Tài liệu Báo cáo khoa học: Single phosphorylation of Tyr304 in the cytoplasmic tail of ephrin B2 confers high-affinity and bifunctional binding to both the SH2 domain of Grb4 and the PDZ domain of the PDZ-RGS3 protein ppt

Single phosphorylation of Tyr304 in the cytoplasmic tail of ephrin B2
confers high-affinity and bifunctional binding to both the SH2 domain
of Grb4 and the PDZ domain of the PDZ-RGS3 protein
Zhengding Su, Ping Xu and Feng Ni
Biomolecular NMR and Protein Research Group, Biotechnology Research Institute, National Research Council of Canada,
Montreal, Quebec, Canada
The B class cell-attached ephrins mediate contact-dependent
cell–cell communications and transduce the contact signals
to the host cells through the binding interactions of their
cytoplasmic domains. Two classes of intracellular effectors
of B ephrins have been identified: one contains the PSD-95/
Dlg/ZO-1 (PDZ) domain (for example PDZ-RGS3), and the
second the Src homology 2 (SH2) domain (e.g. the Grb4
adaptor protein). The interaction with Grb4 requires phos-
phorylation of tyrosine residues on the conserved cytoplas-
mic C-terminal region of B ephrins, while binding to the
PDZ domain is independent of tyrosine phosphorylation.
However, the exact phosphorylation site(s) required for
signaling remained obscure and it is also unknown whether
the two classes of effectors can bind to B ephrins simulta-
neously or if the binding of one affects the binding of the
other. We report here that phosphorylation of Tyr304 in the
functional C-terminal region (residues 301–333) of ephrin B2
confers high-affinity binding to the SH2 domain of the
Grb4 protein. Tyrosine phosphorylation at other candi-
date sites resulted in only minor change of the binding
of Tyr304-phosphorylated ephrin B peptide (i.e. eph-
rinB2(301–333)-pY304) with the SH2 domain.
1
H-
15

N
NMR HSQC experiments show that only the eph-
rinB2(301–333)-pY304 peptide forms a stable and specific
binding complex with the SH2 domain of Grb4. The SH2
and PDZ domains were found to bind to the Tyr304
phosphopeptide both independently and at the same time,
forming a three-component molecular complex. Taken
together, our studies identify a novel SH2 domain binding
motif, PHpY304EKV, on the cytoplasmic domains of B
ephrins that may be essential for reverse signaling via the
Grb4 adaptor protein alone or in concert with proteins
containing PDZ domains.
Keywords: ephrin B; Grb4; SH2; PDZ; phosphorylation.
The ephrin-Eph signaling systems play critical roles in
multiple cell functions including cell migration, tissue border
formation in vascular development and angiogenesis, and
cell–cell communications for axonal guidance and at the
synaptic junction [1]. The Eph molecules resemble classical
receptor tyrosine kinases (PTKs) in that they are transmem-
brane proteins with kinase domains and other binding
motifs projecting into the cytoplasmic space. The plasma
membrane-bound ephrins, however, orchestrate cell move-
ments and morphogenesis by transducing bidirectional
signals into cells expressing the Eph molecules as well as
cells expressing the ephrins [2–6]. The unique capacity of
reverse signaling, by the B-class cell-attached ephrins in
particular, is to communicate the cell contact signals to the
host cells through the association of their cytoplasmic
domains with intracellular effector proteins. So far, two
typesofintracellulartargetsforBephrinshavebeen

identified. Proteins containing PSD-95/Dlg/ZO-1 (PDZ)
domains have been shown to bind to the C-termini of the
B ephrins [7–10]. At least one protein containing an Src
homology 2 (SH2) domain, Grb4, is recruited to the
cytoplasmic tails of B ephrins upon phosphorylation [11].
Although details of these signaling events have yet to be
investigated, many of the molecular interactions down-
stream of the Eph and ephrin molecules appear to lead to the
regulation of the cytoskeleton of the interacting cells [1,2,6].
Phosphorylation of the well-conserved cytoplasmic
domains of B ephrins has been a subject of significant
interest [11–16] as it is required for reverse signaling into
the ephrin B-expressing cells. The short cytoplasmic tails of
the B ephrins contain five tyrosine residues, all of which are
located within the extreme C-terminal 33-residue region
[12,13]. Three tyrosine residues are contained in a 22-residue
segment CPHYEKVSGDYGHPVYIVQEMP(301–322)
for ephrin B2, which was shown to be responsible for
Grb4 binding and is identical in both ephrin B2 and ephrin
B1 [11]. Strikingly, the Grb4 SH2 domain shows strong
binding to tyrosine-phosphorylated ephrin B1, whereas one
Correspondence to F. Ni, Biotechnology Research Institute,
National Research Council of Canada, 6100 Royalmount Avenue,
Montreal, Quebec, H4P 2R2, Canada.
Fax: + 514 4965143, Tel.: + 514 4966729,
E-mail:
Abbreviations: FGF, fibroblast growth factor; HSQC, heteronuclear
single quantum coherence; NOESY, nuclear Overhauser effect
spectroscopy; PDZ, PSD-95/Dlg/ZO-1; SFK, Src family kinase;
SH2, Src homology 2; TOCSY, total correlation spectroscopy.

(Received 23 December 2003, revised 27 February 2004,
accepted 9 March 2004)
Eur. J. Biochem. 271, 1725–1736 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04078.x
of its closest known relatives, the Nck SH2 domain, has little
or no binding [11]. The phosphorylations of Tyr311 and
Tyr316 were detected in both embryonic retinal tissues
and 293 cells stimulated with a multimeric EphB2 [15].
In Xenopus embryos, the activated fibroblast growth factor
(FGF) receptor was found to associate with and induce the
phosphorylation of ephrin B1 on equivalent positions of
Tyr311 and Tyr316, but not on Tyr304 [16]. The region
from residues 308–316 containing two tyrosine residues was
found to be critical for binding interactions with the FGF
receptor. Two other tyrosine residues are at the extreme
carboxyl terminus or residues YY(330–331)KV, constitu-
ting a PDZ-domain binding element [8]. In neural tissues,
Tyr330 was found to be the major phosphorylation site of
ephrin B1 after binding to EphB2, while the phosphoryla-
tion of Tyr331 could not be detected [15]. However, this
phosphorylation of ephrin B1 did not significantly affect
intracellular protein–protein interactions [8]. More recently,
the Src family kinases (SFKs) and the PTP-BL phosphatase
were proposed to be mediators of the phosphorylation state
of the cytoplasmic domain of ephrin B1 whereby the SFKs
may create the docking sites for Grb4 and the phosphatase
may disengage Grb4 binding from B ephrins [17].
Our previous work suggested that the well-conserved
33-residue C-terminal region of residues 301–333 of ephrin
B2 might encode a latent three-dimensional structure [18],
which may be activated through phosphorylation for Grb4

binding. More interestingly, the 22-residue region, i.e. the
ephrinB2(301–322) peptide fragment encoding the Grb4
binding site [11], appears to assume an autonomously folded
structure, independently of the PDZ-binding extreme
C-terminal region, i.e. residues IY330YKV [18]. As well,
cellular functions ascribed to the Grb4 and PDZ-binding
events can work synergistically to coordinate cell migration
and morphogenesis in establishing defined patterns of cell
assemblies [1,2,6]. It is therefore possible, upon phosphory-
lation, for the PDZ and SH2 domains to bind simulta-
neously to distinct regions of the already very short (33
residues) ephrin B cytoplasmic tails. In the current work, we
first identify the phosphorylation site in ephrin B2 that
creates high-affinity binding to the Grb4 SH2 domain using
synthetic ephrinB2(301–333) fragments containing single or
double phosphorylations at five candidate tyrosine residues,
Tyr304, Tyr311, Tyr316, Tyr330 and Tyr331. We then
assess whether the binding of the PDZ domain of PDZ-
RGS3 affects binding of the Grb4 SH2 domain to
phosphorylated peptides, through studies of ternary inter-
actions among the PDZ domain, the phosphotyrosine
peptide and the Grb4 SH2 domain. We also show that
phosphorylations of the ephrinB2(301–333) peptide alter
only the binding interactions and the three-dimensional
structure of the Grb4-binding region, i.e. residues 301–322,
while leaving the PDZ-binding C-terminus essentially
unperturbed.
Experimental procedures
Construction of expression vectors for the Grb4 SH2
and RGS3 PDZ domains

The DNA sequences encoding the Grb4 SH2 and the RGS3
PDZ domains were individually deduced from the amino
acid sequences of murine Grb4 protein and murine PDZ-
RGS3 protein as published previously [7,19] using the
codon preference of Escherichia coli. The synthetic genes
were amplified by PCR from six pairs of overlapping
synthetic primers containing the two restriction sites of NcoI
and BamHI for the SH2 domain and the two restriction sites
of BamHI and EcoRI for the PDZ domain at their two
ends, respectively. The double-digested DNA fragment of
SH2 was subcloned into the pET3215 expression vector,
which was modified from pET32 and pET15 vectors
(Novagen, Madison, WI, USA), removing the original
fusion carrier in the pET32 vector. In order to facilitate
protein purification, a His-tag with six histidine residues
was placed at the N-terminus of the SH2 domain linked
with a thrombin cleavage sequence. The same insert was
also subcloned into the pGFN GST fusion vector, which is
a derivative of the pGEX-4T-1 expression vector (Amer-
sham Biosciences, Piscataway, NJ, USA), replacing the
LVPR thrombin cleavage site with the FNPR sequence [20].
The double digested DNA fragment of the PDZ domain
was subcloned into the pGFN vector. All these expression
constructs were confirmed by DNA sequencing and trans-
formed into the E. coli BL21(DE3) expression host.
Protein expression and purification
The SH2 protein was expressed at 37 °C. The cells were
harvested four hours after induction with isopropyl thio-
b-
D

-galactoside at D
600
¼ 0.8. Labeling with the
15
N
isotope was accomplished using M9 media containing
1.0 gÆL
)1
of [
15
N]ammonium sulfate, 5 gÆL
)1
of glucose plus
a supplement of trace levels of metal ions and vitamins.
Uniform
15
N/
13
C-labeling was accomplished by substitution
of unlabelled glucose with 2.0 gÆL
)1
of [
13
C
6
]glucose. Protein
purification was performed under denaturing conditions
with Ni-nitriloacetic acid agarose beads (Qiagen) in the
presence of 20 m
M

2-mercaptoethanol at pH values of 8.0,
6.3, 5.9 and 4.5 for the binding, two washing, and eluting
steps, respectively. Protein fractions were analyzed using
20% Phast gels (Amersham Biosciences). The fractions
containing the SH2 domain were collected and refolded by
dialyzing 2–3 times against a large volume of 50 m
M
sodium
phosphate buffer plus 20 m
M
2-mercaptoethanol (pH 6.8)
at 4 °C. The pellet was removed by centrifugation and the
supernatant was concentrated by ultrafiltration (Millipore,
Bedford, MA, USA). The protein concentration was
determined at A
280
with a calculated extinction coefficient
of 12 210
M
)1
Æcm
)1
[21].
The expression and purification of GST-SH2 and GST-
PDZ proteins were carried out with standard protocols
provided by the supplier (Amersham Biosciences). Throm-
bin cleavage was performed at room temperature for 2 h in
1 · NaCl/P
i
buffer. The concentration of the PDZ protein

was determined at A
280
with a calculated extinction
coefficient of 12 620
M
)1
Æcm
)1
[21].
Peptide synthesis and purification
All the peptides, either with or without phosphotyrosine
(pY), were synthesized chemically using standard Fmoc
solid-phase chemistry at the Biotechnology Research Insti-
tute’s Peptide Facility. The synthetic peptides were purified
1726 Z. Su et al. (Eur. J. Biochem. 271) Ó FEBS 2004
by HPLC using a reverse-phase C18 semipreparative
column with an acetonitrile gradient of 10–35%. The purity
of the peptides was verified by a reversed-phase analytical
HPLC column and the identity of the final products was
verified by mass spectral analysis and NMR assignments.
Fluorescence polarization measurements
Fluorescein-labeled peptides were prepared through reac-
tion of the ephrinB2(301–333) peptide and its variants
containing a single phosphotyrosine residue (Fig. 3), or
double phosphotyrosine residues with 5-iodoacetamido-
fluorescein (Molecular Probes, Eugene, OR, USA). Upon
completion of labeling, an excess of 2-mercaptoethanol was
added to consume the excess 5-iodoacetamidofluorescein
followed by the removal of small organic compounds using
a C18 September-Pak column. The eluate containing

fluorescein-labeled peptide was further purified by HPLC.
The authenticity of fluorescein labeling was confirmed by
mass spectroscopy.
Fluorescence polarization was performed at 25 °Con
a Perkin-Elmer fluorescence polarization instrument, the
EnVision
TM
multilabel plate reader, which was equipped
with two fluorescence polarizers. All the polarization values
are expressed in mili-polarization units (mP). Fluorescence
measurements were carried out with an excitation wave-
length of 490 nm and an emission wavelength of 520 nm.
For binding studies, each fluorescein-labeled peptide was
dissolvedin50m
M
phosphate buffer (pH 6.8, with 20 m
M
2-mercaptoethanol) to a concentration of 25 n
M
.The
dissociation constants were obtained by fitting the binding
curves using the computer program
ORIGIN
TM
6.0 (Nor-
thampton, MA, USA) based on the following equations:
L þ P ()
K
on
K

off
LÁP
where L and P denote the ligand and protein, respectively.
The fluorescence polarization (DmP) is related to the
dissociation constant K
d
as follows:
DmP ¼
C
0
½L
T
½P
K
d
þ½P
where C
0
is a constant dependent on the properties of
the ligand, [L]
T
is the total ligand concentration, [P]isthe
concentration of free SH2 protein, and K
d
is the equilibrium
dissociation constant. Average K
d
values were determined
from multiple independent measurements.
NMR spectroscopy and resonance assignments

All NMR spectra were collected at 15 °C on a Bruker
Avance 500 MHz or 800 MHz spectrometer using triple-
resonance probes equipped with pulse field gradients.
Protein samples for NMR analysis contained % 0.5 m
M
of
uniformly
15
N- or
15
N-/
13
C-labeled protein. Assignments of
the H/
15
N/
13
C NMR signals from the main-chain atoms
were obtained from a combined analysis of the
1
H-
15
N
HSQC, CBCA(CO)NH and HNCACB experiments [22].
Proton NMR experiments with the peptides, which included
data acquisition, processing and analysis, were the same as
described previously [18,23].
GST pull-down assays
A GST pull-down experiment was employed to recons-
titute the three-component molecular complexes formed

by the ephrin B peptides, the SH2 domain and the PDZ
protein. GST or GST-PDZ proteins bound to glutathione
agarose beads were incubated with 5 lL of the SH2
protein in the binding buffer (50 m
M
sodium phosphate,
pH 6.8) for 2 h at 4 °C in the absence or presence of
ephrin B2 peptides in excess amount. The beads were
washed extensively with the binding buffer and the
samples were boiled for 10 min in the SDS/PAGE sample
buffer and analyzed by SDS/PAGE Phast gel (Amersham
Biosciences).
Results
Characterization of the expressed SH2 and PDZ protein
domains
The SH2 domain (Fig. 1A) of murine Grb4 was
expressed using a synthetic DNA fragment subcloned
for protein production in the E. coli host (Experimental
procedures). The over-expressed SH2 protein was found
in inclusion bodies and purified with Ni-nitiloacetic acid
agarose beads under denaturing conditions. The quality
of the expressed SH2 domain is improved at each step of
the purification procedure (Fig. 1B). A synthetic DNA
fragment encoding the PDZ domain of PDZ-RGS3 was
prepared in the same way as for the SH2 domain based
on the published amino acid sequence of the RGS3
protein [20]. The PDZ domain was over-expressed as a
fusion to the GST carrier protein in a mostly soluble
form and the intact domain can be purified after
digesting the GST-PDZ fusion protein with thrombin

(Fig. 1C). The expressed PDZ domain is reasonably
soluble in solution and is functionally active as it has the
same binding affinity to the ephrin-B2 peptide as the
GST-PDZ fusion protein does (see below).
The
1
H-
15
N HSQC spectrum of the SH2 domain shows
a good dispersion of the
1
H-
15
N correlation peaks
(Fig. 2A), indicative of a well-folded protein. Nearly
complete assignments of the main chain
1
H
N
,
15
N,
13
C
a
,
and
13
C
b

NMR signals of the SH2 domain were
obtainable using triple-resonance heteronuclear NMR
experiments. Figure 2B shows the differences between
the
13
C
a
and
13
C
b
chemical shifts of an
15
N/
13
C-labeled
sample of the SH2 domain. The secondary structure
elements were deduced from the positive or negative
derivations of the
13
C
a
and
13
C
b
chemical shift differences
from random coil values over a number of consecutive
amino acids [24–26]. Overall, secondary structures of the
Grb4 SH2 domain are very similar to these of the Src

SH2 domain (Fig. 1A). However, there are some signifi-
cant differences in the structural regions determining the
binding specificity towards phosphopeptides. For example,
the b-sheet structure after the EF3 segment in the Src SH2
does not exist in the Grb4 SH2 domain while the insertion
after the BG2, BG3 and BG4 structures in the Grb4 SH2
domain forms one additional b-sheet structure (Fig. 1A
and Fig. 2B).
Ó FEBS 2004 Phosphorylation of Tyr304 on ephrin B2 (Eur. J. Biochem. 271) 1727
Identification of the binding site(s) on ephrin B2
for the SH2 domain of Grb4
Six synthetic peptides including ephrinB2(301–333) and
its five individual tyrosine-phosphorylated derivatives,
ephrinB2(301–333)-pY304, ephrinB2(301–333)-pY311,
ephrinB2(301–333)-pY316, ephrinB2(301–333)-pY330 and
ephrinB2(301–333)-pY331 (Fig. 3), were labeled with
fluorescein through the Cys301 residue at their N-termini.
The highly sensitive fluorescence polarization method
was used to detect the binding interactions between the
peptides and the Grb4 SH2 domain. The affinity of
the binding interactions was evaluated by measuring the
changes of fluorescence polarization of the peptides at
each step of titration with the Grb4 SH2 domain, i.e. the
binding isotherms (Fig. 4A). The ephrinB2(301–333)-
pY304 peptide was found to have the strongest binding
to the Grb4 SH2 domain while the other five peptides,
ephrinB2(301–333), ephrinB2(301–333)-pY311, eph-
rinB2(301–333)-pY316, ephrinB2(301–333)-pY330 and
ephrinB2(301–333)-pY331, showed low or no binding
affinity to the SH2 domain. The dissociation constant of

the ephrinB2(301–333)-pY304/Grb4 SH2 complex was
estimatedtobe0.2l
M
from the titration curves
(Table 1). The calculated dissociation constants of the
complexes between Grb4 SH2 and each of the other five
peptides (i.e. ephrinB2(301–333), ephrinB2(301–333)-
pY311, ephrinB2(301–333)-pY316, ephrinB2(301–333)-
pY330 and ephrinB2(301–333)-pY331) were larger than
500 l
M
. These results show that only phosphorylated
Tyr304 can result in high-affinity and specific binding of
ephrinB2(301–333) to the Grb4 SH2 domain. The
fluorescence binding experiments also indicate that the
Cys301 residue may not be required for binding as it is
labeled by the bulky fluorescein group in the peptide
fragments.
Fig. 1. Comparison of amino acid sequences of SH2 domains from Src and the mouse Grb4, and expression and purification of the Grb4 SH2 and
RGS3 PDZ domains. (A) Secondary structural elements are from the X-ray crystallographic structure of the Src SH2 domain in complex with a
pYEEI peptide [35]. The notation used for the binding pockets of the phosphotyrosine peptide is as described previously [35]. Residues determining
the binding specificity are underlined. (B) SDS/PAGE of each purification step of the expressed SH2 domain of Grb4. Lane 1: the soluble part of the
cell lysate; Lane 2: the insoluble part of the cell lysate; Lane 3: the Ni-nitiloacetic acid agarose beads bound with the SH2 protein at pH 8.0; Lane 4:
the Ni-nitiloacetic acid agarose beads with the bound SH2 protein at pH 6.3; Lane 5: the eluted protein at pH 4.5 and Lane M: molecular mass
markers (Amersham Bioscience). The sample of the Ni-nitiloacetic acid agarose beads with the bound SH2 protein at pH 5.9 was not shown here as
the protein was already pure. The position of the SH2 protein is marked by the arrow. (C) SDS/PAGE patterns following each purification step of
the expressed GST-PDZ. Lane 1: the soluble part of the cell lysate. The high-density band at the bottom is lysozyme, which was used to lyse the cells.
Lane 2: the purified GST-PDZ fusion protein by the glutathione-agarose beads. Lane 3: The digestion of the GST-PDZ fusion protein by thrombin
at room temperature for 1 h. The fusion proteins are readily and completely digested within 30 min. Lane 4: the purified PDZ protein by
glutathione-agarose beads followed by ion-exchange chromatography. Lane M: molecular mass markers (Amersham Bioscience).

1728 Z. Su et al. (Eur. J. Biochem. 271) Ó FEBS 2004
We next address the questions of whether secondary
tyrosine phosphorylations would affect the Grb4 SH2
domain binding to the ephrinB2(301–333)-pY304 peptides
and more specifically whether the combined phosphory-
lation of Tyr311 and Tyr316 would replace phosphory-
lation at Tyr304. The choice of Tyr311 and Tyr316 was
because these two residues were reported to be the major
detectable phosphorylation sites in vivo [15] and a peptide
derived from residues 301–322 of ephrinB2 or the
N-terminal 22 residues of ephrinB2(301–333) was found
to contain all the phosphorylation sites for high-affinity
binding [11]. As shown in Table 1, the phosphorylation of
Tyr311 or Tyr316 does not significantly affect the binding
of the ephrinB2(301–333)-pY304 peptide to the Grb4
SH2 domain. Our data also show that the peptide with
double phosphorylations at Tyr311 and Tyr316 has no
significant binding to the Grb4 SH2 domain. Therefore,
the high-affinity Grb4 SH2 binding of the ephrinB2(301–
333) fragment conferred by the Tyr304 phosphorylation is
independent of phosphorylations at the other two tyrosine
residues, Tyr311 and Tyr316.
To further assess the binding specificity of the Grb4 SH2
domain, we synthesized the short phosphorylated peptide,
ephrin B2(301–309) or CPHpY304EKVSG with a number
of substitutions at the KV positions. The single amino
acid substitutions successively transformed the pYEKV
sequence of ephrin B2 to the pYEEI sequence specific for
the Src SH2 domain. As shown in Fig. 4B, only the
CPHpY304EKVSG peptide exhibited high-affinity binding

to the Grb4 SH2 domain. The dissociation constant of the
CPHpY304EKVSG/Grb4 SH2 complex was estimated to
be 0.23 l
M
from the fluorescence titration curve, whereas all
amino acid substitutions C-terminal to the pTyr304 led to
dramatically reduced binding (Table 1).
Two-dimensional NMR spectroscopy was employed to
investigate the specificity of binding of phosphorylated
ephrinB2(301–333) peptides to the Grb4 SH2 domain. The
1
H-
15
N HSQC spectrum of the Grb4 SH2 domain (Fig. 2A)
Fig. 2. Characterization of the Grb4 SH2 by heteronuclear NMR. (A) The
1
H-
15
N HSQC spectrum of the SH2 domain of Grb4 with the
assignment of the amide
1
H-
15
N correlations to specific residues. The measurement was performed at 15 °C and pH 6.8. Sequence specific
assignments of the backbone
15
Nand
13
C resonances were achieved using triple-resonance NMR experiments with a
15

N/
13
C-labeled
SH2 sample. (B) Secondary structure of the Grb4 SH2 protein deduced from the differences of the
13
C
a
and
13
C
b
chemical shifts of the
15
N/
13
C-labeled SH2 domain.
Ó FEBS 2004 Phosphorylation of Tyr304 on ephrin B2 (Eur. J. Biochem. 271) 1729
responded to the addition of the ephrinB2 peptide phos-
phorylated at Tyr304, or ephrinB2(301–333)-pY304
(Fig. 5A). Almost twice the numbers of HSQC peaks were
found at lower peptide concentrations and most of the
peaks redistributed at a molar ratio of 2 : 1 for the peptide-
SH2 concentrations (Fig. 5A, red spectrum). More signifi-
cantly, in a stepwise titration experiment, the HSQC peaks
did not undergo gradual shifting with increased concentra-
tions of the peptide (data not shown), indicating that the
free and peptide-bound SH2 must exchange slowly as
observed for other SH2 domains. In contrast, the nonphos-
phorylated peptide, ephrinB2(301–333), did not induce any
significant changes to the HSQC peaks up to a molar

concentration ratio of 2 : 1 (Fig. 5B, red spectrum). As well,
no significant changes in the HSQC peaks were observed in
the presence of two other single tyrosine-phosphorylated
peptides, ephrinB2(301–333)-pY311 and ephrinB2(301–
333)-pY316, similar to that of the unphosphorylated
ephrinB2(301–333) (spectra not shown). Taken together,
both the NMR and fluorescence polarization experiments
Fig. 4. The effects of tyrosine phosphorylation on ephrin B2 binding to
the SH2 domain of Grb4 analyzed by fluorescence polarization.
(A) Fluorescence binding curves were collected for the unmodified
peptide ephrinB2(301–333) (j) and the three singly phosphorylated
peptides, ephrinB2(301–333)-pY304 (d), ephrinB2(301–333)-pY311
(m) and ephrinB2(301–333)-pY316 (.). Data for two other singly
phosphorylated peptides including ephrinB2(301–333)-pY330 and
ephrinB2(301–333)-pY331 are not shown, only their experimentally
determined binding constants are listed in Table 1 for comparison.
(B) Binding curves were collected for YEKV-pY304 (d), YEKI-
pY304 (j), YEKI-pY304 (m) and YEEI-pY304 (r)(Fig.3).
Fig. 3. Schematic representation of peptides for fluorescence polarization assays. The 33-residue peptide, ephrinB2(301–333), is derived from the
extreme C-terminal sequence of the cytoplasmic domain of ephrin B2. The phosphorylated tyrosine residues are labeled with pY at the five
individual sites, Tyr304, Tyr311, Tyr316, Tyr330 or Tyr331, for the five tyrosine-phosphorylated peptides, ephrinB2(301–333)-pY304, eph-
rinB2(301–333)-pY311, ephrinB2(301–333)-pY316, ephrinB2(301–333)-pY330 and ephrinB2(301–333)-pY331. The consensus binding sequence of
ephrin B2 for the Grb4 SH2 is minimized to a nine-residue segment, ephrinB2(301–309) or CPHpYEKVSG (referred to as YEKV-pY304). Three
variants of the short peptide are generated to determine the binding specificity of the Grb4 SH2 in relation to the closest consensus binding sequence
of the Src SH2 domain (i.e. pYEEI).
Table 1. The effect of tyrosine phosphorylations on the binding affinity
of ephrinB2(301–333) to the SH2 domain of Grb4 and to the PDZ
domain of PDZ-RGS3. In the combination experiments, the two C
terminal tyrosine residues were ignored due to their distant positions.
ND, not determined.

pY position K
d (SH2)
(l
M
) K
d (PDZ)
(l
M
)
EphrinB2(301–333) series
WT >500 3.0 ± 0.17
pY304 0.21 ± 0.05 3.1 ± 0.18
pY311 and pY316 >500 ND
pY311 or pY316 >500 2.9 ± 0.16
pY304 and pY311 0.21 ± 0.06 ND
pY304 and pY316 0.22 ± 0.05 ND
pY330 or pY331 >500 2.8 ± 0.16
EphrinB2(301–309) series
YEKV-pY304 0.23 ± 0.05 –
YEEV-pY304 >500 –
YEKI-pY304 >500 –
YEEI-pY04 >500 –
1730 Z. Su et al. (Eur. J. Biochem. 271) Ó FEBS 2004
show that the phosphorylation of Tyr304 confers high-
affinity and specific binding of ephrinB2(301–333) to the
Grb4 SH2 domain.
The ephrinB2(301–333)-pY304 peptide can bind to the
Grb4 SH2 and RGS3 PDZ domains simultaneously
It has been shown that the cytoplasmic tail of B-class
ephrins constitutes a PDZ domain binding motif, YYKV,

whose binding to PDZ domains is phosphorylation-inde-
pendent [8]. Figure 6A shows that the GST-PDZ fusion
protein binds to the fluorescein-labeled ephrinB2(301–333)
peptide and this binding has an affinity of K
d
¼ 3.0 l
M
(Table 1). The binding affinities of the GST-PDZ fusion
protein to two single tyrosine phosphorylated peptides at
the extreme C-terminus (i.e. the PDZ binding motif), i.e.
ephrinB2(301–333)-pY330 and ephrinB2(301–333)-pY331,
are essentially the same as that of the ephrinB2(301–333)
peptide (Table 1). Therefore, the fluorescein-labeled eph-
rinB2(301–333) peptides with single tyrosine phosphoryla-
tion at Tyr304, Tyr311, Tyr316, Tyr330 or Tyr331 had
essentially the same binding affinities to the GST-PDZ
protein in agreement with previous observations [8]. Binding
experiments with the isolated PDZ domain showed similar
binding affinities to the ephrin B peptides as that of the
GST-PDZ protein. Therefore, the GST-PDZ fusion protein
was used for all other binding experiments including the
GST pull-down experiments (see below).
On the other hand, the SH2 domain of Grb4 binds to the
cytoplasmic tail of ephrin B2 in a phosphorylation-depend-
ent manner. It is therefore interesting to know whether
Grb4 and PDZ-RGS3 can bind to the cytoplasmic tail
of ephrin B2 simultaneously or whether Grb4 binding
can affect or even exclude the binding of the PDZ-RGS3
Fig. 5.
1

H-
15
N-HSQC spectra of the SH2
domain of Grb4 titrated by ephrinB2(301–333).
ThespectraofthefreeGrb4-SH2domain
(black) were overlaid on those in the presence
of ephrinB(301–333) peptides (red). (A)
Titration with ephrinB2(301–333)-pY304.
(B) Titration with ephrinB2(301–333). The
protein concentration was 0.5 m
M
and the
peptide concentration was 1 m
M
.
Ó FEBS 2004 Phosphorylation of Tyr304 on ephrin B2 (Eur. J. Biochem. 271) 1731
protein. We set out to address this question by use of the
SH2 domain of Grb4 and the PDZ domain of the RGS3
protein. We titrated the SH2 protein into the fluorescein-
labeled ephrinB2(301–333)-pY304 after saturation by the
GST-PDZ fusion protein. Consequently, the fluorescence
polarization increased further from that elicited by PDZ
binding with increasing concentrations of the SH2 protein.
It was found that after normalization, the polarization
changes had similar trends as the titration experiments in
the absence of the GST-PDZ protein (Fig. 6B). Therefore,
it appears that binding of the PDZ domain to the
ephrinB2(301–333)-pY304 peptide has no impact on the
binding of the Grb4 SH2 domain.
Formation of three-component complexes among the

SH2 domain, the PDZ domain and ephrinB2(301–333)
peptides shown by GST pull-down experiments
In vitro GST pull-down experiments were also carried out to
verify the ternary interactions among the Grb4 SH2 and
RGS3 PDZ domains and the ephrinB2(301–333) peptides.
First, the Grb4 SH2 domain-binding peptide, eph-
rinB2(301–333)-pY304, was used for the formation of the
three-component complex with the Grb4 SH2 and RGS3
PDZ domains. The purified GST-PDZ protein, which was
bound to the glutathione-agarose beads, was mixed with the
Grb4 SH2 domain and the ephrinB2(301–333)-pY304
peptide. The beads were washed extensively to remove the
unbound proteins. The proteins bound to the bead were
visualized by use of SDS/PAGE (Fig. 7). As indicated by
this kind of pull-down data, both the Grb4 SH2 and
the RGS3 PDZ domains showed strong binding to the
ephrinB2(301–333)-pY304 peptide in a three-component
molecular complex. Control experiments showed the lack of
binding of the Grb4 SH2 domain to the GST-PDZ protein
nor to the GST carrier protein. Second, pull-down experi-
ments were also carried out for the other five peptides
including ephrinB2(301–333), ephrinB2(301–333)-pY311,
ephrinB2(301–333)-pY316, ephrinB2(301–333)-pY330 and
ephrinB2(301–333)-pY331. None of these peptides can link
the Grb4 SH2 and the RGS3 PDZ domains to form a
ternary complex (Fig. 7).
Effects of tyrosine phosphorylation on the conformation
of ephrinB2(301–333)
The unphosphorylated peptide, ephrinB2(301–333), was
shown to form a well-folded b-hairpin structure for the

putative SH2-binding region of residue 301–322 along with
a very flexible C-terminal tail from residues 323–333 [18].
Phosphorylation at Tyr304, Tyr311 or Tyr316 does not
affect the conformational characteristics of these PDZ-
binding C-terminal tail residues. For example, the typical
NOE contact between the aCH proton of Ala327 and the
NH proton of Ile329, and the consecutive H
N
-H
N
NOEs
from Lys329 to Tyr331, existed in all the peptides
independent of phosphorylation (Fig. 8A,B). Thus, the
conformation of the PDZ binding domain of eph-
rinB2(301–333) is essentially independent of tyrosine
phosphorylation within the SH2-binding element of eph-
rinB2(301–333).
On the other hand, tyrosine phosphorylation appears to
have a profound effect on the b-hairpin conformation of the
SH2-binding region. First, all three kinds of single phos-
phorylation at the Tyr304, Tyr311 or Tyr316 site caused
perturbation to the loop region in the b-hairpin structure as
the unique NOEs detected previously [18] completely
disappeared in the NOESY spectrum of the phosphorylated
ephrinB2(301–333) peptides, as shown in Fig. 8A for
ephrinB2(301–333)-pY304. Second, the consecutive back-
bone H
N
-H
N

NOEs from residues Tyr316 to Gln319 were
observed for ephrinB2(301–333)-pY304 and ephrinB2(301–
333)-pY316 (Fig. 8B) but not for ephrinB2(301–333)-
pY311. Third, there is a dramatic reduction of NOE
contacts among the sidechains of aromatic residues pur-
porting to side-chain packing interactions of a b-hairpin
structure (Fig. 8C). Many previously identified long-range
NOEs such as those between Val318 and His303, Gln319
and His303, Lys306 and Tyr316 were absent in the
phosphorylated peptides as shown in Fig. 8C for eph-
rinB2(301–333)-pY304. Interestingly, the long-range NOE
contacts involving residues Lys306 and Tyr316, Glu305 and
Fig. 6. Effect of PDZ binding on the interactions of the Grb4-SH2
domain with the ephrinB2(301–333)-pY304 peptide. (A) The PDZ-
binding curves are for the fluorescein-labeled peptides including eph-
rinB2(301–333) (j), ephrinB2(301–333)-pY304 (d), ephrinB2(301–
333)-pY311 (m), ephrinB2(301–333)-pY316 (.). Corresponding data
for the phosphorylated peptides at Tyr330 and Tyr331 are listed in
Table 1. (B) Binding experiments performed with the fluorescein-
labeled ephrinB2(301–333)-pY304 peptide in the absence of the GST-
PDZ protein (d)orwith75l
M
of the GST-PDZ protein (s).
1732 Z. Su et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Fig. 7. Formation of a three-component complex through GST pull-down. Lane M: molecular mass markers. The molecular sizes of each band are
97.0, 66.0, 45.0, 30.0, 20.1 and 14.4 kDa from the top to the bottom. Lane 1: the three-component complex of GST-PDZ, ephrinB2(301–333)-
pY304 and the SH2 protein. Lane 2: sample prepared similarly as that in Lane 1 except for the absence of ephrinB2(301–333)-pY304. Lane 3:
sample prepared the similarly as that in Lane 2 except the GST was used instead of GST-PDZ. Lane 4–8: samples prepared the same as that in
Lane 1 except that the peptides are ephrinB2(301–333), ephrinB2(301–333)-pY311, ephrinB2(301–333)-pY316, ephrinB2(301–333)-pY330 and
ephrinB2(301–333)-pY331, respectively.

Fig. 8. Homonuclear NOESY spectra of the ephrinB(301–333)-pY304 peptide. (A) NH-aH region of the NOESY spectrum (red) with a mixing time
of 200 ms in H
2
O overlaid with a TOCSY spectrum (dark). The cross indicates the missing medium-range NOEs in ephrinB(301–333)-pY304
compared with those in the unphosphorylated ephrinB(301–333) [18]. (B) The NH-NH NOE connectivities for residues Tyr316-Gln319 and
Lys329-Tyr331. (C) The aromatic region of the NOESY spectrum of the ephrinB(301–333)-pY304 peptide. The NOE experiments were carried out
with a mixing time of 200 ms in H
2
O at pH 6.8 and 288 K. Some key medium- and long-range NOEs are still present, but many long-range NOEs
are absent compared to the unphosphorylated ephrinB2(301–333) peptide [18]. Interestingly, the number of side-chain NOE contacts in the
two other phosphorylated peptides, ephrinB2(301–333)-pY311 and ephrinB2(301–333)-pY316, was further reduced and almost all NOEs have
disappeared.
Ó FEBS 2004 Phosphorylation of Tyr304 on ephrin B2 (Eur. J. Biochem. 271) 1733
Tyr316 remained in ephrinB2(301–333)-pY304 but not in
the two other phosphorylated peptides.
Figure 9 shows a structural model of ephrinB2(301–333)-
pY304, generated from the NOE data of Fig. 8. Compared
to the solution conformation of the nonphosphorylated
peptide [18], phosphorylation at Tyr304 dramatically
increased the flexibility of the b-hairpin structure and
essentially unfolded it by phosphorylation at Tyr311 or
Tyr316. Instead, residues around Tyr316 were found to
form a helical structure with the ephrinB2(301–333)-pY304
peptide (Fig. 9). Phosphorylation of Tyr316 destroys even
this helical structure as typical NOEs determining the short
helix were not observable in the ephrinB2(301–333)-pY316
peptide. In contrast, the short helix found for the PDZ-
binding motif [18] remains unperturbed by any of the
phosphorylations. Taken together, these results indicate
that phosphorylated ephrinB2(301–333) peptides become

much more flexible especially in the N-terminal region
around residue Tyr304 or the PHY304EKV sequence
region. This kind of flexible conformations may be more
favorable for Grb4 SH2 domain binding, as almost all SH2-
binding phosphotyrosine peptides appear to assume exten-
ded conformations in the binding site of the complex [27].
Discussion
Using fluorescence polarization and NMR spectroscopy, we
have identified a sequence segment around Tyr304 or
PHpY304EKV on a C-terminal 33-residue peptide of the
ephrin B2 cytoplasmic region as a high-affinity binding site
for the Grb4 SH2 domain, in which tyrosine phosphory-
lation is critical. We also show that phosphorylation of
two distant tyrosine residues, i.e. Tyr311 or Tyr316, did not
affect the binding of the pTyr304 motif to the Grb4 SH2
domain. Some other SH2 domains, for instance, p85N-SH2
[28], were found to have higher affinity for doubly tyrosine-
phosphorylated peptides. Therefore, our results indicate
that the phosphorylations of Tyr311 and Tyr316 identified
in vivo may have unknown alternative functions other than
physical interactions with the Grb4 protein. Earlier work
already provided some evidence for low levels of in vivo
phosphorylation at an equivalent position of residue Tyr304
in the Xenopus ephrin B1 [16]. Other studies implicate
Tyr311 and/or Tyr316 as the major phosphorylation sites
[15,16]. However, it is not known whether these two
phosphorylated tyrosine residues contribute to high-affinity
binding of the ephrin B cytoplasmic domain to the Grb4
adaptor protein [11]. The equivalent Tyr304 residue of
Xenopus ephrin B1 was shown to be essential for binding to

Grb4 in a recent work employing truncations and residue
substitutions of ephrin B1 expressed in Xenopus oocytes cells
[29]. This latter work also did not find evidence for the
involvement of phosphorylated Tyr311 or Tyr316 in ephrin
B1 binding to the Grb4 protein, in sharp contrast to a
previous claim otherwise [30]. It is now clear that phos-
phorylated Tyr304 alone determines the direct binding to
Grb4 while sequence elements surrounding the Tyr311 site
are important for the interactions of ephrin B with tyrosine
kinases or phosphatases [29].
Structurally, the Grb4 SH2 domain binding motif on B
ephrins, pYEKV, is somewhat different from other SH2
binding sequences [31,32], for example, pYEEI, pYDNV,
pYTDM and pYTDL for Src family SH2 domains;
pYENP, pYTEV and pYMDL for the Abl SH2 domain;
pYDHP, pYKFL and pYNR for the CrK SH2
domain; pYDEP, pYDED and pYDEV for the Nck
SH2 domain; pYLNV, pYLNV, pYIN and pYMN for
the Sem5 SH2 domain; pYMXM, pYVXM, pYIXM and
pYEXM for the N-terminal SH2 domain of p85;
pYXXM for the C-terminal SH2 domain of p85; pYVIP,
pYILI and pYILV for the C-terminal SH2 domain of
PLC-r; pYELE, pYIDI and pYVDV for the N-terminal
SH2 domain of PLC-r; and pYIXV, pYVXI, pYVXL
and pYVXP for the N-terminal SH2 domain of SHPTP2.
The different SH2 domain binding motifs provide diver-
sity to signal transduction through a wide range of SH2
domain-containing proteins [31]. Through the alignment
of the Grb4 and Src SH2 domains (Fig. 1A), it is seen
that the phosphotyrosine binding pocket of the Grb4

SH2 domain is almost identical to that of the Src SH2
domain, being composed of residues in the bA2, bB5,
bD3, bD4, bD6andbD¢1 structural elements. On the
other hand, the local environment of the Grb4 SH2
domain binding pocket is different from that in the Src
SH2 domain, which is composed of residues at the bD5,
bE4, EF1, EF3, BG2, BG3 and BG4 sites. This particular
Fig. 9. Model of the flexible structure of ephrinB2(301–333)-pY304 in
solution. A cluster of solution conformations was generated by use of
NOE data shown in Fig. 7, specifying the secondary structure elements.
Residues for the Grb4 SH2 domain binding, i.e. PHpY304EKV, are
colored in blue, while the tail residues IY330YKV for PDZ domain
binding are colored in red. This representation of the peptide confor-
mation was prepared using
INSIGHT II
(Tripos, San Diego, CA, USA).
1734 Z. Su et al. (Eur. J. Biochem. 271) Ó FEBS 2004
pocket in the Grb4 SH2 domain has reduced negative
charges and increased hydrophobicity, which may be
related to the difference between the Grb4 SH2 domain
binding motif, pYEKV, identified in this study and the
Src SH2 binding motif, pYEEI reported previously
[33,34]. Moreover, the secondary structures around the
pTyr binding pocket of Grb4 SH2 are significantly
different from those in the Src SH2 domain. The b-sheet
after the EF3 segment in the Src SH2 domain appears to
be shifted to the residues behind the BG segments in the
Grb4 SH2 domain. All these structural variations may
determine the binding specificity of the Grb4 SH2 domain
for the pYEKV motif in B ephrins (Fig. 2B and Table 1).

The phosphorylation of Tyr311 or 316 has a significant
influence on the solution conformation of the ephrin B
cytoplasmic tail at least within the ephrinB2(301–333)
fragment. The unphosphorylated ephrin B peptide adopts
a well-folded b-hairpin structure for the Grb4 SH2 domain-
binding region [18]. The phosphorylation of either Tyr311
or Tyr316 largely exposes Tyr304 from a folded b-hairpin
as shown by the disappearance of long-range NOE contacts
specifying the hairpin structure. It is likely that the
phosphorylation of Tyr311 or Tyr316 has significant
contributions to the exposure and positioning of the
Tyr304 residue for binding to tyrosine kinases and phos-
phatases. On the other hand, tyrosine phosphorylations do
not affect the conformation of the PDZ binding motif at the
C-terminus (Fig. 9). Therefore, the 33-residue functional tail
of ephrin B2 appears capable of accommodating two
relatively independent structural subunits carrying bind-
ing motifs for different docking proteins. Indeed, in vitro
GST pull-down experiments showed that a three-compo-
nent complex can be formed among the Grb4 SH2 domain,
the Tyr304-phosphorylated ephrineB2(301–333) peptide
and the GST-PDZ protein from PDZ-RGS3. Fluor-
escence-binding experiments revealed that Grb4 SH2
domain-binding and RGS3 PDZ domain-binding to phos-
phorylated peptides are almost entirely independent of one
another (Table 1). These observations demonstrate that the
flexibly linked SH2-binding and PDZ-binding sequences in
the ephrin B peptide (Fig. 9) can accept the simultaneous
docking of both the Grb4 SH2 and RGS3 PDZ domains. It
is possible that simultaneous binding to the cytoplasmic

domain of B ephrins may also be able to coordinate the two
different types of reverse signaling events mediated by the
Grb4 and PDZ-RGS3 proteins.
Activation of reverse signaling through B ephrins leads to
clustering and tyrosine phosphorylation of the cytoplasmic
tail of B ephrins, and a concomitant recruitment of Grb4
and its SH3-binding protein partners. Subsequently, regu-
lation of the cell cytoskeleton occurs through many
subcellular events such as localization and/or alteration
of tyrosine-phosphorylation levels of other proteins [11].
In vivo and cell-based experiments showed that ephrin B2
or B1 lacking the cytoplasmic tails were no longer able to
exert reverse signaling [14,35]. Taken together, our findings
strongly suggest that the high-affinity binding between
phosphotyrosine304 in ephrinB2 and the Grb4 SH2 domain
is one critical step in the reverse signaling of the Eph/ephrin
B system, that orchestrate the assemblage of Eph/ephrin-
containing cells and tissues into defined functional aggre-
gates and compartments.
Acknowledgements
We thank Drs Dmitri Tolkachev and Surajit Bhattacharjya for their
valuable discussion and Patrice Bouchard and Betty Zhu for technical
assistance. We also thank Andy Ng and Evelyne Copeland for critical
reading of the manuscript. This work was supported by a Genomics
and Health Research Initiative of the National Research Council of
Canada (NRCC Publication No 46198), sponsored by the Government
of Canada.
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