A new high affinity binding site for suppressor of cytokine signaling-3
on the erythropoietin receptor
Michael Ho¨ rtner
1,2
, Ulrich Nielsch
1
, Lorenz M. Mayr
3
, Peter C. Heinrich
2
and Serge Haan
2
1
Bayer Pharma Research Center, Wuppertal, Germany;
2
Institut fu
¨
r Biochemie, Rheinisch-Westfa
¨
lische Technische Hochschule
Aachen, Germany;
3
Novartis Pharma, Basel, Switzerland
Erythropoietin (Epo) is a hematopoietic cytokine that is
crucial for the differentiation and proliferation of erythroid
progenitor cells. Epo acts on its target cells by inducing
homodimerization of the erythropoietin receptor (EpoR),
thereby triggering intracellular signaling cascades. The
EpoR encompasses eight tyrosine motifs on its cytoplasmic
tail that have been shown to recruit a number of regulatory
proteins. Recently, the feedback inhibitor suppressor of

and differentiation of erythroid precursor cells. It acts on
target cells by inducing homodimerization of its specific cell
surface receptor. The erythropoietin receptor (EpoR) is a
member of the cytokine receptor superfamily that includes
receptors for prolactin, IL-3, granulocyte-colony stimula-
ting factor and thrombopoietin (for a recent review on
EpoR signal transduction see [3]). Following ligand binding,
the EpoR associated Janus kinase 2 (Jak2) is activated and
phosphorylates tyrosine residues within the cytoplasmic
region of the receptor. The phosphotyrosine motifs act as
recruitment sites for cytoplasmic proteins like the signal
transducer and activator of transcription 5 (STAT5).
STAT5 itself is then phosphorylated, dissociates from the
receptor and forms active dimers that translocate into the
nucleus where they bind to specific enhancer sequences in
the promoters of responsive genes.
Suppressor of cytokine signaling-3 (SOCS-3), alternatively
referred to as cytokine-inducible SH2-containing protein-3
(CIS-3), belongs to the SOCS family of proteins which have
been shown to be induced by a number of cytokines and
negatively regulate signal transduction in a classical feedback
loop [4–7]. SOCS-proteins share a central src homology)2
(SH2) domain and a C-terminal motif called the SOCS box
[8–10], which is thought to be involved in degradation of the
protein by the ubiquitin-proteasome pathway [11,12]. The
first member of this family, CIS, was cloned as an immediate-
early gene induced by several cytokines. CIS has been
demonstrated to bind to tyrosine-phosphorylated motifs
within EpoR and the IL-3 receptor, thereby inhibiting signal
transduction [4]. Furthermore, CIS was shown to bind to

accepted 5 April 2002)
Eur. J. Biochem. 269, 2516–2526 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02916.x
affinity of SOCS-3 for the double-phosphorylated peptide
containing both pY429 and pY431 than to the respective
single-phosphorylated tyrosine motifs. Surface plasmon
resonance (SPR) analysis, together with in vitro binding
assays and model structures of the SH2 domain of SOCS-3
complexed with EpoR peptides provide evidence for
pY429pY431 being a new high affinity binding site for
SOCS-3 within the EpoR.
MATERIALS AND METHODS
Materials
Biotinylated peptides were purchased from PolyPeptide
Laboratories (Munich, Germany). The amino-acid sequen-
ces of the peptides are shown in Fig. 1. Simian monkey
kidney (COS7) cells were purchased from ATCC (Rock-
ville, MD, USA) (CRL 1651). Cell culture media and
antibiotics were obtained from Life Technologies (Rock-
ville, MD, USA), and fetal bovine serum from Seromed
(Berlin, Germany).
Cloning of human SOCS-3
Constructions were carried out using standard procedures
[21]. Human SOCS-3 cDNA was amplified from
EST#725896 (Research Genetics, Huntsville, AL, USA)
and cloned into the pET32 vector (pET32-hSOCS-3).
Flanking primer sequences for PCR were as follows:
5¢-CCATGGTCACCCACAGCAAGTTT-3¢ and 5¢-TGG
ACCAGTACGATGCCCCGCTTTAATGAATTC-3¢.
For the expression in COS7 cells, human SOCS-3 cDNA
was subcloned into pcDNA3.1 (+) by the use of the BamHI

isopropyl thio-b-
D
-galactoside.
Cells were harvested after 3 h of expression, resuspended in
50 m
M
Tris/HCl, pH 8.0, 10% glycerol, and lysed by
sonication. SOCS-3 was purified on a HiTrap chelating
5 mL column (Amersham-Pharmacia, Freiburg, Germany)
with nickel-iminodiacetic acid as matrix. Native eluted
SOCS-3 was dialyzed into 50 m
M
Tris, 10 m
M
dithiothre-
itol, pH 8.5 and purified to homogeneity by anion exchange
chromatography on a MonoQ column (Amersham–Phar-
macia, Freiburg, Germany). For biosensor measurements
the protein was dialyzed against 50 m
M
Tris/HCl, pH 8.0,
10 m
M
dithiothreitol, 0.05% Chaps. Purity of the recom-
binant protein was monitored by SDS/PAGE.
COS7 and 293T cells were grown in Dulbecco’s modified
Eagle’s medium supplemented with 10% fetal bovine
serum, 50 lgÆmL
)1
penicillin and 100 lgÆmL

ation of the tyrosine residue is indicated as (pY), unphosporylated
tyrosines as (Y).
Ó FEBS 2002 SOCS-3 binds to the pY429pY431 motif of Epo-R (Eur. J. Biochem. 269) 2517
chip surface at a flow rate of 20 lLÆmin
)1
for 1 min. Before
injection of SOCS protein, the sensor chip was flushed with
bovine serum albumin (0.1 mgÆmL
)1
)ataflowrateof
20 lLÆmin
)1
for 1 min. For measurement of the K
d
value
the flow rate was enhanced to 100 lLÆmin
)1
in order to
obtain higher resolution of kinetics. For this type of
experiment SOCS-3 was injected for 3 min, dissociation
time was 5 min, regeneration of the chip between the
measurements in all experiments performed was done at
20 lLÆmin
)1
with 1
M
NaCl in 50 m
M
NaOH for 30 s.
Binding curves were analyzed by using

(5 lgÆmL
)1
), phenylmethanesulfonyl fluoride (1 m
M
)and
Na
3
VO
4
(1 m
M
). Equal amounts of cellular protein and
expressed SOCS-3 in each sample were obtained by mixing
the total cell lysates prior to the precipitation experiment.
SOCS-3 was precipitated by incubation of the total cell
lysates with the immobilized peptides at 4 °Covernight.
Precipitates were then washed three times with 500 lLlysis
buffer. The precipitated proteins were resolved by SDS/
PAGE and transferred to an Immobilon poly(vinylidene
difluoride) membrane (Millipore, Eschborn, Germany)
using a semidry electroblotting apparatus. Human SOCS-
3 was detected with a polyclonal antibody kindly provided
by J. A. Johnston (Queen’s University, Belfast, Northern
Ireland). A polyclonal goat anti-rabbit horse-radish per-
oxidase-conjugated secondary Ig (DAKO, Hamburg,
Germany) was used to visualize the immunoreactive bands
by ECL techniques.
Molecular modeling of the human SOCS-3 SH2 Domain
For molecular modeling and graphic representation of the
protein structures, the programs

inhibitory activity on IL-6 signaling by binding to phos-
photyrosine 759 of gp130 [16,17], which is also the
recruitment site for the phosphotyrosine phosphatase
SHP2 [33]. Moreover, it has been shown that SOCS-3 also
binds to the recruitment site for SHP2 of the erythropoetin
receptor [20]. To determine the binding affinities of SOCS-3
to the EpoR we investigated SOCS-3 binding to tyrosine-
phosphorylated and nonphosphorylated peptides of all
EpoR tyrosine motifs. Figure 1 shows the sequences of the
peptides used in this study. Peptides with two proximate
tyrosine residues were presented as double-phosphorylated
or mutually substituted with phenylalanine to check syner-
gistic effects on SOCS-3 binding. The N-terminal biotinyl-
ated peptides were captured on a SA Biosensor chip and the
interaction with SOCS-3 was analyzed. As control, binding
of SOCS-3 to an unloaded sensor surface was measured in
parallel. Additionally, in all experiments 3.5 l
M
thioredoxin
was injected to rule out nonspecific binding of the fusion
protein. As shown in Fig. 2, we confirmed the binding of
SOCS-3 to phosphotyrosine pY401 recently reported by
Sasaki et al. [20]. We also found that SOCS-3 weakly binds
to a peptide containing pY343, a binding site for STAT5
[34]. Interestingly, SOCS-3 showed high affinity binding to a
phosphopeptide encompassing pY429 and pY431 of the
EpoR (Fig. 2A). Both tyrosines Y429 and Y431 are
phosphorylated after stimulation with Epo [34]. The inter-
action between SOCS-3 and this peptide is phosphoryla-
tion-dependent as a nonphosphorylated peptide Y429Y431

motif bound with a K
d
of 1.1 l
M
(Table 1).
SOCS-3 needs a double phosphorylated Y429Y431
motif for highest affinity binding
The motif pY429pY431 of the EpoR contains two tyrosine
phosphorylation sites, spaced only by one amino-acid
residue (Fig. 1). In order to differentiate between these
two tyrosines in the context of SOCS-binding we deter-
mined binding affinities of SOCS-3 to peptides containing
only phosphotyrosine pY429 or pY431, as well as a double-
phosphorylated peptide pY429pY431. The SPR measure-
ments demonstrated that highest affinity binding of SOCS-3
occurred only if both tyrosine 429 and tyrosine 431 were
phosphorylated (Fig. 3B–D and Table 1).
SOCS-3 specifically binds to the receptor motifs
encompassing tyrosines pY401 and pY429pY431
in COS7 cells
In order to investigate whether SOCS-3 binds to the
receptor motifs containing pY401 and pY429pY431, we
performed a peptide precipitation assay with the biotinyl-
ated EpoR-peptides, which have been shown to interact
with SOCS-3 in the SPR experiments. The nonphosphor-
ylated peptide Y429Y431 was used as control. Equal
amounts of whole cell extracts of COS7 cells expressing
SOCS-3 were incubated with the different EpoR-peptides
immobilized on NeutrAvidin-coupled agarose. Subse-
quently, precipitated SOCS-3 was analyzed by Western

specificities of the SOCS-3 SH2 domain, we modelled the
complex of the SOCS-3 SH2 domain and the receptor
peptides pY401 and pY429pY431 (Fig. 6). For comparison,
Fig. 2. Comparison of SOCS-3 binding to pY343, pY401 and
pY429pY431 of the human EpoR (A) and sensogram showing the
interaction of serial dilutions of SOCS-3 and peptide pY401 (B).
(A) Biotinylated peptides were immobilized on SA chips, the concen-
tration of SOCS-3 was 8.8 l
M
. (B) SOCS-3 was diluted twofold from
8.8 l
M
to 275 n
M
. Purified thioredoxin was taken as control for
specific binding. Steady state binding values were taken for Scatchard-
analysis for the determination of K
d
values.
Table 1. Calculated K
d
values of the EpoR peptides as determined by
Scatchard analysis. ND, not determined due to a lack of interaction
with SOCS-3.
Peptide K
d
(l
M
)
pY343 > 30

Y)2, Y+1 and Y+3.
SOCS-3 binding to the peptide pY429pY431 is represen-
tedinFig.6B.ThepredictedcontactswithintheSH2
domain for the residues at positions Y)2(L)andY+3(L)
of the peptide are similar to pY401. The leucine at Y+1 is
predicted to undergo a hydrophobic contact with the side
chain of K91. In addition, the valine residues at positions
Y+4 and Y+5 contact F136 and P108, respectively. The
Y+2 residue in SH2/peptide interactions is usually exposed
to the solvent and does not contribute to the binding
[35–37]. Most interestingly, in our model the phosphotyr-
osine at Y+2 is able to form a salt bridge with the positively
charged R94 (contact is shown by a red Ô±Õ symbol in
Fig. 6B). This explains our observation that the double
phosphorylated peptide pY429pY431 binds with higher
affinity than a peptide in which pY431 is substituted by
phenylalanine. In addition the side chain of R94 is able to
build up a hydrophobic contact with the aromatic ring of
the phosphotyrosine at position Y+0 (contact shown as a
red ÔhÕ in Fig. 6B). Taken together, the contributions of the
positions Y+2, Y+4 and Y+5 appear to account for most
Fig. 3. Scatchard analysis of SOCS-3 interaction with EpoR peptides pY401 (A), pY429pY431 (B), pY429F431 (C), and F429pY431 (D). Plateau
values of the binding curves with serial dilutions of SOCS-3 (30, 15, 7.5, 3.75, 1.9 and 0.9 l
M
) were taken for calculation of the K
d
values.
Fig. 4. SOCS-3 selectively binds to tyrosine-phosphorylated peptides
corresponding to the pY401 and pY429pY431 motifs of EpoR. COS7
cells were transfected with an expression vector for human SOCS-3

coupled agarose. Subsequently, precipitated SOCS-3 was
analyzed by Western blotting. Figure 7A shows that the
SOCS-3 mutant L58A, which we predicted not to affect
peptide binding, can be precipitated with pY429pY431 to
the same extent as wild-type SOCS-3. The point mutations
R94E, L93A and G53V that we expected to play a role in
peptide recognition all impair SOCS-3 precipitation with
R94E and G53V most strongly affecting the interaction
between SOCS-3 and pY429pY431 (Fig. 7A).
To better assess the binding mode of the peptides
pY429pY431, pY429F431 or F429pY431, we performed a
peptide precipitation assay with wild-type SOCS-3 or
the SOCS-3 R94E mutant (Fig. 7B). Again we find that
the mutation of arginine 94 to glutamic acid strongly affects
the interaction of SOCS-3 with the double phosphorylated
peptide pY429pY431 (pYpY). In comparison, the muta-
tion only marginally reduces the interaction with the
single phosphorylated peptides pY429F431 (pYF) and
F429pY431 (FpY) suggesting that both phosphotyrosines
bind to the phosphotyrosine binding pocket of the SH2
domain, with R94 only playing a minor role in the
binding of these peptides.
DISCUSSION
The cytoplasmic part of the EpoR contains eight tyrosine
residues that serve as recruitment sites for a number of SH2
domain containing proteins. Among these are the protein-
tyrosine phosphatases SHP1 and SHP2 [38,39], the Jak2
and PI3 kinases [40,41] as well as STAT5, CIS and SOCS-3
[4,20,42]. In order to study binding of SOCS-3 to the EpoR
we used a biochemical approach by means of SPR

Residues that are highly conserved within the represented sequences are highlighted (bold characters). Blue and red characters indicate residues
conserved in SH2 domains to at least 30% or 80%, respectively (software:
MULTALIN
v5.4.1 [51]). Residues interacting with the phosphotyrosine as
suggested by the model structure are represented by closed circles. The open arrowhead highlights the amino acid in the aA helix that contacts the
residue Y)2. The amino acids postulated to interact with the peptide residues Y+1, Y+2, Y+3 Y+4 and Y+5 are indicated by the numbers 1, 2,
3, 4 and 5, respectively.
Ó FEBS 2002 SOCS-3 binds to the pY429pY431 motif of Epo-R (Eur. J. Biochem. 269) 2521
was not possible because the sensograms could not be fitted
to an ideal binding model. Confirming results were obtained
from peptide precipitation assays, as we were not able to
precipitate SOCS-3 out of COS7 cells overexpressing
human SOCS-3 (Fig. 4). This indicates that the pY343
motif does not play a role with respect to SOCS-3
recruitment. In the context of IL-6 signaling, SOCS-3 has
been found to act as potential competitor to SHP2 for the
binding of the same tyrosine Y759 in the gp130 receptor
subunit [16,17]. Additionally, it was recently shown that the
binding site of SHP2 in the EpoR, Y401 also recruits
SOCS-3, which results in the down-regulation of the Epo
signaling [20]. In our experiments, we confirm binding of
SOCS-3 to pY401, with a calculated K
d
for this interaction
in the range of 9.5 l
M
(Table 1). Concerning EpoR
signaling, SHP2 is suggested to positively regulate prolifer-
ation [43]. As we found that SOCS-3 binds to the same
phosphotyrosine of gp130 as SHP2, which negatively

blot analysis using a polyclonal SOCS-3 antibody to detect copreci-
pitated SOCS-3. Detection of total cell lysates (TCL) with a SOCS-3
antibody was used to check the expression levels of the different
mutants. (A) Precipitation of SOCS-3 WT or the SOCS-3 point
mutations G53V, L58A, L93A and R94E with the peptide
pY429pY431 (pYpY). (B) Precipitation of SOCS-3 WT or SOCS-3
R94E with the peptides pY429pY431 (pYpY), pY429F431 (pYF) and
F429pY431 (FpY).
2522 M. Ho
¨
rtner et al. (Eur. J. Biochem. 269) Ó FEBS 2002
In Epo signal transduction, SHP1 has been reported to
negatively regulate proliferation and differentiation of
Ba/F3 or SKT6 cells [38,44]. Interestingly, we found a
peptide encompassing the double phosphorylated tyrosine
motifpY429pY431tobindSOCS-3withaK
d
of 1.1 l
M
,a
ninefold higher affinity than determined for pY401. Single
phosphorylated peptides pY429 and pY431 revealed K
d
values in the range of 5 l
M
. We were able to confirm this
finding by the use of a peptide precipitation assay. SOCS-3
was coprecipitated with both the double phosphorylated
peptide pY429pY431 as well as the single phosphorylated
motifs, with pY429pY431 precipitating SOCS-3 most

phosphatases SHP1 and SHP2, the position Y)2ofthe
interacting phosphotyrosine motif seems to play an import-
ant role. Although the two phosphatases bind to different
tyrosine motifs within the EpoR, they are recruited to the
same phosphotyrosine pY612 of the common b chaininthe
context of IL-3 signaling [45]. A common feature of SHP1
and SHP2 recruiting motifs is a hydrophobic residue at
position Y)2 of the binding phosphotyrosine sequence. It
has been shown that this residue is filling a gap created by a
glycine in helix aA within the SH2 domain of the
phosphatase [31,46–48]. The glycine is conserved in the
N- and C-terminal SH2 domains of both SHP1 and SHP2
and is required for the unusual involvement of the residue
Y)2 of the binding phosphotyrosine motif [47]. Most
interestingly, the glycine residue in helix aA of the SH2
domain is conserved in SOCS-3 and has recently been
shown to contribute to the binding of SOCS-3 to gp130 [17].
As illustrated in Table 2, all receptor tyrosine motifs that
have been shown to bind SOCS-3 contain a hydrophobic
residue at position Y)2. The model structure of the SOCS-3
SH2 domain shows that this residue can easily be fitted into
a gap created by G53 of SOCS-3. In regard to the position
Y+1 of the interacting motifs, we suggest a hydrophobic
residue contacting the side chain of K91 or alternatively a
small polar residue like serine or threonine making a
hydrogen bond with the backbone of the b strand D to be
most favourable for peptide recognition. For the positions
Y+3 to Y+5, a hydrophobic residue seems optimal for
SOCS-3 recruitment with Y+3 contributing most to high
affinity binding.

the pY429pY431 motif (contact shown as red Ô±Õ in
Fig. 6B). The point mutation R94E (which should only
marginally affect the interaction ÔhÕ but would impede
the contact Ô±Õ) drastically affected SOCS-3 binding to the
double phosphorylated peptide pY429pY431 in our peptide
Table 2. Sequence comparison of receptor phosphotyrosine motifs
known to recruit SOCS-3. Bold characters highlight residues favour-
able for selective binding to the SOCS-3 SH2 domain. h, hydrophobic
residue.
Receptor pY location sequence
h-gp130 pY759
STV Q pY S T VVHSG
h-EpoR pY401 ASF E pY T I L D P SS
h-EpoR pY429 PHL K pY L pY L V V SD
m-LeptinR pY985 PSV K pY A T LVSND
m-LeptinR pY1077 KSV C pY L G V TSVN
Consensus
sequence
h X pY h X Lhh
SV
T
Position
relative
to pY
)20+1 +2 +3+4+5
Ó FEBS 2002 SOCS-3 binds to the pY429pY431 motif of Epo-R (Eur. J. Biochem. 269) 2523
precipitation assay (Fig. 7A,B). In contrast the binding of
the single phosphorylated peptides pY429F431 and
F429pY431 is only weakly affected by the mutation of
arginine 94 to glutamic acid (Fig. 7B). This indicates that

increasing binding affinity. Interestingly, the EpoR contains
a similar phosphotyrosine arrangement pattern. The plas-
mon resonance studies, peptide precipitation assays as well
as the model structures presented in this report, suggest that
phosphotyrosine pY429 binds into the classical phospho-
tyrosine binding pocket of the SOCS-3 SH2 domain between
helix aA and the central b sheet. pY431 appears to increase
the binding affinity by providing an additional contact with
the SH2 domain of SOCS-3 involving R94. A conforma-
tional change induced by the phosphorylation of tyrosine
Y431 may also contribute to the increase in binding affinity
compared to the single phosphorylated peptide. The posi-
tively charged residue (R94 in SOCS-3) in b strand D is
conserved (R/K) in a large number of SH2 domains. As the
members of the Src family also carry a positively charged
residue at this position, we favour the idea that the enhanced
binding of Src family kinases to the pY579pY581 motif of
the PDGF b-receptor reported by Mori et al. [49] may be
due to the formation of a salt bridge between pY581 and the
lysine residue in b strand D of the SH2 domain of the Src
kinases. The co-operative binding mode that we describe
may thus represent a more general binding mechanism by
which SH2 domains achieve high affinity binding to motifs
with proximal phosphotyrosine residues.
Our data strongly suggest that SOCS-3 binds to more
than one binding site to the EpoR. As shown by SPR
measurements as well as in vitro binding assays the double
phosphorylated motif pY429pY431 in the EpoR seems to
be the preferred binding site for SOCS-3. The implications
of the regulatory proteins SOCS-3, SHP2 and SHP1 sharing

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