From the Programme in Molecular Biology and Cancer,
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada, the
§ Department of Molecular and Medical Genetics, University
of Toronto, Toronto, Ontario M5S 1A8, Canada, and the
Department
of Biology, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139
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ABSTRACT |
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Ephrin B proteins function as ligands for B class
Eph receptor tyrosine kinases and are postulated to possess an
intrinsic signaling function. The sequence at the carboxyl terminus of
B-type ephrins contains a putative PDZ binding site, providing a
possible mechanism through which transmembrane ephrins might interact
with cytoplasmic proteins. To test this notion, a day 10.5 mouse
embryonic expression library was screened with a biotinylated peptide
corresponding to the carboxyl terminus of ephrin B3. Three of the
positive cDNAs encoded polypeptides with multiple PDZ domains,
representing fragments of the molecule GRIP, the protein syntenin, and
PHIP, a novel PDZ domain-containing protein related to
Caenorhabditis elegans PAR-3. In addition, the binding
specificities of PDZ domains previously predicted by an oriented
library approach (Songyang, Z., Fanning, A. S., Fu, C., Xu,
J., Marfatia, S. M., Chishti, A. H., Crompton, A., Chan,
A. C., Anderson, J. M., and Cantley, L. C. (1997)
Science 275, 73-77) identified the tyrosine phosphatase
FAP-1 as a potential binding partner for B ephrins. In
vitro studies demonstrated that the fifth PDZ domain of FAP-1 and
full-length syntenin bound ephrin B1 via the carboxyl-terminal motif.
Lastly, syntenin and ephrin B1 could be co-immunoprecipitated from
transfected COS-1 cells, suggesting that PDZ domain binding of B
ephrins can occur in cells. These results indicate that the
carboxyl-terminal motif of B ephrins provides a binding site for
specific PDZ domain-containing proteins, which might localize the
transmembrane ligands for interactions with Eph receptors or
participate in signaling within ephrin B-expressing cells.
Among the large number of receptor tyrosine kinases identified in
metazoan organisms, the members of the Eph family are unusual in
several respects. Although only one Eph receptor tyrosine kinase is
known to be encoded by the Caenorhabditis elegans genome
(the vab-1 gene product (2)), vertebrates typically possess
up to 14 genes for Eph receptors, suggesting that these tyrosine
kinases may be important in controlling complex cellular interactions (3, 4). Consistent with this possibility, C. elegans VAB-1 regulates morphogenetic cell movements during ventral closure in
the embryo (2) while vertebrate Eph receptors have been implicated in
controlling axon guidance and fasciculation, in specifying topographic
map formation within the central nervous system, in organizing the
movements of neural crest cells during development, in directing fusion
of epithelial sheets in closure of the palate, and in angiogenesis
(5-15).
Early work on the expression patterns of EphB2 (formerly Nuk) suggested
that this receptor is clustered at sites of cell-cell junctions in the
developing mouse midbrain and raised the possibility that Eph receptors
might mediate signals initiated by direct cell-cell interactions (5).
Several lines of evidence support the notion that Eph receptors are
normally activated by ligands that are physically associated with the
surface of an adjacent cell. All known ligands for the Eph receptors
(termed ephrins) are related in sequence but can be divided into two
groups based on their carboxyl-terminal motifs. The ephrin A class of
ligands become modified by a carboxyl-terminal
glycosylphosphatidylinositol moiety, through which the ligand is
anchored to the surface of the ligand-expressing cell (7, 9, 16). In
contrast, B-type ephrins possess a transmembrane element and a highly
conserved cytoplasmic tail comprised of 82-88 carboxyl-terminal
residues (17-22). The Eph receptors can, in turn, be divided into A
and B subgroups based on their sequence similarity and their propensity
to bind soluble forms of either A or B type ephrins, respectively (4,
23, 24). However, although soluble ephrins bind tightly to the relevant receptors, consistent activation of Eph tyrosine kinase activity requires either that the ligands be artificially clustered into oligomers or that receptor-expressing cells be co-cultured with cells
expressing membrane-associated ephrins (18). These data suggest that
the ability of ephrins to aggregate and thereby activate Eph receptors
depends on their attachment to the cell surface, consistent with the
view that Eph receptor signaling involves cell-cell interactions.
During embryonic development in the mouse, Eph receptors and their
ligands are expressed in dynamic but complementary patterns, indicating
that Eph receptors are likely activated at boundaries where Eph and
ephrin-expressing cells are directly juxtaposed to one another (23,
25).
Genetic analysis of Eph receptor function in C. elegans and
the mouse has indicated that Eph receptors have both
kinase-dependent and kinase-independent modes of signaling
and raised the possibility that B-type Eph receptors and ephrins might
mediate bidirectional cell-to-cell signaling (2, 6). Of interest, the
binding of Eph receptors to transmembrane ephrin B1 or ephrin B2, as
well as treatment of ephrin B-expressing cells with platelet-derived growth factor, leads to the phosphorylation of the ephrins on tyrosine
residues within their highly conserved cytoplasmic tails (26, 27).
Furthermore, expression of the cytoplasmic tail of a Xenopus
ephrin B molecule leads to a striking loss of cell adhesion in
Xenopus embryos, an effect that is suppressed by treatment with fibroblast growth factor (28). Taken together, these results have
suggested that the cytoplasmic tails of ephrin B molecules may interact
with cellular proteins. Such ephrin B-interacting proteins could be
important either in localizing the ligands to appropriate sites in the
cell, for binding and activation of Eph receptors on adjacent cells, or
in transmitting a signal within the ligand-expressing cell.
Phosphorylation of tyrosine residues within the ephrin B tail might
then modulate the binding of such proteins in a positive or negative
fashion. Inspection of the sequences of the ephrin B proteins has
revealed a motif at the extreme carboxyl terminus (Tyr-Tyr-Lys-Val)
that contains potential sites of phosphorylation and has features
characteristic of binding sites for
PDZ1 domains. We have
therefore screened for known or novel polypeptides that might interact
with ephrin B ligands through this carboxyl-terminal motif. Here we
report the identification of such ephrin B-interacting proteins.
Peptide Synthesis--
The B ephrin carboxyl-terminal peptide
probe of sequence biotin-Aca-GPPQSPPNIpYYKV, related peptides NIpYpYKV,
NIpYYKV, NIYpYKV, NIYYKV, and DHQpYpYND were synthesized as described
previously (29).
Isolation of PDZ Domain-encoding cDNA Clones--
A Antibodies, Constructs, and Mutagenesis--
Anti-ligand
antibodies (Santa Cruz) were raised against residues 329-346 of ephrin
B1. Anti-FLAG M2 monoclonal antibodies were purchased from Eastman
Kodak Co. The expression construct of ephrin B1 cDNA in vector
pJFE14 has been described (18). Full-length syntenin cDNA was
subcloned in frame into the mammalian expression vector pFLAG CMV2
(Kodak) using standard cloning procedures. For GST fusion constructs,
cDNA sequences of syntenin (full-length: residues 1-299; PDZ 1 + 2: residues 101-299; PDZ1: residues 101-211; PDZ2: residues 172-299)
were cloned into pGEX4T2 (Amersham Pharmacia Biotech). FAP-1
(Fas-associated phosphatase) PDZ3 and FAP-1 PDZ5 constructs have been
described (1). The ephrin B1 Val deletion mutation was constructed by
the removal of nucleotides coding for the carboxyl-terminal Val-346
using a polymerase chain reaction-mediated protocol. The
PpuMI/EcoRI polymerase chain reaction fragment
carrying the mutated region was subcloned into the full-length ephrin
B1 cDNA in pJFE14. This mutation and all fusion constructs were
confirmed by sequencing of both strands of the affected region.
Immunoprecipitation and Western Blot Analysis--
COS-1 cells
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum. Transient transfections were performed
using Lipofectin reagent and Opti-MEM medium (Life Technologies,
Inc.) as outlined by the manufacturer. To reduce phosphorylation of
ephrin B1 by binding to endogenously expressed EphB receptors or by
stimulation with serum growth factors, transfected cells were
transferred from 10-cm to 15-cm plates 24 h after transfection and
serum-starved in Dulbecco's modified Eagle's medium, 0.5% fetal
bovine serum 12 h prior to cell lysis. Transfected cells were
rinsed once in phosphate-buffered saline A and lysed in phospholipase C
lysis buffer (5) with 10 µg/ml aprotonin, 10 µg/ml leupeptin, 1 mM sodium vanadate, and 1 mM
phenylmethylsulfonyl fluoride added. Immunoprecipitations were
performed for 1 h at 4 °C using 1 µg of anti-ephrin B1
antibody or 1 µg of anti-interleukin 3 receptor Fluorescence Polarization Analysis--
Binding constant
determination and peptide competition studies were carried out using
fluorescence polarization on a Beacon 2000 Fluorescence Polarization
System (Pan Vera, WI) equipped with a 100-µl sample chamber.
Fluorescein-labeled probes were prepared through reaction of B ephrin
carboxyl-terminal peptides with 5-(and-6)-carboxyfluorescein,
succinimidyl ester (Molecular Probes) and purified by reverse-phase
high performance liquid chromatography. The authenticity of the
fluorescein-labeled peptides was confirmed by mass spectroscopy. In the
binding studies, the fluorescein-labeled peptide probe was dissolved in
20 mM phosphate, pH 7.0, 100 mM NaCl, and 2 mM dithiothreitol to a concentration of 25 nM
and a known quantity of GST fusion protein was added. The reaction
mixtures were allowed to stand for 10 min at room temperature prior to
each measurement. All fluorescence polarization measurements were
conducted at 22 °C.
Identification of Potential Binding Partners for the Putative PDZ
Binding Site of B Ephrins--
As one approach toward identifying
proteins that interact with the cytoplasmic tails of B-type ephrins, we
initially examined the carboxyl-terminal regions of the transmembrane
ephrins for conserved peptide motifs that might bind modular domains of
intracellular signaling proteins. The extreme carboxyl terminus of the
three known B ephrins has a conserved sequence reminiscent of known or
predicted binding sites for PDZ domains (Fig.
1). We employed two strategies to
identify PDZ domain-containing proteins with the potential to recognize
the B ephrins. Firstly, comparison of the known binding specificities
of PDZ domains, predicted through the use of an oriented peptide
library technique, revealed the fifth PDZ domain of the cytoplasmic
tyrosine phosphatase FAP-1 as a possible ephrin B binding partner (Fig.
2A). FAP-1 (also known as
PTP-bas and PTP-L1) has at least six PDZ domains, an element related to
the Band 4.1 cytoskeletal polypeptide, and a carboxyl-terminal tyrosine
phosphatase domain (31-33). The fifth PDZ domain binds in
vitro to peptides with the consensus E(I/Y/V)Y(Y/K)(V/K/I), which
closely matches the conserved carboxyl terminus of B-type ephrins
(YYKV) (1).
A more direct approach to isolate ephrin B-binding proteins was
undertaken by screening a cDNA expression library from a day 10.5 mouse embryo with a peptide probe based on the putative PDZ domain
binding site of ephrin B3. The probe was a biotinylated peptide,
biotin-Aca-GPPQSPPNIpYYKV, conjugated to streptavidin-alkaline phosphatase. Although this peptide contained a phosphotyrosine residue
at the Syntenin and FAP-1 PDZ5 Bind Ephrin B1 in Vitro--
To determine
whether either syntenin or FAP-1 could interact with ephrin B1 in
vitro, GST fusions containing the fifth PDZ domain of FAP-1 or
full-length syntenin were incubated with lysates of ephrin
B1-transfected COS-1 cells. Recovery of these immobilized GST fusion
proteins and immunoblotting of associated proteins with anti-ephrin B1
antibody revealed that both FAP-1 PDZ5 and full-length syntenin were
able to bind intact ephrin B1 (Fig. 3,
A and C). The region of syntenin required for
binding to ephrin B1 was mapped using GST fusions containing defined
fragments of the syntenin protein. The minimal sequence necessary for a
strong interaction included both PDZ domains of syntenin but not the amino-terminal third of the protein (Fig. 3D).
Interestingly, both PDZ domains of syntenin are also required for
binding to the carboxyl-terminal sequence of syndecans, suggesting that
the involvement of two PDZ domains in the binding of a single target site may be a common feature of syntenin interactions (36). While the
syntenin PDZ1 domain alone was unable to associate with ephrin B1, the
second PDZ domain of syntenin alone exhibited a very weak
interaction.
In these experiments, neither GST alone nor a GST fusion with the third
FAP-1 PDZ domain showed detectable binding to ephrin B1. The identity
of the ~50-kDa band recognized by GST-FAP-1 PDZ3 is not known but
its apparent size does not correlate with any of the three known B
ephrins. Consistent with this finding, the binding specificity of FAP-1
PDZ3, as previously determined using an oriented peptide library, is
significantly different from that of FAP-1 PDZ5 with a preference
toward target sequences such as the QSLV-COOH motif in the Fas antigen
(1, 33). The inability of the FAP-1 PDZ3 domain to bind ephrin B1
indicates a degree of specificity in recognition of ephrin B1 by PDZ domains.
A hallmark of many PDZ domain binding sites is a requirement for a
carboxyl-terminal hydrophobic residue that contacts the PDZ domain
through its side chain and carboxyl-terminal carboxylate group (1, 38,
39). The involvement of the carboxyl-terminal Val of ephrin B1 in
specific binding to syntenin and FAP-1 PDZ5 was initially evaluated by
expressing a deletion mutant of ephrin B1 lacking the terminal Val
residue in COS-1 cells. Removal of the carboxyl-terminal Val from
full-length ephrin B1 abrogated its binding to both syntenin and FAP-1
PDZ5 GST fusion proteins (Fig. 3, B and
C).
As an alternative approach toward investigating the specificity of
ephrin B1 interactions with PDZ domain proteins, we employed a specific
peptide modeled on the carboxyl terminus of B-type ephrins in
competition experiments. For this purpose, lysates of ephrin
B1-transfected cells were incubated with either GST-syntenin or
GST-FAP-1 PDZ5 in the presence or absence of a peptide corresponding in
sequence to the carboxyl-terminal six residues of B ephrins. The
peptide successfully blocked syntenin and FAP-1 PDZ5 binding at a
peptide concentration of 100 µM (Fig.
4, A and B). The
addition of the unrelated peptide, DHQpYpYND, did not decrease binding, indicating the specificity of the peptide competition (Fig.
4A and data not shown).
FAP-1 PDZ5 and Syntenin Display Differential Binding to
Phosphopeptides--
Binding of B ephrins to their cognate Eph B
receptors, expression of an activated Src tyrosine kinase, or treatment
of ligand-expressing cells with platelet-derived growth factor results
in tyrosine phosphorylation of residues in the ephrin cytoplasmic
domain (26, 27). Preliminary evidence based on specific substitutions
of the Tyr residues in the ephrin B1 tail suggests that the two
tyrosines at the
The GST-FAP-1 PDZ5 bound to a fluorescein-labeled NIYYKV peptide with
an affinity of 9.9 ± 1.0 µM, while GST-FAP-1 PDZ3
binding was much weaker (65.0 ± 9.6 µM) (Fig.
5A). This is consistent with
the GST mixing experiments that indicated FAP-1 PDZ3 does not interact
stably with ephrin B1. Similar results were obtained when binding to
the three different phosphorylated peptides was investigated,
indicating that alternative tyrosine phosphorylation states of the B
ephrin carboxyl-terminal sequence had little effect on binding to
GST-FAP-1 PDZ5. Similar binding affinity values of 6.8 ± 0.8 µM, 15.4 ± 3.4 µM, and 8.4 ± 2.5 µM were obtained for the NIpYYKV, NIYpYKV, and
NIpYpYKV peptides, respectively.
Fluorescence polarization experiments measuring GST-syntenin fusion
protein binding to fluorescein-labeled NIYYKV and NIpYYKV peptides
yielded nearly identical binding curves (Fig. 5B). Affinity values of 17.7 ± 1.2 µM and 15.4 ± 0.5 µM were obtained, indicating that phosphorylation at the
Ephrin B1 and Syntenin Can Associate in Cells--
We have pursued
the possibility that B-type ephrins may interact with PDZ domain
proteins in vivo by assaying whether ephrin B1 and syntenin
associate when co-expressed in COS-1 cells. In cells co-transfected
with ephrin B1 and syntenin (tagged at its N terminus with a FLAG
epitope) immunoprecipitation of ephrin B1 specifically co-precipitated
syntenin (Fig. 6). Precipitation with
protein A-Sepharose alone or with an arbitrarily chosen antibody did
not yield detectable syntenin, indicating that the interaction is
specific. Further, co-immunoprecipitation experiments with the ephrin
B1 Val deletion mutant, which fails to interact with PDZ domains
in vitro, showed that ephrin B1 lacking the
carboxyl-terminal Val did not detectably associate with syntenin (Fig.
6). While the truncated protein could be successfully
immunoprecipitated by antibodies against ephrin B1, syntenin could not
be co-immunoprecipitated with the mutant protein. These results
demonstrate that ephrin B1 and syntenin can associate in cells and show
that an intact PDZ domain binding site in ephrin B1 is necessary for
its interaction with syntenin in vivo.
In an effort to identify components of the cytoplasmic domain that
may contribute to ephrin B function, we have demonstrated that the
carboxyl-terminal residues of B ephrins constitute a binding site for
PDZ domains, a class of protein module known to mediate specific
protein-protein interactions. Several lines of evidence indicate that
the carboxyl-terminal YYKV sequence, conserved among all 3 known B
ephrins, represents a PDZ domain binding site. Firstly, a biotinylated
peptide probe with a sequence corresponding to the carboxyl-terminal
residues of ephrin B3 identified cDNAs coding for the known PDZ
domain-containing proteins syntenin and GRIP, as well as a cDNA for
PHIP, a novel PDZ domain-containing protein. In addition, a fourth
PDZ-containing protein, FAP-1, was identified as a binding candidate
based initially on the predicted binding specificity of its fifth PDZ domain.
Secondly, in vitro studies with syntenin and FAP-1 have
demonstrated specific interactions of the PDZ domains of these proteins with the carboxyl terminus of ephrin B1. The finding that the carboxyl-terminal Val residue of ephrin B1 is absolutely required for
these interactions indicates that binding occurs in a manner characteristic of other PDZ domain interactions with carboxyl-terminal target sequences. Similar results were also obtained from in
vitro binding experiments with ephrin B2 (data not shown),
suggesting that PDZ domain interactions may be common to all B ephrins.
In vitro experiments were also performed with separate GST
fusions of GRIP PDZ6 and GRIP PDZ7. Interactions with ephrin B1 or with the fluorescent GNIYYKV peptide were not detected in GST mixing and
fluorescence polarization experiments (data not shown). It is possible
that binding to ephrin B1 may require both PDZ6 and PDZ7 of GRIP in a
fashion reminiscent of the requirement of both syntenin PDZ domains for
binding. We are currently investigating this possibility. Lastly, we
have demonstrated that B ephrin-PDZ domain interactions can occur
in vivo, since syntenin can be successfully co-immunoprecipitated with full-length ephrin B1 but not with ephrin B1
truncated in its PDZ domain target site. Thus far, we have not detected
consistent complexes of ephrin B1 with GRIP or FAP-1 in
vivo, but it remains possible that these are also physiological
binding partners for B-type ephrins.
The effect of the phosphorylation state of two adjacent tyrosines at
positions While we have identified a PDZ domain binding site in B-type ephrins,
the issue of how PDZ domain interactions relate to ephrin B function
remains to be determined. Possible roles for PDZ domain-ephrin B
associations, however, can be proposed based on known functions of PDZ
domains. Several examples have highlighted the importance of PDZ domain
interactions in the proper localization and clustering of transmembrane
proteins (42, 43). For instance, the positioning of NMDA receptors and
K+ channels at post-synaptic termini is likely dependent on
specific interactions of these receptors with PDZ domain-containing
proteins (34, 44-47). In Drosophila larvae, null mutations
of the gene encoding the PDZ protein discs-large result in
mislocalization of the Shaker K+ channel (48). Clustering
of Shaker K+ channels via PDZ domain interactions has also
been demonstrated in COS-7 cells co-expressing the channel with either
of its binding partners, PSD-95 or chapsyn 110 (49).
A requirement for correct localization and clustering figures
prominently in the proposed functions of B class ephrins. Since ephrin
B-EphB interactions involve direct cell-cell contact, ephrins must
presumably be present at sites of contact with receptor-expressing cells. This localization may be mediated by PDZ domain associations with the carboxyl terminus of B ephrins. In this regard, it is of
interest that PHIP is a close relative of PAR-3, a C. elegans protein that regulates asymmetry and polarity in the early
embryo. It is possible that PHIP has a similar function in mammalian
cells in controlling the asymmetric distribution of proteins with PDZ domain-binding motifs.
Studies involving soluble forms of the extracellular domain of ephrins
have revealed a requirement for ligand clustering in receptor
activation. Whereas treatment of receptor-expressing cells with soluble
versions of the ligands does not result in receptor activation and
subsequent autophosphorylation, artificial aggregation of soluble
ephrins by clustering antibodies allows activation of the receptor
(18). Since co-culturing of ephrin-expressing cells with cells
expressing Eph receptors leads to receptor activation, membrane-bound
ligands must also become clustered in some manner. Furthermore, recent
studies in a renal endothelial cell system have indicated that the
state of ephrin B1 oligomerization is important in determining
alternative receptor signaling complexes as well as attachment and
assembly responses in the receptor-bearing cell (50). Although binding
of both ligand dimers and higher order oligomers caused receptor
autophosphorylation, only tetrameric forms of the ligand were able to
induce the attachment response and stimulate the recruitment of a low
molecular weight phosphotyrosine phosphatase to the activated receptor.
Given the known role of PDZ domains in the clustering of transmembrane
proteins, it is conceivable that PDZ domain interactions with ephrin B1
may play a role in the presentation of the ligand in the correct
oligomeric form to elicit specific responses in the receptor-expressing cell.
Another role ascribed to PDZ domain-containing proteins is to act as a
scaffold to organize signaling complexes. This is well illustrated by
the function of the protein InaD in the phototransduction pathway of
the Drosophila compound eye. Key components of this cascade,
including the transient receptor potential calcium channel, the eye
form of protein kinase C and phospholipase C- Further work is required to distinguish among these possible roles for
PDZ domain-ephrin B interactions. Cell culture experiments using the
Val deletion mutant of ephrin B1 that is unable to bind the PDZ domains
of syntenin and FAP-1 are currently in progress to determine the effect
of uncoupling PDZ domain interactions from B ephrins. These studies,
along with the further characterization of the binding candidates
identified in this report, will help to determine the physiological
relevance of PDZ domain interactions in the function of B ephrins.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
EXlox
10.5 day mouse embryo expression library (Novagen) was plated at an
initial density of 10,000 plaque-forming units/15-cm Petri plate.
Library screening was performed using a biotinylated peptide probe
conjugated to streptavidin-alkaline phosphatase following a procedure
similar to that described by Sparks et al. (30). To isolate
more coding sequence for PHIP, an EcoRI/PstI fragment of PHIP cDNA (encoding amino acid residues 462-602) was radiolabeled with [
-32P]dCTP and used to screen the
EXlox 10.5 day mouse embryo library. The DNA sequencing of positive
clones was carried out using the ALF automated DNA sequencer (Amersham
Pharmacia Biotech).
antibody with
protein A-Sepharose. GST mixing experiments were carried out by 1-h
incubation at 4 °C of lysate with 5-10 µg of fusion protein
immobilized on glutathione-Sepharose. For the peptide competition
experiments, peptides were included in the incubation with the GST
fusion proteins at a final concentration of 100 µM. Beads
for both immunoprecipitations and GST mixing experiments were washed
2-3 times in HNTG buffer (20 mM Hepes, pH 7.5, 10% glycerol, 0.1% Triton X-100, 150 mM mM NaCl)
(5). Proteins were separated by 10% SDS-polyacrylamide gel
electrophoresis, transferred to Immobilon-P membrane (Millipore), and
immunoblotted with the appropriate antibody. Blots were developed by
Enhanced Chemiluminescence (Pierce).
RESULTS
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Fig. 1.
Amino acid sequence of the cytoplasmic
domains of the human B ephrins. Conserved residues among the three
B ephrins are highlighted. Asterisks mark
conserved tyrosines that are potential sites of phosphorylation. The
potential PDZ domain binding site is underlined.
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Fig. 2.
Identification of PDZ domain-containing
candidates for ephrin B binding. A, the preferred
binding sequence of FAP-1 PDZ5 is shown below a schematic
representation of the entire FAP-1 protein-tyrosine phosphatase. FAP-1
PDZ5 domain specificity was deduced from an oriented peptide library
technique (1). Residues within the optimal binding sequence that match
the carboxyl-terminal sequence of B ephrins are indicated in
bold. The organization of the PDZ domains of FAP-1 shown in
this figure follows the numbering described by Sato et al.
(33). B, diagrammatic representations of the PDZ
domain-containing proteins identified through an expression screen with
a biotinylated peptide probe of ephrin B3 carboxyl-terminal sequence.
The brackets mark the portions of the protein encoded by the
cDNAs isolated from the screen. PDZ domains are represented by
gray boxes. C, amino acid sequence
alignment of FAP-1 PDZ5 and of the PDZ domains isolated in the
expression screen. The numbering of the PDZ domains is as shown in Fig.
2B. Conserved residues are highlighted. The
alignment was performed with the ClustalW program (55). D,
amino acid sequence alignment of PHIP and PAR-3. Conserved residues are
highlighted, and the PDZ domains are underlined.
The alignment was performed with the Genestream Align program.
3 position relative to the carboxyl-terminal valine, we
anticipated that the alkaline phosphatase used in the screen would at
least partially dephosphorylate the probe, allowing detection of both
tyrosine phosphorylation-dependent and independent binding. The screening of approximately 500,000 cDNA clones yielded four distinct cDNA products that bound to the ephrin B3
carboxyl-terminal peptide, of which 3 were subsequently found to
contain PDZ domains upon sequence analysis (Fig. 2, B and
C). One of these cDNAs encodes a portion of the adaptor
protein GRIP, from the sixth PDZ domain to the carboxyl terminus (amino
acid residues 642-1112). GRIP is an ~180-kDa protein composed of
seven PDZ domains, originally identified by its ability to bind the
carboxyl terminus of AMPA receptors through PDZ domains 4 and 5 (34). A
second cDNA isolated by this approach contained the entire coding
sequence for the PDZ domain-containing protein syntenin. Syntenin was
first reported as a transcript down-regulated during melanoma
differentiation (termed Mda-9) and subsequently shown to interact via
its two PDZ domains with the carboxyl terminus of the transmembrane
syndecan proteins (35, 36). A third clone identified in this screen was
a partial cDNA encoding the carboxyl-terminal fragment of a novel
PDZ domain-containing protein (termed PHIP for ephrin interacting protein). Analysis of the sequence
of the PHIP cDNA fragment revealed the presence of two adjacent PDZ
domains followed by a 50-amino acid carboxyl-terminal stretch. The PHIP
cDNA fragment was subsequently used as a probe to isolate a
transcript from a day 10.5 mouse embryo library. The predicted sequence
of PHIP indicates that it encodes a total of three PDZ domains and is closely related to PAR-3, a C. elegans protein involved in
regulating polarity of the early embryo (Fig. 2D) (37). Of
these candidates, FAP-1 PDZ5 and syntenin were further investigated for
their binding to B ephrins.
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Fig. 3.
FAP-1 PDZ5 and syntenin bind specifically to
ephrin B1 in GST mixes. COS-1 cells were transiently transfected
with either wild-type ephrin B1 (W.T.) or the ephrin B1 Val
deletion (Val ) or were untransfected. Cell lysates were
incubated with the GST fusion proteins as indicated and analyzed by
immunoblotting with anti-ephrin B1 antibody. Immunoprecipitated ephrin
B1 or ephrin B1 Val
were included as a positive control.
A and B, GST mixes with fusion proteins of FAP-1.
C and D, GST mixes with fusion proteins of
syntenin.
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Fig. 4.
FAP-1 PDZ5 and syntenin binding to ephrin B1
can be blocked by addition of peptides corresponding to the
carboxyl-terminal sequence of B ephrins. Peptides of the indicated
sequence were included at a concentration of 100 µM in
incubations of GST fusion proteins with lysates of COS-1 cells
transfected with ephrin B1. Associated proteins were separated on a
10% polyacrylamide-SDS gel and analyzed by immunoblotting with
antibodies against ephrin B1. A, competition of FAP-1 PDZ5
binding to ephrin B1 using the indicated peptides. A peptide of
sequence DHQpYpYND was added at a concentration of 100 µM
as a negative control. Immunoprecipitation of ephrin B1 was included as
a positive control. B, peptide competition of the binding of
full-length syntenin to ephrin B1.
2 and
3 positions within the PDZ domain binding
site are among the phosphorylation
sites.2 To investigate
whether tyrosine phosphorylation of these residues might affect PDZ
domain binding, the carboxyl-terminal peptide used for the peptide
competition described above was also synthesized such that either one
or both of the
2 and
3 tyrosine residues were phosphorylated. The
phosphorylated and unphosphorylated peptides were labeled with
fluorescein and employed in fluorescence polarization experiments to
obtain quantitative measurements of their affinities for FAP-1 and
syntenin PDZ domains.
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Fig. 5.
Fluorescence polarization analysis of
GST-FAP-1 PDZ3, GST-FAP-1 PDZ5, and GST-syntenin binding to
fluorescein-labeled peptides corresponding to the carboxyl terminus of
ephrin B1. A, solutions containing the indicated final
concentration of GST-FAP-1 PDZ3 ( ) or GST-FAP-1 PDZ5 (
) fusion
protein in mixtures containing 25 nM fluorescein-labeled
NIYYKV peptide probe, 20 mM phosphate, pH 7.0, 100 mM NaCl, and 2 mM dithiothreitol were monitored
for fluorescence polarization at 22 °C. The GST-FAP-1 PDZ5 fusion
protein was also measured for binding to the phosphorylated peptides,
NIpYYKV (
), NIYpYKV (
), and NIpYpYKV (
). The fluorescence
polarization values obtained for the peptide in absence of added GST
fusion protein has been subtracted from the polarization values
displayed. B, a binding of a GST fusion of full-length
syntenin to the NIYYKV (
), NIpYYKV (
), and NIpYpYKV (
)
peptides as measured by fluorescence polarization.
3 position tyrosine does not significantly affect the PDZ-domain
interaction. However, the GST-syntenin fusion protein bound the pYpYKV
peptide with a much lower affinity of 151.0 ± 20.9 µM, indicating that phosphorylation at the
2 Tyr can
have a detrimental effect on binding to syntenin. A similar low
affinity interaction was observed for the YpYKV peptide (data not shown).
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Fig. 6.
Co-immunoprecipitation of syntenin-FLAG with
ephrin B1. COS-1 cells were co-transfected with either ephrin B1
and syntenin-FLAG or with the ephrin B1 Val deletion and syntenin-FLAG
as indicated. Cell lysates were immunoprecipitated with antibodies
against ephrin B1 or interleukin 3 receptor or were treated with
protein A-Sepharose only. Immunocomplexes were subjected to
SDS-polyacrylamide gel electrophoresis (10%) and blotted with
anti-FLAG antibodies.
DISCUSSION
2 and
3 relative to the carboxyl-terminal Val of the PDZ
domain target site was examined using a fluorescence polarization
assay. Structural studies of PDZ domains have suggested that
interactions between PDZ domains and residues at the
2 and
3
positions of the carboxyl-terminal target site confer binding specificity (38-40). In one case, modification of residues at these positions by serine phosphorylation has been reported to regulate PDZ
domain binding. The specific association between the second PDZ domain
of PSD-95 and the inward rectifier potassium (K+) channel
Kir2.3 is disrupted by protein kinase A-mediated phosphorylation of a
key serine residue at the
2 position from the carboxyl terminus of
Kir2.3 (41). Our results with B class ephrins and the PDZ domain
proteins FAP-1 and syntenin suggest that the phosphorylation of
residues within the PDZ domain binding site has different effects on
different PDZ domains. The results with FAP-1 PDZ5 suggest that the PDZ
domain residues, which contact the tyrosines in the binding site of B
ephrins are able to accommodate the addition of two phosphate groups.
This is consistent with observations that the single PDZ domain of AF-6
binds an unphosphorylated peptide with the consensus target sequence
AYYV and a corresponding peptide phosphorylated at the
2 Tyr residue
with approximately equal affinity.3 In contrast,
GST-syntenin exhibited significantly decreased binding to peptides
phosphorylated at the
2 residue of the PDZ domain binding site. It
will be of interest to determine whether the lower affinity values seen
in our in vitro studies are significant in vivo.
These data suggest one mechanism through which tyrosine phosphorylation
of ephrin B1 may regulate interactions with modular cytoplasmic proteins.
are bound by the PDZ
domains of InaD to form a compartmentalized signaling complex (51, 52).
Mutations in specific InaD PDZ domains that abolish binding result in
defects in the kinetics of the phototransduction cascade. In the case
of B ephrins, genetic evidence along with biochemical studies
indicating that tyrosine residues in the intracellular domain become
phosphorylated upon receptor binding or platelet-derived growth factor
treatment has led to the hypothesis that the cytoplasmic tail of B
ephrins may have an intrinsic signaling function (2, 6, 26, 27). The
phosphorylated tyrosine residues represent potential docking sites for
proteins with phosphotyrosine recognition modules such as SH2 or PTB
domains. Downstream components of this possible
phosphotyrosine-dependent signaling pathway may be assembled around a PDZ domain-containing protein in a manner similar to the InaD
complex. Furthermore, the PDZ domain-containing protein PSD-95, which
associates with glutamate receptors and K+ channels, also
interacts through its PDZ domains with neuronal nitric-oxide synthase
and a Ras GTPase activating protein (p135 SynGAP) (53, 54). PDZ
domain-containing proteins may thereby serve as adaptors to directly
activate signaling pathways. In this context, it is of interest that
phosphorylation of the Tyr residues in the carboxyl-terminal ephrin B1
motif may regulate interactions with PDZ domains, as suggested by our
results with syntenin.
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ACKNOWLEDGEMENTS |
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We thank S. Holland and R. Dhand for technical advice and critical reading of the manuscript. We thank C. H. Heldin for additional FAP-1 (PTP-L1) reagents and N. Gale and G. Yancopoulos for ephrin B cDNAs.
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Note Added in Proof |
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A recently published mammalian PAR-3 homolog, ASIP, is closely related to PHIP (Izumi, Y., Hirose, T., Tamai, Y., Hirai, S., Nagashima, Y., Fugimoto, T., Tabuse, Y., Kemphues, K. J., and Ohno, S. (1998) J. Cell Biol. 143, 95-106).
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FOOTNOTES |
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* This work was supported by the Medical Research Council (MRC) of Canada and by a Howard Hughes Medical Institute International Research Scholar Award (to T. P.).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.
¶ Supported by a graduate studentship from the MRC of Canada.
** Distinguished Scientist of the MRC of Canada. To whom correspondence should be addressed. Tel.: 416-586-8262; Fax: 416-586-8857; E-mail: pawson{at}mshri.on.ca.
The abbreviations used are: PDZ, PSD95/Dlg, ZO-1; GST, glutathione S-transferase; FAP, Fas-associated phosphatase.
2 G. Mbamalu, S. Holland, and T. Pawson, unpublished results.
3 Z. Songyang, unpublished results.
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REFERENCES |
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