Shared and Unique Determinants of the Erythropoietin (EPO)
Receptor Are Important for Binding EPO and EPO Mimetic Peptide*
Steven A.
Middleton
§,
Francis P.
Barbone
,
Dana L.
Johnson
,
Robin L.
Thurmond
,
Yun
You¶,
Frank J.
McMahon
,
Renzhe
Jin
,
Oded
Livnah
**,
Jennifer
Tullai
,
Francis X.
Farrell
,
Mark A.
Goldsmith

,
Ian A.
Wilson
, and
Linda K.
Jolliffe
From the
R. W. Johnson Pharmaceutical Research
Institute, Raritan, New Jersey 08869, the ¶ Gladstone Institute of
Virology and Immunology, San Francisco, California 94141, the

Department of Medicine, School of Medicine,
University of California, San Francisco, California 94143, and the
Department of Molecular Biology and Skaggs Institute for
Chemical Biology, The Scripps Research Institute,
La Jolla, California 92037
 |
ABSTRACT |
We have shown previously that
Phe93 in the extracellular domain of the
erythropoietin (EPO) receptor (EPOR) is crucial for binding EPO.
Substitution of Phe93 with alanine resulted in a dramatic
decrease in EPO binding to the Escherichia coli-expressed
extracellular domain of the EPOR (EPO-binding protein or EBP) and no
detectable binding to full-length mutant receptor expressed in COS
cells. Remarkably, Phe93 forms extensive contacts with a
peptide ligand in the crystal structure of the EBP bound to an
EPO-mimetic peptide (EMP1), suggesting that Phe93 is also
important for EMP1 binding. We used alanine substitution of EBP
residues that contact EMP1 in the crystal structure to investigate the
function of these residues in both EMP1 and EPO binding. The three
largest hydrophobic contacts at Phe93, Met150,
and Phe205 and a hydrogen bonding interaction at
Thr151 were examined. Our results indicate that
Phe93 and Phe205 are important for both EPO and
EMP1 binding, Met150 is not important for EPO binding but
is critical for EMP1 binding, and Thr151 is not important
for binding either ligand. Thus, Phe93 and
Phe205 are important binding determinants for both EPO and
EMP1, even though these ligands share no sequence or structural
homology, suggesting that these residues may represent a minimum
epitope on the EPOR for productive ligand binding.
 |
INTRODUCTION |
Erythropoietin (EPO),1
the primary cytokine involved in the regulation of red blood cell
production, functions by binding and signaling through a cell surface
receptor (EPOR) on red blood cell precursors (1-5). Signaling through
the EPOR includes the activation of a receptor-associated tyrosine
kinase, JAK2 (3), which leads to the subsequent phosphorylation and
activation of STAT5 (4, 5), a member of the signal transducer and
activator of transcription (STAT) family of transcriptional activators
(reviewed in Ref. 6). Activated STAT5 moves to the nucleus where it
promotes transcription of target genes by binding to specific STAT5
response sequences. In binding studies, the EPOR has been detected on
purified primary human erythroid progenitor cells with one affinity
(Kd = 0.1 nM) or with two affinities
(Kd = 0.1 nM and 0.57 nM)
for 125I-EPO (7, 8). The significance of two affinities of
the EPOR for EPO is not clear. Evidence suggests that there is an accessory component of the EPOR that increases EPO binding affinity (9-11), but this component(s) has yet to be identified. Alternatively, two affinities of the EPOR for EPO may arise from two nonequivalent receptor binding sites on EPO, one of high affinity (~1
nM) and one of low affinity (~1 µM), that
have been reported for the extracellular domain of the EPOR in solution
(12). Structurally, the human EPOR is a 484-amino acid glycoprotein
with a single transmembrane segment located between extracellular and
intracellular domains each of nearly equal size (13). EPOR is a member
of a large family of cytokine and growth factor receptors whose ligand
binding domains contain homologous sequences and are predicted to be
structurally related (14). These ligand binding domains consist of
approximately 200-250 amino acid residues and contain two subdomains,
each predicted to consist of seven
-strands and to be structurally
related to fibronectin type III (FNIII) domains (14, 15). The
amino-terminal FNIII-like domain contains a pair of spatially conserved
cysteine bridges, while the carboxyl-terminal FNIII-like domain
contains a conserved
-strand F and a highly conserved
WSXWS motif that are hallmarks of receptors of the cytokine
receptor family (14).
Previously, we reported the characterization of an EPO-binding protein
(EBP), consisting of amino acids 1-225 of the extracellular domain
of the mature human EPOR, that was expressed in E. coli in
quantity and purity sufficient for site-specific mutagenesis and
crystallographic studies (16). Recently, the crystal structures of the
EBP bound to agonist peptide (EPO-mimetic peptide, EMP1) and an
antagonist peptide (EMP33) have been determined (17, 18). EMP1 is a
20-amino acid peptide that is one of a series of related peptides
discovered by phage display methodology (19). EMP1 exhibits no homology
to EPO, yet binds specifically to the EPOR and mimics the biological
effects of EPO both in vitro and in vivo (19).
The structure of the EBP in the EMP1·EBP complex consists of two
domains, each containing seven
-strands arranged in a FNIII-like
topology (17). This topology for the EBP was also observed in the
recently described crystal structure of EBP (EPObp) bound to EPO (20).
The structure of the EBP shows close homology with the structures of
ligand binding domains of GHR, prolactin receptor, granulocyte
colony-stimulating factor receptor, and GP130 (21-24), all members of
the cytokine receptor family. The EBP also shows structural homology
with the extracellular domains of TF (25, 26) and ligand bound
IFN
R
(27), and even a natural killer cell inhibitory receptor
(28). Tissue factor and IFN
R
are "class 2" cytokine receptors
(14) in which the seven-
-strand FNIII-like fold is maintained but
the relative orientation of the two domains differs.
The interaction between EMP1 and EBP consists of two molecules of EMP1
bound by two molecules of EBP to form a two-fold symmetrical assembly,
where each peptide interacts with its peptide partner and both receptor
molecules (17). As a result of this binding symmetry, EBP residues that
interact with EMP1 are practically identical for each EBP in the
complex. These interactions include hydrophobic contacts with the side
chains of Phe93, Met150, and Phe205
and a hydrogen-bonding interaction with the side chain of
Thr151 of the EBP. The interactions with Phe93,
Met150, and Phe205 are among the most
significant non-polar contacts in the binding interface in terms of the
area of molecular surface buried by each of these residues (17). Prior
to the determination of the EMP1·EBP crystal structure, we
investigated what role Phe93 and Met150 might
have in EPO binding, based on their predicted secondary structural
homologies to the positions of ligand binding determinants in other
receptors of the cytokine receptor family (29). Phe93 was
found to be critical for EPO binding, while Met150 was
found to be relatively unimportant. In the present study, we used the
alanine substitution mutants at Phe93 and
Met150 to determine if these residues contribute
significantly to EMP1 binding. In addition, further alanine
substitution mutants were created at EBP residues Thr151
and Phe205. These residues were selected for mutagenesis,
based on their contacts with EMP1 in the EMP1·EBP crystal structure
and on predicted secondary structural homologies to the positions of
ligand binding determinants in other receptors of the cytokine receptor
family. Furthermore, these residues contact EPO in the recently
reported EPO·EPObp crystal structure (20). Our results indicate that Phe93 and Phe205 are important for both EPO and
EMP1 binding, while Thr151 appears to be unimportant for
the binding of these ligands. In contrast, Met150, which is
relatively unimportant for EPO binding, is critical for EMP1 binding.
Thus, some of the same residues on the EPOR are important for binding
two very different ligands, EPO and EMP1, suggesting that these
residues may be required for productive ligand binding to the EPOR.
 |
EXPERIMENTAL PROCEDURES |
Expression and Mutagenesis of the EBP--
The bacterial
expression, purification, and characterization of the 225-amino acid
extracellular domain of the EPOR (referred to here as an EBP) has been
described in detail elsewhere (16). Biophysical analysis indicated that
the purified EBP contained the expected amino terminus,
disulfide-bridging pattern, and molecular mass. The EBP exhibited a low
nanomolar binding affinity (Kd = 5 nM)
for EPO. Mutagenesis of the EBP was performed as described previously
(29), and the concentrations of purified wild-type and mutant EBP were
estimated using the experimentally determined extinction coefficient of
the wild-type EBP of 2.3 absorbance units/mg/ml at 280 nm (16). Gross
structural characterization of the F205A-EBP by circular dichroism (CD)
was performed as described previously (29).
Assay of the Binding Activity of Wild-type and Mutant
EBP--
Competition binding assays in which wild-type or mutant EBP
compete for 125I-EPO binding with EPOR on the surface of
TF-1 cells were performed as described (30). Equilibrium binding
experiments on the wild-type and mutant EBP were carried out using a
surface plasmon resonance assay on a BIAcore 2000 instrument. Proteins
were coupled to the carboxymethylated dextran surface using amine
coupling chemistry. The derivatization levels were ~3,000 resonance
units. Different concentrations of either EPO or EMP1 were injected
over all four flow cells sequentially at a flow rate of 5 µl/min. The
surfaces were regenerated with a 1-min pulse of 10 mM
sodium acetate, pH 4.0. Each chip had one negative control surface and
one surface containing wild-type EBP. To calculate the dissociation
constants, the change in the equilibrium amount of ligand bound as a
function of the concentration of ligand was fit to the equation for a
simple 1:1 binding model,
|
(Eq. 1)
|
or to a model for two independent binding sites
|
(Eq. 2)
|
where R is the response, Rmax
is the maximum response, and Kd is the dissociation constant.
EMP1-mediated Dimerization of the EBP--
The ability of EMP1
to mediate the dimerization of wild-type and mutant EBP was evaluated
in chemical cross-linking assays using the sulfhydryl-reactive
cross-linking reagent (1,4-di- (3'-(2'-pyridyldithio) propionamido)butane (DPDPB; Pierce). The assay was performed as described previously (17, 30), except that the reaction products were
detected and quantified using high performance-size exclusion chromatography (HP-SEC). Briefly, wild-type or mutant EBP (11 µM) were incubated with a variable concentration of EMP1
(stock prepared in 0.1% trifluoroacetic acid) and 0.5 mM
DPDPB (stock prepared in dimethyl sulfoxide) in 75 µl of
phosphate-buffered saline, pH 7.4, for 4 h at room temperature
then overnight at 4 °C. All reactions and controls contained a final
concentration of 4.4% Me2SO and 0.007% trifluoroacetic
acid to improve the solubility of the cross-linker. The samples were
analyzed by HP-SEC on a Waters 625 HPLC system equipped with a Waters
996 detector. Separations were performed at room temperature on a
7.8 × 300-mm G3000 SWXL column (Supelco, Bellfonte, PA). The
column was equilibrated in 10 mM
Na2PO4, pH 7.2, 150 mM NaCl at a
flow rate of 1 ml/min and was monitored at 220 nm. Under these
conditions, EBP eluted at ~9.6 min, while the dimer product eluted
earlier at ~8.9 min. These elution times correspond to
Mr ~25,000 for EBP monomer and Mr ~52,000 for the dimer product based on
calibration of the column using a commercial HP-SEC standard mixture
(16). The percentage of dimer product formed was calculated by adding
the integrator determined peak area for the monomer and dimer protein
peaks to determine total protein peak area. The dimer area value was
divided by the total protein peak area and multiplied by 100 to yield the percentage of dimer observed in each reaction mixture. The reported
value for each concentration is the average over three different
chromatographic separations, and the error bars are the standard
deviation value for the three experiments.
STAT Activation Assay--
For expression in COS7 cells, the
various mutants were subcloned from the EBP bacterial expression
plasmid pSAM3 (16) to a mammalian expression plasmid encoding the
full-length human EPOR in pSG5 (Stratagene, La Jolla, CA). Transient
transfection of COS7 cells (ATCC, Rockville, MD) was performed using
LipofectAMINE (Life Technologies, Inc.) per the manufacturer's
instructions. Cells were cotransfected with expression plasmids
encoding the wild-type or mutant (F93A, M150A, or F205A) forms of the
human EPOR along with a plasmid encoding STAT5a (pME18S-STAT5a, kindly provided by Dr. Alice Mui, DNAX Research Institute). Stimulations were
performed for 10 min at 37 °C with either EPO or EMP1 at the stated
concentrations. Electrophoretic mobility shift assays were performed
using nuclear extracts prepared from resting or stimulated cells and an
oligonucleotide probe containing the Fc
RI STAT response sequence, as
described previously (31).
 |
RESULTS |
Previously, we expressed the extracellular domain of the human
EPOR as a soluble EBP in E. coli (16). The crystal structure of the EBP bound to an EPO-mimetic peptide (EMP1) revealed several EBP
residues that form significant contacts with the peptide (17). These
include major nonpolar interactions with the side chains of
Phe93, Met150, and Phe205 and a
hydrogen bond with the side-chain hydroxyl group of Thr151
(Fig. 1). Phe93,
Met150, and Phe205 of the EBP along with
TyrP4, PheP8, TrpP13, and
CysP15 of the peptide form a hydrophobic core at the heart
of the interaction between EBP and EMP1 (Ref. 17; Fig. 1). The hydrogen
bond with the hydroxyl group of Thr151 is part of a network
of mostly main-chain hydrogen bonds formed with main-chain atoms of the
type I
-turn of the peptide (Fig. 1).

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Fig. 1.
EBP residues contacting EMP1 in the
EMP1·EBP crystal structure. EMP1 binds EBP with a 2:2
stoichiometry in the crystal structure (17). Each peptide
(blue) forms symmetrical contacts with both receptors
(gray) in the complex. Phe93,
Met150, and Phe205 of the EBP interact with
TyrP4, PheP8, TrpP13, and
CysP15 of the peptide to form the hydrophobic core of the
interaction between EBP and EMP1. The hydrogen bond with the hydroxyl
group of Thr151 is part of a network of mostly main-chain
hydrogen bonds formed with main-chain atoms of the type I -turn of
the peptide. The six loops containing binding determinants in receptors
or the cytokine receptor family are labeled L1-L6. For
clarity, the side chains of PheP8 and CysP15
are not shown.
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|
To determine if Thr151 and Phe205 contribute
significantly to EPO binding, these residues were replaced with alanine
and the resultant mutant proteins were purified and tested for their
ability to compete for 125I-EPO binding with EPOR on the
surface of TF-1 cells. Alanine was chosen as the replacement residue,
since it is likely to result in a loss of interactions from the
original side chain without introducing new interactions or gross
structural changes (32, 33). The T151A-EBP exhibited an
IC50 of 6.5 nM in this assay, nearly identical
to the wild-type value of 3.5 nM, indicating that
Thr151 does not contribute significantly to EPO binding
(Fig. 2). In contrast, the F205A-EBP does
not compete for EPO in this assay except at very high concentrations.
About 45% inhibition of 125I-EPO binding to the cells was
observed at the highest concentration of F205A-EBP tested (10 µM; Fig. 2). Thus, the F205A-EBP exhibits an
IC50 > 10 µM in this assay compared with the
wild-type value of 3.5 nM, suggesting that this mutation
results in a dramatic decrease in the ability to bind EPO. Competition
binding analyses were also performed on the F93A- and M150A-EBP.
Similar to the F205A-EBP, the F93A-EBP exhibited about 45% inhibition
of 125I-EPO binding at 10 µM (Fig. 2) and,
thus, also has an IC50 > 10 µM in this
assay. The M150A-EBP bound EPO as well as wild-type, exhibiting an
IC50 of 3.5 nM. These results suggest that
Phe93 and Phe205 are critical for EPO binding,
while Met150 and Thr151 are relatively
unimportant for EPO binding.

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Fig. 2.
EPO binding activity of the EBP mutants in
competition binding assays. The wild-type and mutant EBP were
tested for their ability to compete for 125I-EPO binding
with full-length EPOR on the surface of TF-1 cells. The percentage of
125I-EPO bound was calculated by dividing the counts/min at
each data point by the counts/min with no added EBP and multiplying by
100. The IC50 values derived from these assays are 3.5 nM for both the wild-type and M150A-EBP, 6.5 nM
for the T151A-EBP, and >10 µM both the F205A- and
F93A-EBP, indicating that the F205A and F93A mutations result in a
large decrease in the binding of 125I-EPO.
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|
We have previously shown that the dramatic reduction in EPO binding in
the F93A-EBP is not a result of gross structural changes caused by the
mutation (29). To investigate the possible structural consequences of
the F205A mutation, CD was used to evaluate the overall secondary
structure and thermal denaturation of the F205A-EBP relative to the
wild type. The F205A-EBP produced a CD spectrum similar to wild type
except for reduced intensity in the 200-220-nm region (Fig.
3A) and resulted in a small
(~5 °C) decrease in thermal stability relative to the wild-type
EBP (Fig. 3B). Taken together, the limited differences in CD
spectrum and decrease in Tm of the F205A-EBP
indicate that this mutation does not cause significant changes in the
global secondary structure or the stability of the EBP.

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Fig. 3.
CD spectrum and thermal stability of the
F205A-EBP. CD spectra (A) were generated at 25 °C
and 0.365 mg/ml protein in phosphate buffered saline. Overall, the CD
spectrum of the F205A-EBP is similar to that of the wild-type EBP
except for reduced intensity from 200 to 220 nm. The F93A mutation
results in essentially no change in the CD spectrum of the EBP (29).
Thermal denaturation curves (B) were generated by plotting
the ellipticity at 228 nm derived from the CD spectra at each
temperature using 0.365 mg/ml protein in phosphate-buffered saline. The
F205A mutation causes a small decrease in the Tm
of the EBP. The Tm of the F93A-EBP is identical
to that of the wild-type EBP (29).
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To determine an EPO binding affinity (Kd) for the
EBP mutants, equilibrium binding analyses were performed on the wild-type and mutant EBP using surface plasmon resonance. As can be
seen in Fig. 4A, the EPO
binding curves for the M150A- and T151A-EBP were similar to wild type.
The data for the wild-type and T151A-EBP fit best to a two-site model
(see "Experimental Procedures"), yielding a high affinity
Kd of ~5 nM and a low affinity
Kd of ~900 nM for EPO. For the
M150A-EBP, however, the two-site fit of the data gave no improvement
over the 1:1 model. This mutant exhibited a single
Kd for EPO of 36 nM, only slightly
increased relative to the high affinity (5 nM)
Kd of the wild-type EBP. In contrast, the F205A-EBP exhibited a dramatically increased Kd of 1 µM relative to wild-type EBP (Fig. 4A). The
difference between the 1 µM Kd and the
>10 µM IC50 for EPO observed for the
F205A-EBP (see above) might be expected, since competitive binding data
(IC50) are not directly comparable to equilibrium binding
data (Kd). Furthermore, in the competition binding
format, the EBP mutants compete for EPO binding with cell surface
wild-type EPOR exhibiting a Kd for EPO of 400 pM (34). Less active mutants might compete poorly if at all
with the high affinity EPOR, leading to increased IC50
values relative to Kd values from a direct binding
assay. In any case, the 1 µM Kd of the F205-EBP represents a decrease in binding affinity of about 200-fold from the 5 nM Kd of the wild type,
confirming the results of the competition binding experiments that
Phe205 is an important EPO binding determinant. The
F93A-EBP showed no binding to EPO over the concentration range studied
(1 nM to 10 µM; Fig. 4A), confirming that
Phe93 is also an important EPO binding determinant of the
EPOR (29).

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Fig. 4.
Analysis of EPO and EMP1 binding to the
wild-type and mutant EBP by surface plasmon resonance.
A, equilibrium binding analyses of EPO binding to the
wild-type and mutant EBP. The lines are the best fit of the
data to Equations 1 or 2 (see "Experimental Procedures" and
"Results"), and the symbols and vertical
bars are the average and standard error, respectively, of
three independent experiments. The dissociation constants
(Kd) for EPO derived from these data are 5 nM (high affinity) and 900 nM (low affinity)
for both the wild-type and T151A-EBP, and 36 nM (single
affinity) for the M150A-EBP. The Kd for the
F205A-EBP is 1 µM and the F93A-EBP did not show
detectable EPO binding in this assay, indicating that
Phe205 and Phe93 are important EPO binding
determinants. B, sensorgrams showing the response of the
wild-type and mutant EBP to 1 µM EMP1. The T151-EBP bound
EMP1 about as well as the wild type. The M150A-, F93A-, and F205A-EBP
did not show any binding to EMP1 at this concentration, exhibiting
responses similar to the background observed for a blank surface or
surfaces coated with the unrelated proteins leptin and leptin
receptor.
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Surface plasmon resonance was also used to examine EMP1 binding
affinities of the wild-type and mutant EBP. The sensorgrams for EMP1
binding at a concentration of 1 µM are shown in Fig. 4B. The F93A-, M150A-, and F205A-EBP did not show specific
binding to EMP1 above the background seen for an underivatized surface or surfaces coated with unrelated proteins (leptin and leptin receptor). Only the T151A-EBP bound EMP1, showing a response similar to
that of wild type (Fig. 4B). In binding curves generated at various concentrations of EMP1 (0.1-10 µM), the
wild-type and T151A-EBP had comparable affinities for EMP1 of ~350
nM (data not shown). This value is similar to the
half-maximal response (EC50) of 400 nM reported
for EMP1 in cell proliferation assays (19). The F93A-, M150A-, and
F205A-EBP showed slight binding only at µM concentrations
of EMP1 (from 6 to 10 µM); however, the response was not
high enough above controls to be attributed with certainty to specific
binding (data not shown). Therefore, if the peptide binds at all to
these mutants, it is with a much lower affinity (>10 µM)
than for wild type (0.35 µM). These data indicate that
Phe93, Phe205, and Met150 are all
critical binding determinants for EMP1.
The ability of EPO and EMP1 to bind and signal through full-length
wild-type, F93A-, M150A-, and F205A-EPOR was tested in a STAT
activation assay. The T151A mutation was not tested in this assay,
since this mutation did not appear to have a significant effect on
either EPO or EMP1 binding. COS cells cotransfected with full-length
receptor and STAT5a constructs were treated with EPO or EMP1, and
active STAT5a dimer bound to an oligonucleotide containing a
STAT-responsive element was detected in gel shift assays. Initial
experiments were performed with concentrations of ligand that elicited
a good response for the wild-type EPOR. At these concentrations (0.24 or 1.23 nM EPO and 5 or 20 µM EMP1), only the
M150A-EPOR showed a response to EPO and none of the mutants showed a
response to EMP1 (Fig. 5A). A
faint band was observed for the wild-type EPOR at 0.24 nM
EPO, but no band was detectable at this concentration for the
M150A-EPOR, suggesting that this mutant may be slightly less sensitive
to EPO than wild type. The wild-type and mutant EPOR were also tested
at the relatively high concentrations of 1.23 µM EPO and
200 µM EMP1 (Fig. 5B). At these concentrations, the F205A-EPOR gave a faint response to EPO but still
did not respond to EMP1. The F93A-EPOR, however, exhibited the opposite
pattern. It still did not respond to EPO but demonstrated a significant
response to EMP1. Finally, the M150A-EPOR did not show a response to
EPO or EMP1 at the high concentrations. The loss of a response to EPO
at 1.23 µM for the M150A-EPOR is probably due to
supersaturating concentrations of ligand occupying the receptor binding
site at a 1:1 ratio, thereby driving the receptor to monomerization and
reducing signaling. This effect was seen for the wild-type EPOR at 1.23 µM EPO and at 20 and 200 µM EMP1 (Fig. 5,
A and B). Evidently, the M150A-EPOR binds EPO
well enough to exhibit this effect. To be sure that supersaturating
concentrations of ligand were not inhibiting the signaling of the other
mutants, intermediate concentrations of 49 nM EPO and 100 µM EMP1 were tested. Under these conditions, none of the
mutants showed a response to EPO and only the F93A-EPOR showed a
response to EMP1, which was less than that observed at 200 µM EMP1 (data not shown).

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Fig. 5.
Effects of the F93A, M150A, and F205A
mutations on EPO and EMP1-mediated STAT5a activation through the
EPOR. Lysates prepared from COS cells expressing full-length
wild-type or mutant EPOR and STAT5a treated with standard
(A) or high (B) concentrations of EPO or EMP1
were run in electrophoretic mobility gel shift assays as described
under "Experimental Procedures." The arrow indicates the
position of active STAT5a bound to an oligonucleotide probe containing
the Fc RI STAT response sequence in response to stimulation with EPO
or EMP1.
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To investigate further the effects of the EBP mutations on the
interaction with peptide, an EBP cross-linking assay was used to
evaluate the ability of EMP1 to mediate the dimerization of the EBP
mutants (17, 30). Wild-type and mutant EBP were incubated with various
concentrations of EMP1, and the reaction products were detected by
HP-SEC. The percentage of dimer formed, calculated from the areas under
the peaks, was plotted at each concentration of peptide (Fig.
6). The maximum amount of dimer formed by
the T151A-EBP was about 80% at 100 µM EMP1, similar to
the wild-type EBP, which showed a maximum of about 95% dimer at 100 µM peptide. The F205A-EBP also exhibited maximal
dimerization of about 80%, although at a higher concentration of EMP1
(200 µM) than wild type. Thus, the T151A and F205A
mutations do not appear to have a large effect on EMP1-mediated
dimerization of the EBP. In the case of the F93A-EBP, peak dimerization
occurred at 100 µM peptide, similar to wild type, but the
maximal amount of dimerization achieved was only around 35% (Fig. 6).
More dramatically, dimerization of the M150A-EBP was almost
undetectable at 100 µM peptide and only about 5% dimer
was formed at the highest concentration of EMP1 tested (400 µM). These data indicate that the F93A mutation and
especially the M150A mutation result in a large decrease in the ability
of EMP1 to facilitate the dimerization of the EBP. Taken together with
the results of the equilibrium binding and STAT activation assays,
these results indicate that Phe93, Phe205, and
Met150 are all important for the binding and/or agonist
activity of EMP1.

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Fig. 6.
Effects of the EBP mutations on the
EMP1-mediated dimerization of the EBP. Wild-type and mutant EBP
were incubated with various concentrations of peptide in the presence
of the DPDPB cross-linker and analyzed by HP-SEC. The percentage of
dimer formed was calculated from the areas under the peaks and plotted
for each concentration of peptide. The symbols and
vertical bars are the average and standard error,
respectively, of three independent chromatographic separations.
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 |
DISCUSSION |
In this study, we investigated the effects of alanine substitution
of EPOR residues Phe93, Phe205,
Met150, and Thr151 on binding to both EPO and
an EPO-mimetic peptide (EMP1). These residues were chosen for
investigation based on several criteria. First, all of these residues
interact with EMP1 in the crystal structure of EMP1 bound to the EBP
(17), suggesting that they may contribute significantly to EMP1
binding. Second, Phe93 has been shown to be critical for
EPO binding (29), suggesting that residues that interact with peptide
in the EMP1·EBP crystal structure might also be involved in EPO
binding. Third, many cytokine receptors contain ligand binding
determinants in positions homologous to Phe93,
Met150, and Thr151 (29) and Phe205
(35-42), further suggesting that these residues may be involved in
ligand binding to the EPOR. In addition, all these residues are buried
in the interface with EPO in the EPO·EPObp crystal structure
(20).
Although both Phe93 and Phe205 are important
binding determinants for EPO, their relative contributions to the
binding of this ligand differ. For example, the F205A-EPOR exhibits
slight activity at 1.23 µM EPO in the STAT activation
assay, while the F93A-EPOR does not. The activity seen for the
F205A-EPOR at 1.23 µM EPO might be expected, based on the
1 µM Kd for EPO of the F205A-EBP in
the equilibrium binding assay. In the same vein, the F93A-EPOR might
not be expected to respond to 1.23 µM EPO in the STAT
activation assay, since the F93A-EBP did not exhibit detectable binding
to concentrations of EPO as high as 10 µM in the
equilibrium binding assay. It is unlikely that the lack of activity of
the F93A-EPOR in the STAT assay results from poor expression on the
surface of the COS cells used in the assay, since this mutant exhibited
expression on COS cells comparable to wild type (29). Combined, these
results suggest that Phe93 is more important for EPO
binding than Phe205. The 200-fold increased
Kd for EPO of the F205A-EBP compared with the
1,000-fold increased IC50 for EPO reported for the F93A-EBP
(29) adds further support to this hypothesis.
The data presented here indicate that Phe93,
Phe205, and Met150 are all important binding
determinants for EMP1, since none of the respective mutant proteins
exhibit detectable binding to concentrations of EMP1 up to 10 µM in the equilibrium binding assay, while
Thr151 is relatively unimportant for EMP1 binding in this
assay. In addition, the F205A- and M150A-EPOR do not respond to peptide in the STAT activation assay, while the F93A-EPOR did show a response, but only at high concentrations of EMP1 (100 and 200 µM).
These effects could be the result of the reduced binding of EMP1 or aberrant dimerization and/or signaling of the mutant receptors. In any
case, the results of the STAT activation assay indicate that
Phe93 is not as important as Phe205 and
Met150 for activity with EMP1. The results of the
EMP1-mediated dimerization (cross-linking) assay suggest that
Phe93 and especially Met150 are important for
the dimerization of the EBP. Phe205 appears to be
relatively less important for dimerization, since the F205A-EBP
exhibits significant dimerization in the presence of peptide, albeit at
higher concentrations. Apparently, the ability of EMP1 to mediate the
dimerization of the F205A-EBP does not translate to activity in the
STAT assay. The presence of the cross-linker capturing a transient
dimerization of this mutant at the high concentrations of EBP used in
the cross-linking assay might explain this discrepancy. The relatively
small effect of the F205A mutation in the cross-linking assay does not
mean that Phe205 is unimportant for EMP1 binding, since the
amount of dimerization observed for the EBP mutants may not be related
to relative binding affinities due to the high concentrations of
protein used in the assay. In addition, the presence of the
cross-linking reagent complicates interpretation of the results, since
different modes of binding and/or dimerization resulting from the
mutations may affect the cross-linking reaction.
Of the 45 amino acid residues (20%) of the EBP that we have examined
by alanine substitution to date (29, 43), only Phe93 and
Phe205 were found to be critical for EPO binding. The
remaining residues had relatively little or no role in EPO binding or
were important for structure. These results are consistent with the
suggestion that only a few residues make significant contributions to
binding in protein-protein interactions (functional epitope), even
though a large number of residues may be involved in the contact
interface (structural epitope; Refs. 44-46). In the GH-GHR
interaction, two tryptophans (Trp104 and
Trp169) accounted for the majority of the binding free
energy of the interaction, constituting a hot spot for GH binding to
GHR (44). Alanine substitution of Trp104 and
Trp169 resulted in the largest decreases in binding
affinity by far, estimated to be greater than 2,500-fold, relative to
wild-type GHbp (44, 47). Our results suggest that Phe93 and
Phe205 of the EPOR may be functionally analogous to
Trp104 and Trp169 of the GHR in providing the
majority of the binding energy for the interaction with ligand. When
the EPO·EPObp crystal structure became available (20), we found we
had already examined the function of 40% of the residues involved in
H-bonds or salt bridges and nearly 50% of the residues involved in
non-polar interactions with EPO (29, 43). The data presented here,
combined with the fact that Phe93 and Phe205
dominate the non-polar contacts with EPO site 1 and site 2 (20), indicate that these residues are an important component of the hot spot
for EPO binding.
In the EPO·EPObp crystal structure, Met150 contacts only
a single residue (Phe48) in binding site 1 on EPO and is
more buried in site 2, contacting three EPO residues (20). Our results
indicate that the single contact with site 1 is unimportant for EPO
binding and the more extensive contacts with site 2 are apparently also
relatively unimportant for EPO binding, since the response of the
M150A-EPOR to EPO in the STAT activation assay was similar to that of
wild type. In the case of EMP1 binding to the EPOR, Met150
buries significant surface area with EMP1 (~60 Å2; Ref.
17) and is critical for EMP1 binding. However, Thr151 also
forms hydrophobic interactions with peptide in the EMP1·EBP crystal
structure. In addition, the side-chain hydroxyl of Thr151
forms a hydrogen bond with peptide (see Fig. 1). Despite these interactions, Thr151 does not contribute significantly to
EMP1 binding. Apparently, the deletion of a single hydrogen bond in the
network of hydrogen bonds with peptide in the region around
Thr151 (see Fig. 1) is not sufficient to disrupt peptide
binding. These results support the conclusion that the amount of
surface area buried by a contact residue does not always correlate with
binding affinity (44-46) and emphasize the importance of mutagenesis
studies to verify structural based predictions of the function of
specific residues.
In conclusion, the fact that residues important for binding EPO
(Phe93 and Phe205) are also important for
binding the unrelated peptide, EMP1, suggests that these residues may
represent a minimum epitope for productive ligand binding to the EPOR
and has important implications for the design of small molecule
mimetics. In addition, our results suggest that, as is the case for
GHR, only a few residues of the EPOR contribute significantly to ligand
binding (Phe93 and Phe205 for EPO binding and
Phe93, Phe205, and Met150 for EMP1
binding). Thus, our results provide support for the notion that the
functional epitope for ligand binding is relatively small compared with
the structural epitope. Indeed, this may account for the existence of a
relatively small peptide mimetic of EPO (EMP1) in the first place and
indicates that it may be possible to discover non-peptide small
molecule mimetics of EPO and other biologically important cytokines.
 |
ACKNOWLEDGEMENTS |
We thank Minmin Yu for preparation of Fig. 1
and Elizabeth Howard for technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant GM49497 (to I. A. W.) and Grant GM54351 (to
M. A. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: R. W. Johnson
Pharmaceutical Research Inst., 1000 Route 202, P. O. Box 300, Raritan, NJ 08869. E-mail: smiddlet{at}prius.jnj.com.
**
Current address: Dept. of Biological Chemistry, Institute of Life
Sciences, Wolfson Center of Applied Structural Biology, Hebrew
University of Jerusalem, Jerusalem 91904, Israel.
 |
ABBREVIATIONS |
The abbreviations used are:
EPO, erythropoietin;
EPOR, erythropoietin receptor;
EBP, EPO binding protein consisting of
the extracellular domain of the EPOR expressed in E. coli;
EMP, EPO mimetic peptide;
GH, growth hormone;
GHR, growth hormone
receptor;
TF, tissue factor;
IFN
R
, interferon-
receptor-
;
FNIII, fibronectin type III;
STAT, signal transducer and activator of
transcription;
HP-SEC, high performance-size exclusion chromatography;
DPDPB, 1,4-di- (3'-(2'-pyridyldithio) propionamido)butane.
 |
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