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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2667-2675
By
From the Departments of Medicine and Cell Biology, Washington
University School of Medicine, St Louis, MO.
Erythropoietin (EPO) is required for the survival and expansion of
red blood cell progenitor cells and supports continued differentiation
of these committed progenitors to mature red blood cells. After binding
to its cognate receptor, EPO promotes receptor homodimerization,
activation of receptor-associated JAK2, subsequent receptor tyrosine
phosphorylation, and transduction of signal. EPO is also internalized
and degraded in lysosomes. The contribution of EPO-induced receptor
internalization to modulation of EPO signals has not been determined.
To examine this question, we generated a panel of hematopoietic cell
lines containing progressively truncated isoforms of the erythropoietin
receptor (EPO-R) and determined the rate and extent of EPO
internalization and receptor downregulation. We demonstrated that a
membrane-proximal domain of the cytoplasmic tail of the EPO-R was the
minimal region required for EPO-induced receptor internalization. This
cytoplasmic domain is also the minimal domain required for activation
of JAK2, a cytosolic tyrosine kinase essential for the function of the
EPO-R. However, neither EPO activation of cytosolic JAK2 tyrosine
kinase activity nor tyrosine phosphorylation of the EPO-R cytoplasmic
tail was required for EPO-induced receptor downregulation. Both
functional and nonfunctional cell surface receptor isoforms were
internalized equally. These results suggest that, for downregulation of
cell surface ligand occupied EPO-R and possibly for signaling receptors
of the cytokine receptor superfamily in general, internalization of
cell surface ligand occupied receptors may follow a pathway distinct
from signaling receptors of the receptor tyrosine kinase (RTK) family.
ERYTHROPOIETIN (EPO) is a primary
cytokine or growth factor regulating red blood cell
development.1 EPO promotes survival of committed erythroid
progenitors, leading to an expansion in their numbers, and supports the
continued differentiation of these cells to mature red blood cells. Its
action is mediated by a specific high-affinity cell surface receptor
(EPO-R), which is expressed predominantly in mature committed erythroid
progenitors cells present in the adult bone marrow.
The EPO-R is a member of the hematopoietic cytokine receptor
superfamily. These signaling receptors are related structurally to one
another and are encoded by genes that are members of an evolutionary
conserved superfamily. All are type I transmembrane glycoproteins; the
N-terminal extracellular domain of each is responsible for ligand
recognition and binding. The C-terminal cytoplasmic domains of these
receptors are largely divergent in primary sequence; however, 2 characteristics are shared among most. Most contain partially conserved
proline-rich peptide sequences known as box1 and box2 adjacent to the
transmembrane segment.2 Second, the cytoplasmic tails
contain multiple tyrosine residues; some are phosphorylated in response
to ligand binding and, thus, serve as potential docking sites for
intracellular proteins containing phosphotyrosine-binding domains of
the SH2-type.3-7
Ligand binding promotes multimerization of cytokine receptor subunits
as either homodimers (eg, erythropoietin receptor and growth hormone
receptor [GH-R]) or as heteromultimers (eg, the receptors for
interleukin-6 [IL-6]: IL-6R and gp130). EPO-induced EPO-R
dimerization leads to juxtaposition of their cytoplasmic domains
to which a latent intracellular tyrosine kinase, JAK2, is
tethered and hence leads to initiation of signaling through activation
of the kinase activity of JAK2.8 Biochemical and genetic
evidence indicates that JAK2 activation by the EPO-R is essential for
the development of red blood cells.8,9 Tyrosine phosphorylation of individual tyrosine containing sequences within the
receptor cytoplasmic tail, and JAK2 itself (presumably directly via
JAK2 and possibly other cytosolic kinases) generates new docking sites
for cytosolic signaling proteins or adapter proteins, latent cytosolic transcription factors (eg, STAT proteins), and negative regulatory proteins.3-7 Although the sites of
tyrosine phosphorylation in the EPO-R cyto- plasmic tail, in
response to EPO, have not been identified, recent evidence indicates
that tyrosine phosphorylation of the cytoplasmic tail is required for
optimal receptor function in vivo.10,11
After binding to its receptor, EPO has been shown to be internalized
and degraded in lysosomes12; however, the contribution of
receptor internalization and degradation to the modulation of EPO
signals has not been well studied. Other classes of growth factor
receptors (eg, the receptor tyrosine kinase [RTK] family), which are
also important in the regulation of blood cell development, undergo
regulated cellular trafficking after ligand binding. Mutations that
inactivate the signaling capacity of these receptors block ligand-induced receptor downregulation, altering their functional capacity.13 Whether hematopoietic cytokine receptors, such
as the EPO-R, also require activation of associated cytoplasmic
tyrosine kinases or other signaling intermediates for receptor
downregulation and signal attenuation is not known.
To determine whether EPO-induced EPO-R internalization and
downregulation contribute to the modulation of receptor activity, we
generated a series of hematopoietic cell lines containing progressively truncated cytoplasmic tail isoforms of the EPO-R. We demonstrate that a
membrane proximal domain of the cytoplasmic tail of the EPO-R is the
minimal cytoplasmic domain required for internalization of bound EPO.
This domain is also the minimal cytoplasmic domain of the EPO-R
required to activate JAK2 kinase activity. However, in contrast to RTK
growth factor receptors, activation of JAK2 kinase and tyrosine
phosphorylation of the EPO-R were not required for EPO-induced receptor
downregulation. In addition, mitogenically inactive full-length
isoforms of the EPO-R were internalized similar to wild-type receptors
after EPO binding, indicating that receptor activation is not a strict
requirement for receptor internalization. Thus, in contrast to other
classes of growth factor receptors, internalization of cell surface
receptors appears to be regulated independently from EPO signaling.
Cell lines, antiserum, and reagents.
IL-3-dependent myeloid 32D cells and pro-B-cell BaF3 cells were
maintained in RPMI, 10% fetal calf serum (FCS), and 15% WEHI-3B culture supernatant (as a source of IL-3). Polyclonal rabbit antimouse EPO-R antiserum has been described.14 Polyclonal rabbit
antimouse JAK2 antiserum was obtained from Santa Cruz Labs (Santa Cruz, CA). Antiphosphotyrosine antiserum was obtained from Transduction Labs
(Lexington, KY). Purified carrier-free human EPO (hEPO)
was supplied by Abbott Laboratories (Abbott Park, IL). Vibrio
cholerae neuraminidase was from Calbiochem,
Diplococcus pneumoniae Generation of 32D cells containing EPO-R constructs.
Construction of EPO-R cytoplasmic tail deletion mutants was
accomplished by insertion of premature termination codons (via polymerase chain reaction [PCR]) into the EPO-R coding sequence. Mutant receptors were subcloned into the eukaryotic expression plasmid
pMEX and sequenced. pMEX.EPO-R constructs were transfected into 32D
cells by electroporation using a Bio-Rad gene pulser (Bio-Rad,
Hercules, CA). After transfection, individual clones were
selected in IL-3 and G418. Expression of the EPO-R constructs in 32D
cell clones was determined by 125I-EPO binding and
immunoblotting of cell extracts using an antibody to the N-terminus of
the EPO-R.
Proliferation analysis.
The proliferation of 32D transfectants in response to IL-3 or EPO was
determined by methylthiazol-tetrazolium (MTT) analysis. Cells were plated in a 96-well microtiter plate at 1 × 103 cells/well containing the indicated concentrations of
EPO or Wehi-conditioned media (IL-3 source) for 72 to 86 hours at
37°C/5% CO2. The number of cells was then measured by
the addition of 0.04 mL of 5 mg/mL MTT
(dimethylthiazol-w-yl-2,5-diphenyltetrazolium; Sigma) for 4 to 8 hours
at 37°C/5% CO2. The cells were then lysed by the
addition of 0.1 mL of 0.4 N HCl in 2-propanol. The optical density was
measured by an enzyme-linked immunosorbent assay (ELISA) plate reader at a wavelength of 600 nm. Cell proliferation in response
to EPO was normalized by dividing the optical density (OD) at a given EPO concentration by the OD obtained in
15% Wehi conditioned medium (ie, in IL-3).
Immunoprecipitations and immunoblotting.
Cells were starved for 6 hours in serum-free OPTI-MEM (GIBCO-BRL, Grand
Island, NY) at 37°C. The cells were then stimulated with EPO (20 to 50 U/mL) or left unstimulated for 7 minutes at 37°C. The cells were then added to 5 vol of ice-cold
phosphate-buffered saline (PBS), pelleted, and immediately lysed in a
lysis buffer containing 1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L
TrisCl, pH 7.4, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.5% NP40, 0.2 mmol/L
sodium vanadate, 1 mmol/L phenylmethylsulfonyl fluoride, and 0.5 TIU/mL aprotinin. Immunoprecipitations were performed as previously
described.15 Immunoprecipitates or a fraction of the cell
lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) under reducing conditions and transferred to
a Hybond nitrocellulose membrane (Amersham), and immunoblotting
with specific antiserum was performed. Detection was by the enhanced
chemiluminescence method (ECL) system (Amersham).
Iodination of EPO.
EPO was iodinated using IODOGEN (Pierce, Rockford, IL).
IODOGEN (10 mg) was incubated with 1 mCi of Na 125I for 7 minutes at 4°C in a final volume of 40 µL of water. EPO (1.75 µg) in 0.25 mol/L sodium phosphate buffer (pH 7.1) was then added to
the reaction vial and incubated for an additional 10 minutes at
4°C. The reaction was stopped by transferring the liquid into a
centrifuge tube containing 1 mg tyrosine powder. Free iodine was
separated from the radiolabeled ligand by passing it through a Sephadex
G-10 column (Pharmacia Biotech, Uppsala, Sweden) equilibrated first by
PBS/0.02% Tween-20. The specific activity of 125I-labeled
EPO was generally between 6 and 9 × 106 cpm/pmol.
EPO-binding studies.
Cells were washed in RPMI 1640 supplemented with 10% fetal bovine
serum (FBS), resuspended at a concentration of 6 × 107 cells/mL, incubated at room temperature for 30 minutes,
and then pelleted. Aliquots of 3 × 106 cells in
triplicate were incubated with a range of concentrations of iodinated
EPO at 4°C overnight in binding buffer (RPMI/10% FCS/50 mmol/L
HEPES, pH 7.2) in the absence or presence of 100-fold excess unlabeled
EPO. After overnight incubation, cells were determined to be greater
than 90% viable by trypan blue exclusion analysis. Free EPO was
separated from cell-bound EPO by centrifugation through a 100% FBS
cushion, and the cell pellets were counted in a gamma counter.
The data were plotted according to the method of Scatchard. Dissociation constant (kd) and cell surface receptor
numbers were then determined through Scatchard analyses.
EPO internalization studies.
32D cell lines expressing either wild-type EPO-R or mutant EPO-R were
maintained in RPMI, 10% FCS, 15% Wehi 3B supernatant, and G418 (400 mg/mL) at a concentration of 1 × 106 cells/mL. Cells
are harvested, washed twice in RPMI, and starved in binding buffer
(RPMI, 25 mmol/L HEPES, 10% FCS) for 30 minutes at 37°C/5%
CO2. After harvesting, cells were then aliquoted at 3 × 106 cells/1.5 mL into microcentrifuge tubes in
triplicate. The cells were incubated on ice for 2 hours with 5 nmol/L
125I-EPO in the presence or absence of 100-fold unlabeled
EPO. After incubation, cells were washed twice in RPMI to remove
unbound EPO, and then the cells were incubated at 37°C for varying
amounts of time, generally from 0 to 80 minutes. The reaction was
stopped by the addition of 9× vol of ice-cold binding buffer. The
pelleted cells were resuspended in citrate-phosphate buffer (0.1 mol/L citrate-phosphate, 150 nmol/L NaCl, pH 2.6) and were incubated for 3 minutes on ice to release the remaining surface-bound EPO. The cells
were then centrifuged for 30 seconds. The supernatant that contained
the 125I-EPO removed from the surface was separated from
the pellet that contained the 125I-EPO that was
internalized. The radioactivity in each fraction was measured in a
gamma-counter (Coulter, Hialeah, FL). Specific binding
was determined by subtracting the total cell-associated radioactivity
from the cell-associated radioactivity bound in the presence of excess
unlabeled EPO. The initial rate of internalization was measured by
calculating the slope of the linear portion of the curve, between 0 and
10 minutes. Each cell line was tested a minimum of 3 times.
Determination of cell surface erythropoietin receptor
internalization relative to total cell surface glycoproteins
nonspecific pinocytosis.
Cell surface glycoproteins were labeled with tritiated galactose as
described.16 Briefly, cells were washed and treated with
neuraminidase (0.02 U/mL) and To determine if the cytoplasmic domain of the EPO-R regulates EPO
internalization, a series of cytoplasmic tail truncated EPO-R variants
were constructed (Fig 1A). Plasmids
encoding the wild-type and truncated forms of the EPO-R were
transfected into the IL-3-dependent myeloid cell line 32D. The
transfected cells were selected for growth in IL-3 and G418. Individual
clones were isolated and were maintained in culture under these
conditions. Expression of wild-type EPO-R in these cells allows them to
proliferate in response to EPO; yet, cells were exposed to EPO only for
experimental manipulation. EPO binding studies were performed on
multiple clones containing each construct. Parental 32D cells
transfected with the parental pMEX plasmid (no EPO-R) and selected for
growth in G418 and IL-3 (ie, 32D.Neo cells) did not specifically bind
EPO, and EPO added to culture did not support proliferation of these cells. The number of EPO-binding sites (ie, cell surface EPO-Rs) and
the dissociation constant for EPO binding are presented in Table 1. EPO binding curves were plotted,
and saturation of all EPO-binding sites was achieved for all cell lines
over the concentrations of EPO used. The kds for EPO binding to cells
expressing truncated EPO-Rs were equivalent to that obtained for the
wild-type receptor, except for cells containing EPO-R(1-252), in which
the affinity for EPO was 2- to 3-fold lower. Despite this difference,
the relative off rates for bound EPO did not significantly differ
between clones (data not shown). The numbers of cell surface receptors
were also roughly equivalent, except when the complete cytoplasmic tail was removed. Cells containing EPO-R(1-252) expressed 5-fold greater cell surface receptors than cells expressing wild-type receptors. Immunoblot analysis of detergent soluble extracts from the cell lines
demonstrated that each expressed an EPO-R protein of the expected size
and that the level of total cell expression did not greatly differ (Fig
1B). The capacity of each cell line to proliferate in response to added
EPO was determined. In contrast to other reports using Ba/F3
cells,17 none of the 32D clones expressing truncated forms
of the EPO-R were hypersensitive to EPO (not shown). Indeed,
EPO-hypersensitivity of various truncated EPO-R isoforms expressed in
Ba/F3 cells has been suggested to result from residual IGF-1 in
serum.18 32D.EPO-R(1-321) cells, a variant of the EPO-R
that contains only Box1 and Box2, survive and proliferated weakly only
in supraphysiologic concentrations of EPO (>10 U/mL; not shown),
whereas 32D.EPO-R(1-252) cells did not proliferate or survive at
concentrations of EPO up to 50 U/mL.
The membrane proximal domain of the EPO-R cytoplasmic tail was the
minimal domain necessary for internalization of surface-bound EPO.
EPO internalization and EPO-R downregulation was first studied in 32D
cells containing wild-type EPO-R and in 32D.Neo cells not containing an
EPO-R. A saturating amount (5 nmol/L) of radio-iodinated EPO was added to the cells at 4°C. Cells were then washed and warmed to 37°C for various periods of time. At various time points, the radioactivity that was internalized, released into the medium, or
remaining on the cell surface was measured. Multiple clones were
examined. The initial rate of EPO internalization was determined from
the slope of the linear portion of the uptake curve. As shown in
Fig 2, 32D.EPO-R(wt or 1-483) internalized
EPO efficiently during the 80-minute time course. After 40 minutes,
54% of bound EPO was internalized (solid squares), and the amount of
cell surface bound EPO was downregulated by 69% (open squares). After
20 minutes, tricarboxylic acid-soluble counts were
detectable in the medium, indicating that internalized EPO had been
degraded and that 125I was being released from cells into
the medium. In the presence of Na Azide, which prevents receptor
internalization at 37°C, we did not observe any degradation of
bound EPO (ie, all surface counts released after acid wash were
precipitated by the addition of TCA), indicating that internalization
was required for EPO degradation (not shown). These cells internalized
EPO at an initial rate of 3.2% per minute
(Table 2). Parental 32D cells transfected with the control vector pMEX showed neither binding nor
internalization of EPO (Fig 2, open circles). This result demonstrated
that a functional EPO-R complex capable of internalizing bound EPO was reconstituted in transfected 32D cells.
JAK2 tyrosine kinase activation was not required for internalization
of EPO-R-bound EPO.
In the case of the EGF-R, receptor kinase activation and tyrosine
phosphorylation of the receptor and cellular substrates can regulate
endocytosis of the receptor.13,19 The
membrane proximal region of the EPO-R required for internalization of
bound EPO contains the Box1 and Box2 motifs, which are the minimal
region of the EPO-R required for mitogenic responses and JAK2 binding site.20,21 To determine whether EPO-induced JAK2 tyrosine
kinase activation is required for internalization of bound EPO, we
expressed a mitogenically inactive form of the EPO-R, EPO-R(W282R), in
32D cells (Fig 1B). As shown in Fig 5,
cells expressing this mutant receptor do not survive or proliferate in
response to EPO. In addition, there was no change in the
tyrosine-phosphorylated protein profile of 32D.EPO-R(W282R) cells upon
addition of EPO (Fig 6A), and neither the
EPO-R(W282R) (Fig 6B) nor JAK2 (Fig 6C) was tyrosine phosphorylated in
response to EPO. Finally, we were unable to detect any activation of
JAK2 kinase activity in 32D.EPO-R(W282R) cells in response to EPO,
confirming the results of Witthuhn et al8 (not shown).
Despite the absence of JAK2 tyrosine kinase activity in these cells,
the initial rate of EPO internalization and extent of EPO
internalization were similar to that of cells containing wild-type
receptors (Fig 7 and Table 2). Thus,
EPO-stimulated JAK2 kinase activity was not required for
internalization of EPO-R-bound EPO.
Tyrosine phosphorylation of the EPO-R was not required for
internalization of EPO-R-bound EPO.
To determine if tyrosine phosphorylation of the EPO-R was required for
internalization of receptor-bound EPO, we generated 32D cells
containing a full-length EPO-R in which all 8 cytoplasmic tyrosine
residues were changed to phenylalanine residues (Fig 1). The
32D.EPO-R(YF) cells did not proliferate in response to EPO
concentrations as high as 10 U/mL; however, they did survive in
cultures containing 10 U/mL of EPO (Fig 5). Despite these altered growth parameters, JAK2 was tyrosine phosphorylated in response to EPO
(Fig 6C) at a level comparable to cells expressing wt EPO-R, as has
been previously reported.4 As expected, EPO-R(YF) was not
tyrosine phosphorylated in response to EPO (Fig 6B). This mutant
receptor bound EPO with a normal affinity (kd, 109 pmol/L) and
internalized receptor-bound EPO as well as cells containing wild-type
receptors (Fig 7 and Table 2). Similarly, EPO-R(W282R), which also was
not tyrosine phosphorylated in response to EPO, internalized
receptor-bound EPO as well as cells containing wild-type EPO-R (Fig 7
and Table 2). Thus, tyrosine phosphorylation of the EPO-R
was not required for internalization of EPO-R-bound EPO.
We have shown that a membrane proximal domain of the cytoplasmic tail
of the EPO-R was the minimal domain required for internalization of
receptor-bound EPO. This region contains the Box1 and Box2 motifs,
which are conserved among most cytokine receptor family members and
function as the site of receptor-JAK2 tyrosine kinase association.2,8,21 It is the minimal cytoplasmic region required for activation of JAK2 kinase and mitogenic
signaling.8 In contrast to what has been found with other
families of signal-transducing growth factor receptors (eg, EGF-R),
activation of tyrosine kinase activity, specifically JAK2, and tyrosine
phosphorylation of the EPO-R itself were not required for
internalization of EPO.
The authors thank Drs S. Watowich and M. Goldsmith for providing
EPO-R(W282R) and EPO-R(YF) plasmids, respectively; Abbott Laboratories
for purified EPO; Dr Sally York for helpful discussions; and Drs S. Kornfeld and L. Traub for review of the manuscript and helpful suggestions.
Submitted January 25, 1999; accepted June 9, 1999.
Supported in part by American Cancer Society Grant No. ACS-IRG 36-37 (G.D.L.) and by National Institutes of Health Grant No. CA75315
(G.D.L). G.D.L. was a Scholar of the James S. McDonnell Foundation.
D.L.B. was supported as a Lucille P. Markey Pathway postdoctoral
fellow. Support for L.L.L. was provided by a grant to Washington
University from the Howard Hughes Medical Institute through the
Undergraduate Biological Sciences Education Program.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Gregory D. Longmore, MD, Division of
Hematology, Washington University School of Medicine, Campus Box
8125, 660 S Euclid Ave, St Louis MO 63110; e-mail: longmorg{at}medicine.wustl.edu.
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TOP
ABSTRACT
INTRODUCTION
-galactosidase was from
Sigma (St Louis, MO), and bovine milk
galactosyltransferase was from Fluka (Turku, Finland).
UDP-(3H)galactose and Na 125I were from
Amersham (Arlington Heights, IL).
) Wild-type
EPO-R(1-483); (
) EPO-R(1-411); (
) EPO-R(1-373); (
)
EPO-R(1-321); and (
) EPO-R(1-252).
EPO); (
