics of endocytosis and degradation of bound EPO. The second class of mutations, termed
constitutive (cEPO-R), confers on the cell the ability to grow
autonomously in the absence of EPO or other growth factors.
Two different mutant cEPO-Rs have been identified.26One
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
STRUCTURE, FUNCTION, AND ACTIVATION OF EPO-R
contains a single C to T substitution in an otherwise wildtype cDNA, resulting in the substitution of a Cys for Arg at
codon 129 in the exoplasmic domain (EPO-R R 129C). Another is a double mutant containing the carboxy-terminal
deletion of tEPO-R as well as the R129C mutation.
The physiologic effects of the R 129C mutation mimic precisely the activation of the wild-type receptor by gp55.26,48
In
addition to conferring factor-independent growth, the mutation causes accumulation of the receptor in the endoplasmic
reticulum, and prevents the rapid intracellular degradation
characteristic of the wild-type receptor. As noted above, the
R 129C mutant exists as disulfide-linked dimers on the cell
surface as well as in the endoplasmic reticulum, an effect
directly attributable to the mutant Cys at codon 129.46Thus,
although there is no direct evidence that EPO induces dimerization of the cell-surface receptor (Fig 2), the disulfidelinked R 129C homo-dimer is thought to mimic dimerization
of the wild-type EPO-R triggered by EPO binding.
To determine whether the R129C mutant could contribute
to the development of leukemia, a recombinant SFFV was
generated in which the gp55 was replaced with the R129C
EPO-R.48When injected into adult mice, the virus caused
polycythemia and splenomegaly. Clonal, growth factor-independent erythroid cell lines arrested at the proerythroblast
stage were isolated from infected mice expressing the R129C
EPO-R. Injection of these cells back into mice resulted in
rapid development of erythroleukemia. This was the first
demonstration of a truly oncogenic point mutation in a
member of the cytokine receptor superfamily.
Unlike infection with wild-type SFFV, infection with recombinant SFFV expressing the R129C EPO-R showed a
delay in the appearance of early polyclonal erythrocytosis ( 1
to 2 weeks v 5 weeks); a large increase in the number of
megakaryocytes in the spleen, comparable with wild-type
SFFV, but also associated with a transient increase in circulating platelet numbers; and expression of the R129C EPOR in splenic megakaryocytes (Longmore G, Neumann D,
Lodish HF: submitted). Infection of fetal liver cells with this
mutant SFFV also causes proliferation of CFU-E, BFU-E,
and CFU-GM cells.65However, there is no obvious arrest in
differentiation of these cells due to expression of the R 129C
EPO-R. These results demonstrate that SFFV is capable of
infecting and expressing its genes in cells of multiple hematopoietic lineages. Why, then, is SFFV-induced disease by
viruses expressing the R129C EPO-R restricted to erythroid
cells?
Early in the course of SFFV virus infection there is a polyclonal erythroblastosis, without any increase in the self-renewal capacity or abrogation of differentiation potential of
the
After 4 to 6 weeks clonal, growth factor-independent, leukemogenic cells evolve, which are arrested at the
proerythroblast stage of dif"rentiation.'0'~''2 Activation of
the EPO-R by gp55 is thought to be responsible for the early
erythroblastosis, which could facilitate accumulation of subsequent genetic mutations that result in the promotion of a
leukemic clone altered in its differentiation program. Similarly, the structurally activated EPO-R R 129C could supplant
gp55 as an oncogenic agent in Friend virus-induced erythroleukemia by promoting uncontrolled erythroblastosis, re-
2229
sulting in the acquisition of further genetic changes that culminate into frank leukemia. As with gp55, the role of EPOR R 129C, if any, in the later stages of the disease are unclear.
Later events thought to contribute to the development of
immortal, leukemic cells in SFFV-induced erythroleukemia
include insertional activation of the putative oncogene spi1 ,Io3 and/or inactivation of the suppressor oncogene ~ 5 3 . ~ ~ ~ 9 ' ~ ~
Assuming that many types of mutations are acquired during
active DNA replication, the expression of EPO-R R I29C in
nonerythroid cells may not induce a sufficient degree of cell
proliferation to allow accumulation of secondary mutations
necessary for leukemogenesis. Alternatively, distinct batteries
of secondary mutations may be required for the development
of tumors derived from different types of hematopoietic progenitors.
Another major function of EPO is the prevention of programmed cell death (apoptosis) in late-stage erythroid progenitor~.~"'~
An alternatively spliced form ofthe human EPOR that causes truncation of the cytoplasmic domain was found
to be the most abundant mRNA species in early erythroid
progenitors, whereas the full-length species was found to predominate in late progenit~rs.''~Although the truncated receptor transduced a mitogenic signal, it was significantly less
competent than the full-length receptor in preventing apoptosis of transfected cells. These findings suggest that stagespecific posttranscriptional changes in the expression of the
EPO-R can give rise to receptors with distinct functional roles.
GENETIC LOCUS OF THE EPO-R
Soon after the cloning of the cDNAs for the mouse EPOR, the complete genomic sequences encoding the EPO-R and
the regulatory elements that govern its transcription were
characterized.
More recently, genomic sequences of the
human homologue'10'1'3of the murine receptor and those of
two other members of the cytokine receptor superfami l ~ ' ' ~ have
- " ~ also been determined. Several important observations have emerged from these studies.
Chromosomal localization and Southern analysis showed
that both the mouse and human EPO-R genes are present
in a single copy per haploid genome.108~11~'13
The gene for
the mouse EPO-R is on the proximal arm of chromosome
9, near the centromere and closely linked to the low-density
lipoprotein receptor locus, while the human gene is on chromosome 19p.'''~''' These assignments do not show any obvious linkages to pathologic disorders of erythropoiesis. Furthermore, unlike the impressive clustering of other growth
factor genes and their receptors on human chromosome 5q,
there is no similar clustering of members of this family of
receptors or their ligands. For example, the gene for the human IL-2-R /3-chain is on chromosome 22,"491'5the IL-4-R
gene is on human chromosome 16 and mouse chromosome
7,'" and the mouse IL-5-R gene on chromosome 6."' The
chromosomal locations of the ligands are also widely dispersed
(eg, the gene for human EPO is on chromosome 7). Although
the genes for IL-3 and GM-CSF are syntenic (chromosome
5q), those for their receptors as well as those for other members of the cytokine superfamily are not syntenic.
Despite the lack of chromosomal clustering, a remarkable
conservation exists at the level of genomic organization of
1083109
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
YOUSSOUFIAN ET AL
2230
these receptors. Both the mouse and human EPO-R genes
are relatively small, spanning 5 to 6.5 kb, with 8 exons and
7 introns.'08~109~111~112
The sizes of individual exons and introns
and the intron-exon junctions show an extremely high degree
of conservation. The overall identity of the nucleotide sequence of the coding portions of the mouse and human genes
is 81.6%. Another region of homology is in the upstream
sequences neighboring the transcriptional start sites. In the
3' region of the mRNA, the homology ends abruptly after
the stop codon. Therefore, it appears that this relatively small
locus defines a complete transcriptional unit (Fig 3).
The conservation ofgenomic structure also extends to two
other genes of this receptor superfamily characterized recently, those for the human IL-2-R p-~hain"~,"'and the
human and mouse IL-7-R1I6 genes. Like the EPO-R gene,
the IL-7-R gene also contains 8 exons and 7 introns. However,
the IL-2-R &chain gene has 10 exons: the first encodes 5'
untranslated sequences, and the fourth and fifth exons together correspond to the third exon of the EPO-R. The
grouping of these two exons into one facilitates the alignment
of the coding regions of these receptors and leads to the following observations. First, the extracellular domain of these
receptors is encoded by five exons, except for the human IL2-R &chain gene which contains the "split" exon. Second,
each pair of conserved Cys residues is encoded by a separate
exon. Third, the transmembrane domain is encoded by a
single exon. This organization sequesters the exon encoding
the WSXWS motif from that encoding the transmembrane
domain. Fourth, the cytoplasmic domain of each receptor is
encoded by two exons, a small exon encoding the membraneproximal region, and a much longer one encoding the bulk
of the cytoplasmic domain as well as all of the 3' noncoding
sequence. These observations clearly show the evolutionary
relationship of genes encoding these cytokine receptors, and
suggest the intriguing possibility of an ancestral gene that
gave rise to some, if not all, members ofthe cytokine receptor
superfamily.
EXPRESSION OF THE EPO-R GENE
Inasmuch as the expression ofthe EPO-R by hematopoietic
cells represents one of the earliest events after committment
to the erythroid lineage, identification of its regulatory elements could shed light on the molecular events of early
erythropoiesis. Transcriptional activation of the EPO-R gene
occurs when murine embryonic stem cells differentiate in
vitro into hematopoietic precursors.'" However, the specific
erythropoietic stage at which this initial gene activation takes
place is unknown. As described earlier, certain cultured
erythropoietic cells exhibit a dramatic increase in the number
of cell-surface EPO-Rs upon progression from the BFU-E to
the CFU-E stage.‘'^^ This second increase in the level of the
EPO-R is at least partly the result of an increase in transcription of the EPO-R gene. Additional evidence for such a second
transcriptional activation step comes from a comparison of
two murine cell lines arrested at different stages of erythropoiesis. CB5 cells are thought to be arrested at the BFU-E
stage of erythropoiesis. 12' By Northern analysis, EPO-R
expression in these cells is about one fifth to one tenth the
level in the more mature MEL cells (H. Youssoufian, submitted). Hence, the transcription of the EPO-R gene, in addition to being tissue specific, is also temporally regulated
during erythropoiesis.
What are the cis- and trans-acting elements responsible
for these regulatory events? Transcriptional initiation of both
the mouse and human EPO-R genes occurs in the 5' flanking
region about 150 bp upstream of the ATG initiation codon.108~109~1"3'12
In the vicinity of the transcription initiation
sites there are no TATA or CAAT boxes characteristic of
many tissue-specific genes. However, there are potential
binding sites for the ubiquitous Sp-1 and the erythroid-specific
GATA-1 transcription factors (Fig 3).12*The location of these
elements is well conserved relative to the transcription initiation sites in the mouse and human genes. It is possible
that GATA-I is involved in transcriptional activation and
3'
5'
I
/
/
I
I
1Kb
/
/
I
/
/
/
i
-)i
0.5Kb
I
e?.
u
Enhancer
2
u
Inhibitor
U
Minimum Promoter
Enahncer
Fig 3. Schematic diagram of the structure and transcription of the mouse EPO-R gene. The structure of the mouse EPO-R gene is shown
at the top of the figure. Coding (black boxes) and noncoding portions of exons (white boxes), introns and flanking sequences (lines), and
the transcriptional start site (bent arrow) are indicated. The lower figure shows the positions of cis-acting sequences that regulate the
transcriptional activity of the gene. DNase I-hypersensitive sites (vertical arrowheads) in the EPO-R genomic locus are noted in both
erythroid and nonerythroid cells; different investigators have noted high levels of EPO-R expression associated with sites A and B (H.
Youssoufian, submitted) or site D.lZ7Binding sites for transcription factors that constitute the minimum promoter are indicated (CACCC,
GATA-1, Sp-1). Sequences associated with enhancer and inhibitoractivities are shown. RER (striped box) denotes a rodent-specific, highly
repetitive sequence, which is transcribed in the direction shown by the overlying arrow.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
STRUCTURE, FUNCTION, AND ACTIVATION OF EPO-R
transcription initiation site determination, possibly through
association with TFIID or other transcription factors, and in
erythroid-specific genes a functional GATA- 1 binding site
may supplant the more usual TATA element. A 452-bp fragment derived from the segment immediately 5‘ of the transcription initiation site of the mouse EPO-R gene directs erythroid-specifictranscription of a linked reporter gene.’” This
region includes the Sp- 1 and GATA- 1 binding sites, both of
which bind their cognate proteins.lZ3Single-base mutations
at either site greatly diminish the promoter activity of this
fragment. Mutations in a CACCC site about 40 bp upstream
of the GATA- 1 binding site also greatly diminish promoter
activity (H. Youssoufian, unpublished observations, June
1992). Thus, the minimum promoter for the mouse EPO-R
gene includes binding sites for Sp-I, GATA-1, and the
CACCC-binding protein (Fig 3). Furthermore, the EPO-R
promoter is transactivated by coexpression of GATA-1 in
heterologous cells (NIH3T3, COS) that ordinarily express
neither gene.’23sL24
Signals generated by the EPO-R may, in
turn, enhance the expression of the GATA-1 gene, creating
a positive feedback
These observations point to the
central contribution of the erythroid-specific GATA- 1 transcription factor to EPO-R promoter activity.
Biochemical studies of EPO-induced terminal differentiation of CFU-Es and proerythroblasts have identified a set of
erythroid proteins that are induced (eg, band 3 and band 4.1)
or enhanced (eg, spectrin) by exposure to EP0.’’’ Considering
that GATA- 1 may also activate the transcription of its own
gene,’” these studies, taken together, begin to define some
of the constituents of the genetic activation cascade of mammalian erythropoiesis (Fig 4). The dual positive feedback
loops that culminate in the activation of the EPO-R gene
may help to reinforce the sequential activation of genetic
information along the pathway of erythroid differentiation,
and thus maintain the erythroid-committed state.
Although the activation of the “minimum” 452-bp promoter may account for the “basal” level of transcription that
is seen at the BFU-E stage, it probably cannot account for
the second activation phase during differentiation to the CFUE stage. The contribution of other potential regulatory sequences to the activity of the EPO-R promoter is now being
studied. Sequences further upstream of the minimum promoter seem to have both positive and negative regulatory
functions. The latter includes a transcriptionally active repetitive element that interferes with the activation of transcription by the downstream EPO-R promotor.126The in vivo
role of this element, if any, is not known. In both erythroid
and nonerythroid cells, there are DNase 1hypersensitive sites
in the 5’ flanking region (H. Youssoufian, unpublished observations, June 1992)’” and in intron 1 of the EPO-R
gene,’27some of which appear to be erythroid specific (Fig
3). Such sites are the hallmarks of “open” chromatin, and
are thought to make the neighboring genes and promoter
elements more accessible to transcription factors. One of these
sites (site D, Fig 3) within intron 1 appears to have enhancer
Another site (site A, Fig 3) is associated with stagespecific enhancer activity, because both the hypersensitive
site and the associated enhancer activity are present in MEL
cells but not in CB5 cells (H. Youssoufian, unpublished ob-
223 1
Accessory Factors
Fig 4. Genetic activation cascade of erythropoiesis. Expression
of GATA-1 in concert with other regulatory factors induces the
expression of the EPO-R, which in turn induces the expression of
genes that characterize terminally differentiated erythroid cells. Two
positive feedback loops insure the unidirectional activation of the
genetic program along the erythroid differentiation pathway.
servations, June 1992). Others have not discerned stage-specific differences in DNase I-hypersensitive sites of the EPOR gene.I2’ Further analysis of these elements should yield a
reasonably complete picture of the transcriptional activation
of this receptor.
Finally, while it is clear that GATA-1 is necessary for the
activation of the EPO-R promoter, its expression in concert
with constitutive transcription factors may not be sufficient
to activate the EPO-R gene (H. Youssoufian and G. Longmore, unpublished observations, July 1992). A preliminary
survey of a number of hematopoietic cell lines, both erythroid
and nonerythroid, by Northern analysis shows some discordance in the expression of GATA- 1 and EPO-R. It is possible
that in some cells the level of GATA-I protein is below the
threshold required to activate the EPO-R gene.’22It is also
possible that additional transcription factors (eg, SCL) may
be required to activate the EPO-R locus.’22Alternatively, the
EPO-R gene may be activated in certain cell types by GATAI -independent mechanism^.'^'
THE EPO-R AND HUMAN DISEASE?
The finding that the EPO-R can be activated for proliferation by a single point mutation,26partial deletion,27or by
insertional activation of the EPO-R by the LTR of SFFV‘283129
in murine systems has stimulated the search for genetic defects
in the EPO-R in human hematopoietic disorders. To date,
one such defect has been described, a 3’ end deletion of an
EPO-R gene in the human erythroleukemia cell line TF- 1.
The cell line overexpresses EPO-R mRNA, which otherwise
appears to be structurally normal.’30This finding is consistent
with the animal leukemia model described earlier, whereby
excessive activity of the EPO-R leads to leukemogenesis. In
another study, multiple transcripts of the EPO-R were identified in a human erythroleukemic cell line,I3’ although the
precise genetic defect responsible for this phenomenon is not
known.
A candidate disorder for EPO-R defects is polycythemia
vera (PV), a clonal myeloproliferative disorder that presents
primarily with erythrocytosis, although elevations in granulocytes and platelets also occur. The circulating serum levels
of EPO are lower than normalL3’or normal,’33and ex vivo
cultures of erythroid progenitors derived from patients with
PV grow and differentiate independently of EP0.134Such
cells may also be hypersensitive to EP0135or to other growth
factors such as IL-3’36or insulin-like growth factor I.’37 As
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2232
observed in MEL cells, CFU-Es from patients with PV have
a single class of low-affinity EPO-Rs (kd = 720 pmol/L),138
and cross-linking experiments have identified surface proteins
similar in size (90 and 100 Kd) to those of normal erythroid
pr0genitors.6~This situation is reminiscent of murine model
of EPO-R activation by either the R129C mutation or interaction of the wild-type EPO-R with gp55. The effect of an
activated EPO-R need not be restricted to erythroid cells65;
its expression in megakaryocytes may provide an explanation
for the associated thrombocytosis (Longmore G, Neumann
D, Lodish HF: submitted). The implication of a hyperfunctioning EPO-R as the etiology of granulocytosis is more difficult to justify; EPO-R may have a subtle effect on CFUGM growth,65or it may be expressed ectopically in the context
of a neoplastic cell. It must be emphasized that these points
are purely speculative, but appropriate molecular diagnostic
techniques should help resolve the role of EPO-R in this disorder.
Another candidate disorder is familial erythrocytosis, a
heterogeneous group of hereditary conditions manifested by
increases in RBC volume. Many of these conditions are
caused by mutant hemoglobin molecules that have an increased affinity for oxygen. However, in a few families the
disorder appears to be intrinsic to erythroid precursors that
are hypersensitive to EP0.'39J40This observation suggests
possible alterations in the signal transduction pathway(s) of
the EPO-R. However, analysis of the number, affinity, and
gross molecular structure of the EPO-R in three families afflicted with this disorder failed to show any differences from
the wild-type EPO-R.I4' The possibility that there are specific
point mutations that constitutively activate the EPO-R remains to be tested (J.T. Prchal, personal communication,
June 1992).
Diamond-Blackfan anemia (DBA) is a congenital RBC
aplasia with severe normochromic-macrocytic anemia. The
BM is deficient in erythroid precursors that appear to be
arrested in differentiation; other hematopoietic lineages
are normal. Serum EPO levels in DBA tend to be elevated,
and BM CFU-E and BFU-E growth ex vivo is defective. 142,143 These observations suggest that EPO-induced
growth of erythroid progenitors, presumably via the EPOR, is defective in DBA. The growth of CD34+ cells, in
vitro, from the marrow of DBA patients suggests that the
defect lies in the inability of multipotent progenitors (CFUGEMM) to undergo erythroid d i f f e r e n t i a t i ~ n .The
' ~ ~ addition of SCF to EPO and 1L-3 can overcome this
and clinical trials with IL-3 appear to ameliorate the disease.'46,147There are no abnormalities in the structure or
function of c-kit or its ligand SCF.148These data suggest
defects in EPO-R structure or signaling. Molecular studies
of the EPO-R may shed light on the pathogenesis of this
disorder.
Finally, in a number of disorders characterized by high
levels of circulating EPO, such as RBC aplasia, in vitro
erythropoiesis is decreased, and erythroid progenitors
are poorly responsive to exogenous EP0.149Abnormalities
in EPO binding or EPO-signaling remain to be elucidated.
YOUSSOUFIAN ET AL
FUTURE PROSPECTS
Molecular elucidation of the structure and function of the
EPO-R is vital to our understanding of mammalian erythropoiesis. In addition to providing us with a more profound
view of this hematopoietic lineage, such insights also can
facilitate the extension of basic knowledge to clinical circumstances. For example, ligand-receptor interactions are now
attractive targets of therapeutic intervention. The design of
compounds that mimic the action of EPO or, conversely,
block excessive activity of the EPO-R in some forms of leukemia or myeloproliferative disorders may provide clinicians
with powerful therapeutic tools. The structural subunits required to generate a high-affinity receptor are likely to be
solved in the near future. At the level of transcriptional activation, the interactions of GATA- 1 or other transcriptional
factors with the EPO-R promoter should improve our understanding of erythroid-specific gene regulation. Finally, the
mechanism of signal transduction by the EPO-R continues
to be dissected. It seems plausible that several members of
the cytokine receptor family share a common pathway for
intracellular signaling, perhaps involving noncovalent associations of the same or closely related Tyr kinases with the
cytoplasmic domains of these receptors. These molecules are
likely to mediate fundamental activities involved in cell
growth, differentiation, and apoptosis. The eventual identification of such molecules and characterization of their natural substrates should significantlyadvance our understanding
of the molecular regulation of hematopoiesis.
ACKNOWLEDGMENT
We thank D. Hilton, J.T. Prchal, and S. Watowich for permission
to include unpublished data, A. de Vos for Fig 2, H.F. Bunn for
thoughtful comments, and Annie Youssoufian for proofreading.
REFERENCES
I . Emerson SG, Yang YC, Clark SC, Long MW: Human recombinant granulocyte-macrophage colony stimulating factor and interleukin-3 have overlapping but distinct hematopoietic activities. J Clin
Invest 82:1282, 1988
2. Emerson SG, Sieff CA, Wang EA, Wong GG, Clark SC, Nathan
DG: Purification of fetal hematopoietic progenitors and characterization of recombinant multipotential colony-stimulating activity. J
Clin Invest 76:1286, 1985
3. Dai CH, Krantz SB, Zsebo KM: Human burst-forming unitserythroid need direct interaction with stem cell factor for further
development. Blood 78:2493, 1991
4. Sawada K, Krantz SB, Dai CH, Koury ST. Horn ST, Click
AD, Civin CI: Purification of human blood burst-forming units-erythroid and demonstration of the evolution of erythropoietin receptors.
J Cell Physiol 142:219, 1990
5. Gregory CJ, Eaves AC: Three stages of erythropoietic cell differentiation distinguished by a number of physical and biologic properties. Blood 51527, 1978
6. Gregory CJ, Eaves AC: Human marrow cells capable of erythropoietic differentiation in vitro: Definition ofthree erythroid colony
responses. Blood 495355, 1977
7. Koury MJ, Bondurant MC: Maintenance by erythropoietin of
viability and maturation of murine erythroid precursor cells. J Cell
Physiol 137:65, 1988
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
STRUCTURE, FUNCTION, AND ACTIVATION OF €PO-R
8. Sawada K, Krantz SB, Sawyer ST, Civin CI: Quantitation of
specific binding of erythropoietin to human erythroid colony forming
cells. J Cell Physiol 137:337, 1988
9. Landschulz KT, Noyes AN, Rogers 0,Boyer SH: Erythropoietin
receptors on murine erythroid colony-forming units: Natural history.
Blood 73:1476, 1989
IO. Fukamachi H, Saito T, Tojo A, Kitamura T, Urabe A, Takaku
F Binding of erythropoietin to CFU-E derived fetal mouse liver cells.
Exp Hematol 155333, 1987
1 I . Sawyer ST, Krantz SB, Goldwasser E: Binding and receptormediated endocytosis of erythropoietin in Friend virus-infected erythroid cells. J Biol Chem 2625554, 1987
12. Broudy VC, Lin N, Brice M, Nakamoto B, Papayannopoulou
T Erythropoietin receptor characteristics on primary human erythroid cells. Blood 77:2583, 1991
13. Jones SS, D’Andrea AD, Haines LL, Wong GG: Human
erythropoietin receptor: Cloning, expression, and biologic characterization. Blood 76:3 I, 1990
14. DAndrea AD, Lodish HF, Wong GG: Expression cloning of
the murine erythropoietin receptor. Cell 57:277, 1989
15. DAndrea AD, Fasman GD, Lodish H F Erythropoietin receptor and interleukin 2 receptor beta chain: A new receptor family.
Cell 58: 1023, 1990
16. DAndrea AD, Fasman GD, Lodish H F A new hematopoietic
growth factor receptor superfamily. Curr Opin Cell Biol2:648, 1990
17. Cosman D, Lyman SD, Idzerda RL, Beckmann MP, Park LS,
Goodwin RG, March CJ: A new cytokine receptor superfamily.
Trends Biosci 15:265, 1990
18. Bazan J F A novel family of growth factor receptors: A common binding domain in the growth hormone, prolactin, erythropoietin and IL6 receptors, and the P75 IL-2 receptor beta chain. Biochem
Biophys Res Commun 164:788, 1989
19. Bazan JF: Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci USA 87:6934, 1990
20. Gearing DP, Thut CJ, VandenBos T, Gimpel SD, Delaney
PB, King J, Price V, Cosman D, Beckmann MP: Leukemia inhibitory
factor receptor is structurally related to the IL-6 signal transducer,
gp130. EMBO J 10:2839, 1991
21. Davis S, Aldrich TH, Valenzuela D, Wong V, Furth ME,
Squint0 SP, Yancopoulos GD: The receptor for ciliary neurotrophic
factor. Science 25359, 1991
22. Souryi M, Vignon I, Penciolelli J-F, Heard J-M, Tambourin
P, Wendling F A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors. Cell 63: 1137, 1990
23. Vigon I, Mornon J-P, Cocault L, Mitjavila M-T, Tambourin
P, Gisselbrecht S, Souyri M: Molecular cloning and characterization
of MPL, the human homolog of the v-mpl oncogene: Identification
of a member of the hematopoietic growth factor receptor superfamily.
Proc Natl Acad Sci USA 895640, 1992
24. Li J-P, DAndrea AD, Lodish HF, Baltimore D: Activation
of cell growth by binding of Friend spleen focus forming virus gp55
glycoprotein to the erythropoietin receptor. Nature 343:762, I990
25. Miura 0, DAndrea AD, Kabat D, Ihle JN: Induction of tyrosine phosphorylation by the erythropoietin receptor correlates with
mitogenesis. Mol Cell Biol I1:4895, 1991
26. Yoshimura A, Longmore G, Lodish H F Point mutation in
the exoplasmic domain of the erythropoietin receptor resulting in
hormone-independent activation and tumorigenicity. Nature 348:
647, 1990
27. DAndrea AD, Yoshimura A, Youssoufian H, Zon LI, Koo
J-W, Lodish HF: The cytoplasmic region of the erythropoietin receptor contains nonoverlapping positive and negative growth-regulatory domains. Mol Cell Biol I 1:1980, 1991
2233
28. Quelle FW, Wojchowski DM: Localized cytosolic domains of
the erythropoietin receptor regulate growth signaling and downmodulate responsiveness to granulocyte-macrophage colony-stimulating factor. Proc Natl Acad Sci USA 88:480 1, 1991
29. Hatakeyama M, Mori H, Doi T, Taniguchi T: A restricted
cytoplasmic region of IL-2 receptor p chain is essential for growth
signal transduction but not for ligand binding and internalization.
Cell 592337, 1989
30. Yoshimura A, Lodish HF: In vitro phosphorylation of the
erythropoietin receptor and an associated protein, ppl30. Mol Cell
Biol 12:706, 1992
31. Yoshimura A, Zimmers T, Neumann D, Longmore G, Yoshimura Y, Lodish H F Mutations in the WSXWS motif of the
erythropoietin receptor abolish processing, ligand binding, and activation of the receptor. J Biol Chem 267:11619, 1992
32. Miyazaki T, Maruyama M, Yamada G, Hatakeyama M, Taniguchi T: The integrity of the conserved ‘WS motif‘ common to
IL-2 and other cytokine receptors is essential for ligand binding and
signal transduction. EMBO J 10:3191, 1991
33. De Vos AM, Ultsch M, Kossiakoff AA: Human growth hormone and extracellular domain of its receptor: Crystal structure of
the complex. Science 255:306, 1992
34. Hatakeyama M, Tsudo M, Miyamoto S, Kono T, Doi T, Miyata T, Miyasaka M, Taniguchi T: Interleukin-2 receptor /3 chain
gene: Generation of three receptor forms by cloned human a and p
chain cDNA’s. Science 244551, 1989
35. Hibi M, Murakami M, Saito M, Hirano T, Taga T, Kishimoto
T: Molecular cloning and expression of an IL-6 signal transducer,
gp130. Cell 63:1149, 1990
36. Hayashida K, Kitamura T, Gorman DM, Arai K, Yoshida T,
Miyazima A: Molecular cloning of a second subunit of the receptor
for human granulocyte-macrophage colony-stimulating factor (GMCSF): Reconstitution of a high-affinity GM-CSF receptor. Proc Natl
Acad Sci USA 87:9655, 1990
37. Takai S, Mita S, Kitamura T, Yonehara S, Yamaguchi N,
Tominaga A, Miyajima A, Takatsu K Identification of the second
subunit of the murine interleukin-5 receptor: Interleukin-3 receptorlike protein, AIC2B is a component of the high affinity interleukin5 receptor. EMBO J 10:2833, 1991
38. Kitamura T, Sat0 N, Arai K-I, Miyajima A Expression cloning
of the human IL-3 receptor cDNA reveals a shared p subunit for the
human IL-3 and GM-CSF receptors. Cell 66:1165, 1991
39. Tavemier J, Devos R, Comelis S, Tuypens T, Van der Heyden
J, Fiers W, Plaetinck G: A human high affinity interleukin-5 receptor
is composed of an IL-5-specific a chain and a /3 chain shared with
the receptor for the GM-CSF. Cell 66: I 175, 1991
40. Takeshita T, Asao H, Ohtani K, Ishii N, Kumaki S, Tanaka
N, Munakata H, Nakamura M, Sugamura K: Cloning of the y chain
of the human IL-2 receptor. Science 257:379, 1992
41. Ip NY, Nye SH, Boulton TG, Davis S, Taga T, Li Y, Birren
SJ, Yasukawa K, Kishimoto T, Anderson DJ, Stahl N, Yancopoulos
GD: CNTF and LIF act on neuronal cells via shared pathways that
involve the IL-6 signal transducing receptor component gp 130. Cell
69:1121, 1992
42. Gearing DP, Comeau MR, Friend DJ, Gimpel SD, Thut CJ,
McGourty J, Brasher KK, King JA, Gillis S, Mosley B, Ziegler SF,
Cosman D: The IL-6 signal transducer, gp130: An oncostatin M
receptor and affinity converter for the LIF receptor. Science 255:
1434, 1992
43. Sawyer ST: Receptors for erythropoietin. Distribution, structure and role in receptor-mediated endocytosis in erythroid cells, in
Hams JR (ed): Blood Cell Biochemistry, vol I. New York, NY,
Plenum, 1990, p 365
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2234
44. Sawyer ST: The two proteins of the erythropoietin receptor
are structurally similar. J Biol Chem 264:13343, 1989
45. Mayeux P, Lacombe C, Casadevall N, Chretien S, Dusanter
1, Gisselbrecht S: Structure of the murine erythropoietin receptor
complex. J Biol Chem 266:23380, 1991
46. Watowich S, Yoshimura A, Longmore G , Hilton D, Yoshimura Y, Lodish H F Homodimerization and constitutive activation
of the erythropoietin receptor. Proc Natl Acad Sci USA 89:2140,
1992
47. Fukunaga R, Ishizaka-Ikeda E, Nagata S: Purification and
characterization of the receptor for the murine granulocyte colonystimulating factor. J Biol Chem 265:14008, 1990
48. Longmore GD, Lodish H F An activating mutation in the
murine erythropoietin receptor induces erythroleukemia in mice: A
cytokine receptor superfamily oncogene. Cell 67: 1089, 1991
49. Lippincott-Schwartz J, Bonifacino JS, Yuan LC, Klausner RD:
Degradation from the endoplasmic reticulum: Disposing of newly
synthesized proteins. Cell 54:209, 1988
50. Sawyer ST, Krantz SB Erythropoietin stimulates"Ca2' uptake
in Friend virus-infected erythroid cells. J Biol Chem 259:2769, 1984
5 I . Bonanou-Tzedaki SA, Sohi MK, Amstein HR: The role of
CAMPand calcium in the stimulation of proliferation of immature
erythroblasts by erythropoietin. Exp Cell Res 170:276, 1987
52. Miller BA, Scaduto RC Jr, Tillotson DL, Botti JJ, Cheung
JY: Erythropoietin stimulates a rise in intracellular free calcium concentration in single early human erythroid precursors. J CIin Invest
82:309, 1988
53. Imagawa S, Smith BR, Palmer-Crocker R, Bunn H F The
effect of recombinant erythropoietin on intracellular free calcium in
erythropoietin-responsive cells. Blood 73: 1452, 1989
54. Bonanou-Tzedaki SA, Setchenska MS, Amstein HR: Stimulation of the adenylate cyclase activity of rabbit bone marrow by
erythropoietin and haemin. Eur J Biochem 155:363, 1986
5 5 . Setchenska MS, Bonanou-Tzedaki SA, Amstein HR: Independent activation of adenylate cyclase by erythropoietin. Mol Cell
Endocrinol56:199, 1988
56. Kuramochi S, Sugimoto Y, Ikawa Y, Todokoro K: Transmembrane signaling during erythropoietin- and dimethylsulfoxideinduced erythroid cell differentiation. Eur J Biochem 193:163, 1990
57. Fukumoto H, Matsui Y, Obinata M: Mechanism of erythropoietin action on the erythroid progenitor cells induced from murine
erythroleukemia cells (TSAS). Development 105:109, 1989
58. Mason-Garcia M, Clejan S, Tou JS, Beckman BS: Signal
transduction by the erythropoietin receptor: Evidence for the activation of phospholipases A2 and C. Am J Physiol 265:Cl197, 1992
59. DAndrea AD, Zon LI: Erythropoietin receptor; subunit
structure and activation. J Clin Invest 86:681, 1990
60. Carroll MP, Spivak JL, McMahon M, Weich N, Rapp UR,
May W S Erythropoietin induces raf- 1 activation and raf- I is required
for erythropoietin-mediated proliferation. J Biol Chem 266: 14964,
1991
61. Hoatlin ME, Kozak SL, Lilly F, Chakraborti A, Kozak CA,
Kabat D: Activation of erythropoietin receptors by Friend viral gp55
and by erythropoietin and down-modulation by the murine Fv-2'
resistance gene. Proc Natl Acad Sci USA 87:9985, 1990
62. Nagata S, Fukunaga R: Granulocyte colony-stimulating factor
and its receptor. Prog Growth Factor Res 3: 131, 199I
63. Idzerda RL, March CJ, Mosley B, Lyman SD, Van den Bos
T, Gimpel SD, Din WS, Grabstein KH, Widmer MB, Park LS, Cosman D, Beckmann MP: Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily. J
Exp Med 171:861, 1990
64. Pierce JH, Ruggiero M, Fleming TP, Di Fiore PP, Greenberger
JS, Varticovski L, Schlessinger J, Rovera G, Aaronson SA: Signal
YOUSSOUFIAN ET AL
transduction through the EGF receptor transfected in 113-dependent
hematopoietic cells. Science 239:628, 1988
65. Pharr PN, Hankins D, Hofbauer A, Lodish HF, Longmore
GD: Expression of a constitutively active erythropoietin receptor in
primary hematopoietic progenitors abrogates erythropoietin dependence and enhances erythroid colony-forming unit, erythroid burstforming unit, and granulocyte/macrophage progenitor growth. Proc
Natl Acad Sci USA 90:938, 1993
66. Ullrich A, Schlessinger J: Signal transduction by receptors with
tyrosine kinase activity. Cell 61:203, 1990
67. Fung MR, Scearce RM, Hoffman JA, Peffer NJ, Hammer SR,
Hosking JB, Schmandt R, Kuziel WA, Haynes BF, Mills GB, Greene
W C A tyrosine kinase physically associates with the P-subunit of the
human IL-2 receptor. J Immunol 147:1253, 1991
68. Noguchi T, Fukumoto H. Mishina Y , Obinata M: Differentiation of erythroid progenitor (CFU-E) cells from mouse fetal liver
cells and murine erythroleukemia (TSAS) cell without proliferation.
Mol Cell Biol 8:2604, 1988
69. Todokoro K, Kanazawa S, Amanuma H, Ikawa Y: Specific
binding of erythropoietin to its receptor on responsive mouse erythroleukemia cells. Proc Natl Acad Sci USA 84:4126, 1987
70. Watanabe T, Shiraishi T, Sasaki H, Oishi M: Inhibitors for
protein-tyrosine kinases, ST638 and genistein, induce differentiation
of mouse erythroleukemia cells in synergistic manner. Exp Cell Res
183:335, 1989
71. Waneck G, Rosenberg N: Abelson leukemia virus induces
lymphoid and erythroid colonies in infected fetal cell cultures. Cell
26:79, 1981
72. Anderson SM, Klinken SP, Hankins W D A murine recombinant retrovirus containing the src oncogene transform erythroid
precursor cells in vitro. Mol Cell Biol 5:3369, 1985
73. Quelle FW, Wojchowski DM: Proliferative action of erythropoietin is associated with rapid protein tyrosine phosphorylation
in responsive B6SUt.EP cells. J Biol Chem 266:609, 1991
74. Spangler R, Bailey SC,Sytkowski AJ: Erythropoietin increases
c-my mRNA by a protein kinase C-dependent pathway. J Biol Chem
266:68 I , I99 I
75. Spivak JL, Fisher J, Isaacs MA, Hankins WD: Protein kinases
and phosphatases are involved in erythropoietin-mediated signal
transduction. Exp Hematol 20500, 1992
76. Mason-Garcia M, Weill CR, Beckman BS Rapid activation
by erythropoietin of protein kinase C in nuclei of erythroid progenitor
cells. Biochem Biophys Res Commun 168:490, 1990
77. Storm SM, Cleveland JL, Rapp UR: Expression of raf family
proto-oncogenes in normal mouse tissues. Oncogene 5:345, 1990
78. Carroll M, Clark-Lewis PI, Rapp UR, May WS: Interleukin3 and granulocyte-macrophage colony-stimulating factor mediate
rapid phosphorylation and activation of cytosolic c-rat J Biol Chem
265:19812, 1990
79. Turner B, Rapp U, App H, Greene M, Dobashi K, Reed J:
Interleukin 2 induces tyrosine phosphorylation and activation of p7274 Raf-l kinase in a T-cell line. Proc Natl Acad Sci USA 88: 1227,
1991
80. Dusanter-Fourt I, Casadevall N, Lacombe C, Muller 0, Billat
C, Fischer S, Mayeux P Erythropoietin induces the tyrosine phosphorylation of its own receptor in human erythropoietin-responsive
cells. J Biol Chem 267:10670, 1992
81. Ishibashi T, Koziol JA, Burstein SA: Human recombinant
erythropoietin promotes differentiation of murine megakaryocytes
in vitro. J Clin Invest 79:286, 1987
82. Fraser JK, Tan AS, Lin F-K, Benidge MV: Expression of a
specific high-affinity binding sites for erythropoietin on rat and mouse
megakaryocytes. Exp Hematol 17:10, 1989
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
STRUCTURE, FUNCTION, AND ACTIVATION OF EPO-R
83. Berridge MV, Fraser JK, Carter JM, Lin F-K: Effects of recombinant human erythropoietin on megakaryocytes and platelet
production in the rat. Blood 72:970, 1988
84. McDonald TP, Cottrell MB, Clift RE, Cullen WC, Lin F-K
High doses of recombinant erythropoietin stimulate platelet production in mice. Exp Hematol 15:7 19, 1987
85. Eschbach JW, Abdulhadi MH, Browne JK, Delano BG,
Downing MR, Egrie JC, Evans RW, Friedman EA, Graber SE, Haley
NR, Korbet S, Krantz SB, Lundin AP, Nissenson AR, Ogden DA,
Paganini EP, Rader B, Rutsky EA, Stivelman J, Stone WJ, Teschan
P, Van Stone JC, Van Wyck DB, Zuckerman K, Adamson JW: Recombinant human erythropoietin in anemic patients with end-stage
renal disease. Results of a phase 111 multicenter clinical trial. Ann
Intern Med 111:992, 1989
86. Sawyer ST, Krantz SB, Sawada K-I: Receptors for erythropoietin in mouse and human erythroid cells and placenta. Blood 74:
103, 1989
87. Boussios T, Bertles JF, Goldwasser E Erythropoietin: Receptor
characteristics during the ontogeny of hamster yolk sac erythroid
cells. J Biol Chem 264:16017, 1989
88. Anagnostu A, Lee ES, Kessimian N, Levinson R, Steiner M:
Erythropoietin has a mitogenic and positive chemotactic effect on
endothelial cells. Proc Natl Acad Sci USA 87:5978, 1990
89. Koury MJ, Bondurant MC, Graber SE, Sawyer ST: Erythropoietin messenger RNA levels in developing mice and the transfer
of '251-erythropoietinby the placenta. J Clin Invest 82:154, 1988
90. Mostov K The polymeric immunoglobulin receptor. Semin
Cell Biol 2:41 I , 1991
91. Tambourin P, Gallien-Lartigue 0, Wendling F, Hualme D:
Erythrocyte production in mice infected with polycythemia-inducing
Friend virus or by the anemia-inducing Friend virus. Br J Haematol
24311, 1973
92. Ruta M, Bestwick R, Machida C, Kabat D: Loss of leukemogenicity caused by mutations in the membrane glycoprotein
structural gene of Friend spleen focus-forming virus. Proc Natl Acad
Sci USA 80:4704, 1983
93. Li J-P, Bestwick RK, Machida C, Kabat D: Role of a membrane glycoprotein in Friend virus erythroleukemia: Nucleotide sequences of nonleukemogenic mutant and spontaneous revertant viruses. J Virol 57:534, 1986
94. Ruscetti SK, Janesch NJ, Chakraborti A, Sawyer ST, Hankins
WD: Friend spleen focus-forming virus induces factor independence
in an erythropoietin-dependent erythroleukemia cell line. J Virol 64:
1057, 1990
95. Li JP, Bestwick RK, Spiro C, Kabat D: The membrane glycoprotein of Friend spleen focus-forming virus: Evidence that the
cell surface component is required for pathogenesis and that it binds
to a receptor. J Virol 61:2782, 1987
96. Casadevall N, Lacombe C, Muller 0,Gisselbrecht S, Mayeux
P: Multimeric structure of the membrane erythropoietin receptor of
murine erythroleukemic cells (Friend cells). J Biol Chem 266: 16020,
1991
97. Chung S-W, Wolff L, Ruscetti SK: Transmembrane domain
of the envelope gene of a polycythemia-inducingretrovirus determines
erythropoietin-independentgrowth. Proc Natl Acad Sci USA 86:7957,
1989
98. Zon LI, Moreau J-F, Koo J-W, Mathey-Prevot B, D'Andrea
A D The erythropoietin receptor transmembrane region is necessary
for activation by the Friend spleen focus-forming virus gp55 glycoprotein. Mol Cell Biol 12:2949, 1992
99. Hankins WD, Troxler D.Polycythemia and anemia-inducing
erythroleukemia viruses exhibit differential erythroid transforming
effects in vitro. Cell 22:693, 1980
2235
100. Li J-P, Baltimore D: Mechanism of leukemogenesis induced
by mink cell focus-forming murine leukemia viruses. J Virol 65:
2408, 1991
10I . Mager D, Mak TW, Bemstein A: Quantitative colony method
for tumorigenic cells transformed by two distinct strains of Friend
leukemia virus. Proc Natl Acad Sci USA 78:1703, 1981
102. Wendling F, Moreau-Gachelin F, Tambourin P: Emergence
of tumorigenic cells during the course of Friend virus leukemias.
Proc Natl Acad Sci USA 73:3614, 1981
103. Moreau-Gachelin F, Tavitian A, Tambourin P: Spi-1 is a
putative oncogene in virally induced murine erythroleukaemias. Nature 331:277, 1988
104. Ben-David Y ,Bemstein A: Friend virus-induced erythroleukemia and the multistage nature of cancer. Cell 66:831, 1991
105. Lane DP, Benchimol S: p53: oncogene or anti-oncogene?
Genes Dev 4: 1, 1990
106. Koury MJ, Bondurant MC: A survival model of erythropoietin action. Science 279:398, 1990
107. Nakamura Y, Komatsu N, Nakauchi H: A truncated erythropoietin receptor that fails to prevent programmed cell death of
erythroid cells. Science 257:1138, 1992
108. Youssoufian H, Zon LI, Orkin SH, DAndrea AD, Lcdish
HF: Structure and transcription of the mouse erythropoietin receptor
gene. Mol Cell Biol 10:3675, 1990
109. Kuramochi S, Ikawa Y , Todokoro K Characterization of
murine erythropoietin receptor genes. J Mol Biol 216:567, 1990
110. Winkelmann JC, Penny LA, Deaven LL, Forget BG, Jenkins
RB: The gene for the human erythropoietin receptor: Analysis of the
coding sequence and assignment to chromosome 19p. Blood 76:24,
1990
1 11. Maouche L, Tournamille C, Hattab C, Boffa G, Cartron
J-P, Chretien S: Cloning of the gene encoding the human erythropoietin receptor. Blood 78:2557, 199 I
112. Noguchi CT, Bae KS, Chin K, Wada Y , Schechter AN, Hankins WD: Cloning of the human erythropoietin receptor gene. Blood
78:2548, 1991
I 13. Penny LA, Forget BG: Genomic organization of the human
erythropoietin receptor gene. Genomics 11:974, 1991
1 14. Gnarra JR, Otani H, Ge Wang M, McBride OW, Sharon M,
Leonard WJ: Human interleukin receptor @-chaingene: Chromosomal localization and identification of 5' regulatory sequences. Proc
Natl Acad Sci USA 87:3440, 1990
115. Shibuya H, Yoneyama M, Harada H, Hatakeyama M, Minamoto S, Kono T, Doi T, White R, Taniguchi T: The human interleukin-2 receptor fl-chain gene: Genomic organization, promoter
analysis and chromosomal assignment. Nucleic Acids Res 18:3697,
1990
I 16. Pleiman CM, Gimpel SD, Park LS, Harada H, Taniguchi T,
Ziegler S F Organization of the murine and human interleukin-7
receptor genes: Two mRNAs generated by differential splicing and
presence of a type I-interferon-inducible promoter. Mol Cell Biol I 1:
3052, 1991
117. Budarf M, Huebner K, Emanuel B, Croce CM, Copeland
NG, Jenkins NA, D'Andrea A D Assignment of the erythropoietin
receptor (EPOR) gene to mouse chromosome 9 and human chromosome 19. Genomics 8:575, 1990
118. Pritchard MA, Baker E, Whitmore SA, Sutherland GR, Idzerda RL, Park LS, Cosman D, Jenkins NA, Gilbert DJ, Copeland
NG, Beckmann MP: The interleukin-4 receptor gene (IL4R) maps
to 16~11.2-16~12.1
in human and to thedistal regionofmouse chromosome 7. Genomics 10:801, 1991
I 19. Gough NM, Rakar S: Localization of the IL-5 receptor gene
to the distal half of murine chromosome 6 using recombinant inbred
strains of mice. Genomics 12:855, 1992
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2236
120. Schmitt RM, Bruyns E. Snodgrass H R Hematopoietic development of embryonic stem cells in vitro: Cytokine and receptor
gene expression. Genes Dev 5:728, 1991
121. Shibuya T, Mak T W Isolation and induction of erythroleukemic cell lines with properties of erythroid progenitor burst-forming
cell (BFU-E) and erythroid precursor cell (CFU-E). Proc Natl Acad
Sci USA 80:3721, 1983
122. Orkin SH: GATA-binding transcription factors in hematopoietic cells. Blood 80575, 1992
123. Zon LI, Youssoufian H, Mather C, Lodish HF, Orkin SH:
Activation of the erythropoietin receptor by transcription factor
GATA-I. Proc Natl Acad Sci USA 88:10638, 1991
124. Chiba T, Ikawa Y, Todokoro K: GATA-1 transactivates
erythropoietin receptor gene, and erythropoietin receptor-mediated
signals enhance GATA-1 gene expression. Nucleic Acids Res 19:
3843, 1991
125. Koury MJ, Bondurant MC, Mueller TJ: The role of erythropoietin in the production of principal erythrocyte proteins other
than hemoglobin during terminal erythroid differentiation. J Cell
Physiol 126:259, 1986
126. Youssoufian H, Lodish H F Transcriptional inhibition of the
murine erythropoietin receptor gene by an upstream repetitive element. Mol Cell Biol 13:98, 1993
127. Heberlein C, Fischer K-D, Stoffel M, Nowock J, Ford A,
Tessmer U, Stocking C: The gene for erythropoietin receptor is expressed in multipotential hematopoietic and embryonal stem cells:
Evidence for differentiation stage-specific regulation. Mol Cell Biol
12:1815, 1992
128. Lacombe C, Chretien S, Lemarchandel V, Mayeux P, Romeo
PH, Gisselbrecht S. Cartron JP: Spleen focus-forming virus long terminal repeat insertional activation of the murine erythropoietin receptor gene in the T3C1-2 friend leukemic cell line. J Biol Chem 266:
6952. 1991
129. Hino M, Tojo A, Misawa Y, Morii H, Takaku F, Shibuya
M: Unregulated expression of the erythropoietin receptor gene caused
by insertion of spleen focus-forming virus long terminal repeat in a
murine erythroleukemia cell line. Mol Cell Biol I1:5527, 1991
130. Ward JC, Harris KW, Penny LA, Forget BG, Kitamura T,
Winkelmann JC: A structurally abnormal erythropoietin receptor
gene in a human erythroleukemia cell line. Exp Hematol 20:371,
1992
13I . Ehrenman K, St John T: The erythropoietin receptor gene:
Cloning and identification of multiple transcripts in an erythroid cell
line OCIMI. Exp Hematol 19:973, 1991
132. Cotes PM, Dore CJ, Yin JAL, Lewis SM, Messinezy M,
Pearson TC, Reid C: Determination of serum immunoreactive
erythropoietin in the investigation of erythrocytosis. N Engl J Med
315:283, 1986
133. Koeffler HP, Goldwasser E: Erythropoietin radioimmunoassay in evaluating patients with polycythemia. Ann Intern Med
94:44, 198 I
YOUSSOUFIAN ET AL
134. Eridani S, Dudley JM, Sawyer BM, Pearson T C Erythropoietic colonies in a serum-free system: Results in primary proliferative polycythemia and thrombocythemia. Br J Haematol67:387,
1987
135. Casadevall N, Vainchenker W, Lacombe C, Vinci G , C h a p
man J, Breton-Conus J, Varet B: Erythroid progenitors in polycythemia vera. Demonstration of their hypersensitivity to erythropoietin
using serum-free cultures. Blood 59:447, 1982
136. Dai CH, Krantz SB, Means RT, Horn ST, Gilbert HS: Polycythemia vera blood burst-forming units-erythroid are hypersensitive
to interleukin-3. J Clin Invest 87:391, 1991
137. Correa P, Axelrad AA: Circulating erythroid progenitors in
polycythemia vera are hypersensitive to IGF-I. Blood 78:38 la, 199I
(abstr, suppl I )
138. Means RT, Krantz SB, Sawyer S, Gilbert H: Erythropoietin
receptors in polycythemia vera. J Clin Invest 84: 1340, 1989
139. Prchal JT, Crist WM, Goldwasser E, Penine G, Prchal J F
Autosomal dominant polycythemia. Blood 66: 1208, 1985
140. Juvonen E, lkkala E, Fyhrquist F, Ruutu T: Autosomal
dominant erythrocytosis caused by increased sensitivity to erythropoietin. Blood 78:3066, 1991
141. Emanuel PD, Eaves CJ, Broudy VC, Papayannopoulou T,
Moore MR, DAndrea AD, Prchal JF, Eaves AC, Prchal J T Familial
and congenital polycythemia in three unrelated families. Blood 79:
3019, 1992
142. Nathan DG, Clarke BJ, Hillman DG,Alter B P Erythroid
precursor in congenital hypoplastic (Diamond-Blackfan) anemia. J
Clin Invest 61:489, 1978
143. Lipton JM, Kudish M, Gross R, Nathan DG: Defective erythroid progenitor differentiation system in congenital hypoplastic
(Diamond-Blackfan) anemia. Blood 67:962, 1986
144. Bagnara GP, Zauli G, Vitale L, Rosito P, Vecchi V, Paolucci
G , Avanci GC, Ramenghi U, Timeus F, Gabutti V In vitro growth
and regulation of bone marrow enriched CD34+ hematopoietic progenitors in Diamond-Blackfan anemia. Blood 78:2203, 1991
145. Abkowitz JL, Sabo KM, Nakamoto B, Blau CA, Martin FH,
Zsebo KM, Papayannopoulou T: Diamond-Blackfananemia: In vitro
response of erythroid progenitors to the ligand for c-kil. Blood 78:
2198, 1991
146. Campos L, Bastion Y, Felman P, Coiffier B: Stem cells stimulation with interleukin-3 (IL-3) treatment in patients with DiamondBlackfan anemia. Blood 78:94a, 1991 (abstr, suppl 1)
147. Olivieri N, Bemman AM, Davis S, Garrison L, Shore R,
Feltis A, Feig S, Freedman MH: Response to the hematopoietic growth
factor IL-3 in patients with Diamond-Blackfan anemia. Blood 78:
153a, 1991 (abstr, suppl 1)
148. Abkowitz JL, Broudy VC, Bennett LG, Zsebo KM, Martin
F Absence of abnormalities ofc-kit or its ligand in two patients with
Diamond-Blackfan anemia. Blood 79:25, 1992
149. Erslev AJ: Erythropoietin. N Engl J Med 324:1339, 1991
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1993 81: 2223-2236
Structure, function, and activation of the erythropoietin receptor
H Youssoufian, G Longmore, D Neumann, A Yoshimura and HF Lodish
Updated information and services can be found at:
http://www.bloodjournal.org/content/81/9/2223.citation.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.