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Serum Form of the Erythropoietin Receptor Identified
by a Sequence-Specific Peptide Antibody
By Roy D. Baynes, G. Kesava Reddy, Yuan J. Shih, Barry S. Skikne, and James D. Cook
The present investigation was undertaken to searchfor soluble forms of the erythropoietin receptor in human serum
using polyclonal antibody against an amino terminal peptide sequence in the extracellular domain. This sequence
was located adjacent to the amino terminus at residues
25-38. When this antibody was used for Western blots of
solubilized membranes from nucleated bone marrow cells,
a protein consistent with native erythropoietin receptor
was seen. Purified soluble ectodomain of the erythropoietin receptor displayed appropriate reactivity with this antibody. When sera from normal subjects and patients with a
range of hematologic disorders were examined by Western
blotting, a protein with a molecular mass of 34 Kd was
detected in sera from patients with enhanced erythropoiesis including sickle cell anemia, thalassemia, and megaloblastic anemia. This protein was rarely detected in normal
serum but appeared when normal subjects were treated
with recombinant erythropoietin and disappeared after full
treatment of patients with megaloblastic anemia due t o
vitamin B,, deficiency. The protein was not detected after
myeloablation for bone marrow transplantation but appeared with marrow engraftment. Reactivity of this protein with the peptide antibody was competitively inhibited
by the amino terminal peptide sequence. An additional 48
Kd protein was detected that showed minimal variation in
intensity with differing degrees of erythropoietic activity.
Detection of this protein could not be inhibited by the addition of synthetic peptide. Our findings indicate the presence of a soluble form of the erythropoietin receptor related to the extracellular domain that is highly correlated
with enhanced erythropoiesis.
0 1993 by The American Society of Hematology.
E
taining 236 amino acid residue^.'^-'^ The human cDNA encodes a 66 Kd protein. The EPOR shows striking similarities to other receptors belonging to the hemopoietin/growth
factor superfamily including those for IL-2,14 IL-3,I5IL-4,I6
IL-6,” IL-7,18 GM-CSF,” G-CSF, growth hormone,20 and
prolactin.2’ These similarities pertain in the extracellular
domain to four closely spaced cysteine residues and a highly
(WSXWS)
conserved tryptophan-serine-x-tryptophan-serine
motif in the region adjacent to the cell membrane.
Despite the rapid advances in our knowledge of the structure and function of EPOR, clinical applications have not
yet been identified. Structural abnormalities in the EPOR
have been postulated to occur in polycythemia vera resulting in an exaggerated sensitivity to EPO and there are a
number of hematologic disorders with failed or disordered
erythropoiesis and elevated serum EPO levels that may be
attributable to quantitative or qualitative abnormalities of
EPOR. One aspect of EPOR physiology that would be of
potential clinical interest is the presence in serum of secreted or soluble forms. For example, measurement of
serum transferrin receptor has provided a clinically valuable
indirect measure of total e r y t h r o p o i e ~ i s Several
. ~ ~ ~ ~ ~receptors in the hemopoietin superfamily including IL-4,16 IL5,24 IL-6,25 IL-7,I8 G-CSF,26 and GM-CSFZ7 have been
shown to have naturally occurring alternatively spliced
mRNA encoding for potentially secreted soluble forms. A
soluble form of the leukemia inhibitory factor (LIF) receptor has been identified in murine serum.28In addition, the
growth hormone receptor, IL-2 receptor, IL-6 receptor, and
the prolactin receptor have all been shown to have naturally
occurring soluble forms in serum that provide clinically useful information. For example, the soluble IL-2 recept0t.2~
has been shown to correlate with tumor mass in lymphoreticular malignan~y,~’the soluble growth hormone recepto?0,3‘,32
has been used to categorize growth hormone deficiency states,33and the soluble form of the IL-6 receptor
found in the serum of human immunodeficiency virus
(HIV)-infected patients has been suggested to have an immune modulatory function.34The present study was undertaken to determine whether the presence and nature of a
RYTHROPOIESIS is dependent on the sequential maturation of stem cells to mature red blood cells. This
erythropoietic process includes passage through at least
three progenitor stages, the early burst forming unit (BFUE), a subsequent colony forming unit (CFU-E), and the
stage of late progenitors. These maturation stages are subject to regulatory influences of a number of hemopoietic
growth factors, including stem cell factor, interleukin-3 (IL3), granulocyte macrophage-colony stimulating factor
(GM-CSF), insulin-like growth factor I, and insulin’” but
one of the most important regulatory factors is erythropoietin (EPO). The effect of EPO is highly dependent on the
stage of the erythropoietic development. EPO provides a
proliferative signal to BFU-E, a differentiation signal to the
CFU-E4 and a signal to maintain cellular viability ofthe late
progenitor^^.^ by limiting a p o p t o ~ i s .These
~ . ~ different influences of EPO are mediated by interaction with specific erythropoietin receptors (EPOR) on the erythroblast surface.
Molecular studies have afforded a rapid advance in our
knowledge of the structure and function of EPOR. The full
length sequence of both murine and human EPOR has been
determined by molecular ~loning.’.’~
The full length human
erythropoietin receptor (EPOR-f) is a transmembrane protein containing 508 amino acids. The protein is about
equally divided by a small 22 amino acid membrane spanning region into an extracellular amino terminal domain
containing 250 amino acids and a cytoplasmic domain con-
From the Division of Hematology, Department ofMedicine, Kansas University Medical Center, Kansas City.
Submitted February 3, 1993; accepted June 15. 1993.
Address reprint requests to Roy D. Baynes, MD, PhD, Division of
Hematology, Kansas University Medical Center, 3901 Rainbow
Blvd, Kansas City, KS 66160-7402.
The publication costs of this article were defvayed 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.
0 1993 by The American Society of Hematology.
0006-49 71/93/820 7-0024$3.00/0
2088
Blood, Vol 82,No 7 (October l ) , 1993:pp 2088-2095
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2089
SERUM FORM OF THE ERYTHROPOIETIN RECEPTOR
human EPOR’5.36was kindly provided by Dr Simon Jones ofGenetics Institute, Boston.
Preparation qfpeptide antihodv. A peptide sequence was selected from that deduced for the full length EPOR” on the basis of
regional hydr~philicity,~’
proline richness,3sand a paucity of intrapeptide lysines. The peptide sequence excluded those in the region
of the conserved cysteines, the WSXWS motif, other conserved
region^,'^ and the truncation site in the IL-2 receptor amino acid
sequence aligned with the murine EPOR sequence.40The selected
peptide sequence consisted of 14 amino acid residues. The sequence APPPNLPDPKFESK, designated S25, was located at positions 25-38 immediately adjacent to the amino terminus at the
cleavage point of the hydrophobic leader sequence. The terminal
lysine was chosen to facilitate linkage to KLH by meansofglutaraldehyde.4’ The S25 sequence was synthesized, purified. and used to
produce polyclonal antisera as previously described?*
To characterize the reactivity of the peptide antibody, human
bone marrow was obtained from subjects undergoing harvest for
allogeneic bone marrow transplantation. Nucleated cells were obtained by density separation on Ficoll-Hypaque. A cell membrane
fraction of these cells was obtained by sonication in ice-cold HBSS
containing several protease inhibitors including 1 mmol/L PMSF,
50 pmol/L EP475, 50 pmol/L pepstatin A. and I mmol/L I. I O
phenanthroline. The cell membranes were pelleted by ultracentrifugation and solubilized in either sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer or HBSS containing 0.001 mol/L tris and 1% teric (HBSSTT). A whole cell
fraction was also obtained by solubilizing nucleated bone marrow
226
119
96
81
62
45
38
1
2
3
211
119
Fig 1. Westem blot of nucleated marrow cell membranes solubilized either in HBSSIT (lane 2) or sample running buffer (lane 3)
after electrophoresis under nonreducing conditions and immunostaining with S25 amino terminal peptide antibody. Apparent molecular mass indicators (Kd) are present in lane l .
98
81
64
circulating form of EPOR may be helpful in the clinical
assessment of erythropoiesis.
45
39
MATERIALS AND METHODS
Materials. Hank’s buffered salt solution (HBSS), HEPES,
phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DIFP), I , I O phenanthroline, pepstatin A, tris [hydroxymethyllaminomethane (tris), prestained molecular mass markers,
4-chloro- I-napthol, keyhole limpet hemocyanin (KLH), polyoxyethylene 9 lauryl ether (teric), and Freund’s adjuvant were obtained
from Sigma (St Louis, MO). EP475 was kindly provided by Dr H.
Nagase of the Department of Biochemistry and Molecular Biology
at the University of Kansas Medical Center. Additional prestained
molecular mass markers were obtained from Bio-Rad Laboratories
(Richmond, CA). Peroxidase conjugated goat IgG to rabbit IgG was
obtained from Dako (Carpinteria, CA). Polyacrylamide gradient
gels and nitrocellulose membranes were obtained from Bio-Rad
Laboratories. A purified recombinant soluble ectodomain of the
Fig 2. Westem blot of supematant from nucleated bone
marrow cells solubilized in
HBSSTT (lane 2) with S25
amino terminal peptide antibody after electrophoresisunder
nonreducing conditions. Apparent molecular mass indicators
(Kd) are present in lane 1.
1
2
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BAYNES ET AL
2090
B
-A106.0
80.0
106.0
80.0
49.5
32.5
49.5
32.5
27.5
27.5
18.5
18.5
1
2
1
3
2
5
4
3
6
7
8
9
10
Fig 3. SDS-PAGEand Western blot of purified recombinant EPOR ectodomain. (A) Electrophoretic mobility of purified EPOR ectodomain
(2 jig) under nonreducing (lane 2) and reducing (lane 3)conditions. The gel was stained with Coomassie blue. (B)Western blot with S25
peptide antibody of purified EPOR ectodomain (1 jig) after electrophoresis under nonreducing conditions. Individual lanes had included
during the primary antibody incubation incremental amounts of peptide against which t h e antibodies were produced. The concentrations of
peptide (ng/mL)added to respective lanes in parentheses were 0 (2). 100 (3). 250 (4).500 (5), 1,000 (6).5,000 ( 7 ) .20,000 (8). 50,000 (9).
and 100,000 (10).Apparent molecular mass indicators (Kd) are present in lane 1 of both panels.
peroxidase (diluted 1:5.000) in 0.1% nonfat dry milk in PBS for 2
hours. After washing the membranes, immunoreactive material
was detected following the addition of4-chloro-I-napthol as a substrate for the peroxidase. Commercial molecular weight markers
were used and apparent molecular weights are indicated on the
figures. The staining intensity of one of the proteins detected by
Western blotting vaned markedly in different human sera. To compare the results with different sera. a scoring system based on staining intensity was used. No visible band was scored 0, a maximum
reaction was scored 2, and values of 0.5, I , and 1.5 were used to
represent intermediate intensities.
Specificity of the Western blot findings was established by competitive blots during which incremental amounts of the synthetic
peptide were added in the primary antibody incubation step. This
approach was used to study both the purified EPOR ectodomain
and human serum.
cells at 4°C in HBSSTT containing the protease inhibitors listed
above. Following overnight agitation, the particulate matter was
removed by ultracentrifugation. To further characterize the reactivity of the peptide antibody, purified recombinant EPOR ectodomain35.36
was used.
Biochemical procedirre.7. Various marrow cell fractions, purified recombinant EPOR ectodomain. and human sera were subjected to SDS-PAGE on 4% to 20% gradient gels as described by
Laemmli.43The gels were electroblotted as described by Burnette."
The nitrocellulose membranes were blocked by incubation with I %
nonfat dry milk in phosphate-buffered saline (PBS) for I hour and
incubated with peptide antibody (whole antisera diluted 1:200 for
all studies except 1500 for the recombinant ectodomain evaluation
and for the competitive binding studies with the serum) in 0.1%
nonfat dry milk in PBS for 2 hours. After washing, the membrane
was incubated with goat antirabbit IgG conjugated to horseradish
226
119
96
81
61
45
38
-
."p-
r
,
1
I
I
1
2
3
4
5
6
7
8
Fig 4. Western blot with S25 amino-terminal
peptide antibody of serum from normal subjects
before (lanes 2 and 4) and after (lanes 6 and 8 ) the
administration of EPO in therapeutic concentrations. Apparent molecular mass indicators (Kd) are
present in lanes 1,3,5, and 7. The 34 Kd protein in
lanes 6 and 8 were scored 1%.
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2091
SERUM FORM OF THE ERYTHROPOIETIN RECEPTOR
!q m m - -
- T ” ”
106.0
80.0
Fig 5. Western blot with S25 peptide antibody
of serum from a patient with sickle cell anemia
known to contain t h e 34 Kd protein. Apparent molecular mass indicators (Kd) are present in lane 1.
Individual lanes had included during t h e primary
antibody incubation incremental amounts of peptide against which the antibodies were produced.
The concentrations of peptide added (ng/mL)to respective lanes in parentheses were 0 (2). 100 (3),
49.5
32.5
I
27-5
1
18.5
200 (4). 500 (5). 1,000 (6). 2,000 (7), 5,000 (8).
10,000 (9).20,000 (10). 50,000 (11). and
100,000 (12).
2
1
Amino acid sequence analysis was performed after initial electrophoresis on 7.5% to 20% gradient sequencing grade gels. The samples were then transblotted onto polyvinylidine difluoride membranes by the method of Matsudaira.4’ Amino acid sequencingwas
performed at the Protein Sequencing Facility of the Department of
Biochemistry and Molecular Biology at the University of Kansas
Medical Center.
Clinical sumple.y. Sera were obtained from normal volunteer
subjects and patients with a variety of hematologic disorders. All
samples were obtained from participants in investigations that had
been approved by the human subjects committee at the University
of Kansas Medical Center. Informed consent was provided in accordance with the Declaration of Helsinki. In 21 normal subjects,
sera were obtained before and following the administration of 100
U/kg recombinant human EPO subcutaneously on IO days over a
2-week period. Studies were performed on sera from anemic patients with iron deficiency (17), chronic renal failure on maintenance dialysis (9), chronic inflammatory disease (18), sickle cell
anemia ( I I), fl thalassemia major (3). and megaloblastic anemia
due to vitamin B,, deficiency (6). In the latter patients, follow-up
sera were obtained after correction of the deficiency state. Sera were
A
’
3
4
5
6
7
8
9
10
11
12
also obtained from eight patients undergoing bone marrow transplantation. The initial samples were obtained immediately following the myeloablation procedure and subsequent samples following
marrow administration. All sera were diluted 1:2 in normal saline
before analysis by Western blotting. Two p L of diluted serum was
electrophoresed for each sample.
RESULTS
When antisera were reacted with solubilized membranes
from nucleated bone marrow cells, a protein with a molecular mass of 65 Kd was detected. An example of the reaction
of the S25 antisera with membrane solubilized in either
HBSSTT or sample running buffer is shown in Fig 1.
The supernatant obtained by solubilization of whole nucleated bone marrow cells differed from the results seen
with cell membranes. The supernatant contained a protein
with a molecular mass of 34 Kd that showed a strong reaction with the S25 antisera (Fig 2). When this material was
subjected to amino acid sequence analysis, its identity with
B
C
226
119
96
81
62
45
38
1
2
3
4
‘5
1
2
3
4
1
2
3
4
Fig 6. Western blot using S25 antibody of serum from patients with various disorders. (A) Four patients with the anemia of chronic
disease (lanes 2-5).In all lanes, the 34 Kd protein was scored 0. (B) A patient with the anemia of chronic renal insufficiency before (lane 2).
after the initiation of (lane 3). and after some duration (lane 4) of EPO therapy. Scores for the 34 Kd protein were 0 (lane 2).2 (lane 3). and 1
(lane 4). (C) Three patients with sickle cell anemia, not during crisis (lanes 2-4). All lanes were scored a s 2. Apparent molecular mass
indicators (Kd) are present in lanes 1.
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BAYNES ET AL
2092
119
96
81
62
45
38
1
2
3
4
5
Fig 7. Westem blot with 525 amino terminal peptide intibody
of sera from subjects with pemicious anemia before (lanes 2 and 4)
and after (lanes 3 and 5) successful therapy with Vitamin B,*. Apparent molecular mass indicators (Kd) are shown in lane 1. Lanes 2
and 4 were scored as 2 and lanes 3 and 5 as 0.
the EPOR ectodomain was confirmed as indicated below
where EPOR residues 25-34 are shown along with the first
I O amino acids of the protein:
Solubilized cells A X P P N L P X P K
EPOR
***
A P PPNLPDPK***
(X
=
non-identified residues)
In addition, a small peptide was detected with a sequence
identical to that of human EPO at amino acid position 1 18121.46
Solubilized cells
EPO
S P P D
*** A I S P P D A A S A ***
The purified e~todomain’~
on SDS-PAGE migrated as a
predominant 30 Kd moiety and a minor 60 Kd protein
under nonreducing conditions and as a single 30 Kd protein
on reduction (Fig 3A). S25 peptide antibody showed strong
reactivity with this purified EPOR ectodomain. This reactivity could be effectively blocked by the addition of synthetic
peptide during the primary antibody incubation (Fig 3B).
This confirms the specificity of S25 for the peptide sequence
in the EPOR ectodomain.
In all human sera examined, a protein with a molecular
mass of 48 Kd was readily detected (Fig 4). A second smaller
protein band with a molecular mass of 34 Kd was detected
in sera from patients with enhanced erythropoietic activity.
This band was absent from normal sera but could be readily
seen when normal subjects were administered a course of
recombinant EPO to enhance erythropoietic activity (Fig
4). When sera containing this protein band were analyzed
on SDS-PAGE under reducing conditions, no change in the
apparent molecular mass was seen (data not shown). When
competitive Western blots were performed, detection of the
48 Kd protein was not inhibited by the addition ofsynthetic
peptide during the primary antibody incubation (Fig 5). Detection of the 34 Kd protein by S25 antiserum was progressively inhibited by the incremental addition of synthetic
amino-terminal peptide (Fig 5).
The 34 Kd protein was rarely detected in normal sera and
was similarly absent in most patients with iron deficiency
anemia (data not shown), the anemia ofchronic disease (Fig
6A), and the anemia of chronic renal failure (Fig 6B). When
a patient with the anemia of chronic renal failure was
treated with recombinant EPO, this protein became markedly apparent (Fig 6B). This 34 Kd protein band was very
prominent in patients with enhanced erythropoiesis including those with sickle cell anemia (Fig 6C). thalassemia major
(data not shown), and untreated vitamin B,, deficiency (Fig
7). Interestingly, the band disappeared in the latter when
normal erythropoiesis was restored following a course of
parenteral vitamin B,, (Fig 7).
The results of the scoring system to estimate the quantitative reactivity of the 34 Kd protein band in different clinical
states is shown in Fig 8. An absent band was scored 0, a
maximum reaction was scored 2, and values of 0.5, 1, and
1.5 were used to represent intermediate intensities. Of 29
normal sera, a faint reactivity was observed in only 5. In 18
patients with the anemia of chronic disease, the protein
band could be detected in 5 and in 3 of these the band was
highly visible. In contrast with these findings in normal subjects and patients with hypoproliferative anemia, an intense
reactivity of the 34 Kd peptide was invariably observed in
patients with enhanced erythropoiesis.
Serial measurements on sera from patients undergoing a
bone marrow transplant permitted studies of the effect of
erythropoiesis on anti-EPOR antibody reactive protein
within the same patient. The 34 Kd protein was not visible
immediately following the marrow ablation procedure, first
appearing some weeks following transplantation at the time
of engraftment (Fig 9). The patient shown was also evaluated with serum transfemn receptor concentrations. These
were measured by our monoclonal enzyme-linked immunosorbent assay (ELISA)” and are known to accurately reflect
the erythroid progenitor mass.23The appearance ofa prominent 34 Kd protein at day 16 post-transplant coincided with
a brisk increase in the serum transfemn receptor concentration. Indeed, in the other transplant cases studied, there was
a strong correlation between the 34 Kd protein and the
serum transfemn receptor concentrations (data not shown).
DISCUSSION
The key observation in the present investigation was the
detection of a 34 Kd protein in human serum that is highly
correlated with enhanced erythropoiesis. This protein was
detected with antibody against a sequence of the amino terminus of the receptor ectodomain in the region most remote from the cell membrane. This antibody was shown in
a number of ways to be specific for EPOR, including appro-
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SERUM FORM OF THE ERYTHROPOIETIN RECEPTOR
2093
2.0
.
1.5
..
=:
:
:
s
..
s:
?!
8
v)
W
zn 1.0
e.
e
0..
W
n.
Y
-F?Y
0.5
0
e
.e
..
REk!
m
Normal
Chronic
renal
failure
...
Post
ablation
.e
ttttnm
Iron
deficiency
"."
Anemia 01
chronic
disease
Sickle
cell
disease
Marrow
engraftment
Effective
Normal
Hypoproliferative
Thalassemia
812
deficiency
Ineffective
Hyperproliferative
Fig 8. Scatterplot of the visually determined intensity of the 3 4 Kd protein on Westem blot with S25 amino terminal peptide antibody of
sera of normal subjects and those with a number of conditions characterized by altered erythropoietic activity.
priate reactivity with nucleated bone marrow cells and purified recombinant EPOR ectodomain, which could be inhibited by the synthetic amino terminal peptide sequence.
Detection of the 34 Kd protein in serum could be competitively inhibited by the synthetic peptide sequence to which
the antibody was produced. The reason why the 34 Kd peptide was detected only in states ofenhanced erythropoiesis is
unclear but it is likely attributable to the differences in the
quantity of the material in human serum. In some normal
1
2
3
4
5
6 ' 7
8
serum, small quantities could be detected and this was also
true in various hypoproliferative anemias (Fig 8). Very high
quantities were observed in hyperproliferative states characterized by either effective erythropoiesis as in patients with
sickle cell anemia or in normal subjects or those with the
anemia of chronic renal failure following recombinant EPO
or by ineffectiveerythropoiesisas seen in patients with megaloblastic anemia or thalassemia.
Recent molecular studies of EPOR in normal bone
9 1 0 11 12 13
14 15 16
17 18 19 20
Fig 9. Westem blot with S25 amino terminal peptide antibody of sera obtained longitudinally from a patient undergoing bone marrow
transplantation. With the day of transplant designatedday 0, the days studied were 1 (lane2). 0 (lane3). 1 (lane 4). 2 (lane 5). 5 (lane6). 6
(lane 7).7 (lane8). 8 (lane 9). 9 (lane 10). 12to 1 4 (lanes 12to 14). 1 6 (lane 15). 19 (lane 16). 21 (lane 17). 27 (lane 18). 29 (lane 19). and 33
(lane 20).Molecular mass indicators are shown in lanes 1 and 11. From top to bottom, these represent 226,119,96,81,62,45, and 38 Kd.
respectively. Scores from lanes 15 to 2 0 of the 34 Kd protein were 2, %, 1%, 1, %, 1%.
-
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BAYNES ET AL
2094
marrow shed some light on the possible nature of the soluble form of EPOR detected in human serum in the present
investigation. When bone marrow cells were sorted after
incubation with monoclonal antibodies to an early stem cell
marker (CD34) and the transferrin receptor (CD7l), two
distinct populations were seen in relation to EPOR expression.' Early progenitors (CD34+,CD7 1-) express predominantly a membrane-bound EPOR with a truncated cytoplasmic domain containing only 56 amino acids (EPOR-t).
This form of the receptor apparently conveys a mitogenic
signal but not a signal for protection against programmed
cell death.' This arises as a consequence of a 95 bp @ insert
corresponding to the intron between exon VI1 and VIII.
Late progenitors (CD34-, CD7 1+) express predominantly a
membrane-bound EPOR with a full-length cytoplasmic domain (EPOR-f) that conveys both a mitogenic signal and a
signal protecting against apoptosis.' Of relevance to the present study is the finding of a third form of receptor mRNA
that encodes a potentially secreted form of the receptor
termed EPOR-s. This mRNA is expressed in fixed proportion to the total EPOR mRNA and is produced as a consequence of a 104 bp a insert due to the presence of an imperfect splice acceptor site in the 5' flanking region of exon V
resulting in an additional 46 amino acids after position
195.8,47It would be predicted that the ectodomain of all
three forms of EPOR would be detected by the S25 antisera
used in the present study because the amino terminus of
EPOR-t, EPOR-f, and EPOR-s are all the same. Therefore,
the current data are unable to distinguish between a protein
product of EPOR-s mRNA and a proteolytic cleavage fragment of either EPOR-f or EPOR-t. This distinction must
await isolation of this circulating protein and carboxy terminal amino acid sequence analysis. Likewise, the origins of
the 34 Kd moiety detected in solubilized marrow also awaits
further biochemical identification.
One limitation of the present study was the reliance on a
semiquantitative visual estimate of the amount of the clinically relevant 34 Kd protein. A clearer definition of the role
of this protein in issessing erythropoiesis could be obtained
by the development of a quantitative ELISA for the serum
form of EPOR. The development of such an assay could
potentially provide a quantitative measure of different
stages of erythropoietic development. The recent introduction of the assay2*that detects the truncated transferrin receptor4' in serum has provided a useful index of the size of
the late progenitor
Assuming that the 34 Kd peptide
derived from EPOR provides a quantitative marker of either early progenitor or total progenitor activity, the use in
tandem of measurements of the serum transferrin receptor
and serum EPOR may be a useful tool to assess the number
and stage of maturation of red blood cell precursors. In this
regard, a recent preliminary report using a polyclonal antibody developed to a fusion molecule of the EPOR-ectodoin an ELISA system
main with gl~tathione-S-transferase~~
has described the presence of immunodetectable EPOR in
human sera.50
ACKNOWLEDGMENT
The authors thank Lilla Khalifah for technical assistance, Leslie
Kuharich for assistance with preparation ofthe manuscript, and Dr
Simon Jones of Genetics Institute for kindly providing the purified
recombinant soluble ectodomain of the EPOR.
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2. Dai CH, Krantz SB, Zsebo KM: Human burst forming unitserythroid need direct interaction with stem cell factor for further
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1993 82: 2088-2095
Serum form of the erythropoietin receptor identified by a sequencespecific peptide antibody [see comments]
RD Baynes, GK Reddy, YJ Shih, BS Skikne and JD Cook
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