* Institut für Medizinische Virologie and Elektronenmikroskopisches Zentrallabor, Universität Zürich; § Institut für
Neuroinformatik, Universität Zürich/Eidgenössische Technische Hochschule, CH-8028 Zürich, Switzerland; and ¶ Weizmann
Institute for Science, Rehovot 76100, Israel
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Abstract |
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The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized sites of cell-cell junctions. To identify potential receptor-binding targets of AF-6 we screened the PDZ domain of AF-6 against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.
Key words: postsynaptic clustering; PDZ domains; receptor tyrosine kinases; neuron physiology; Ras-binding protein ![]() |
Introduction |
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MANY cellular processes rely in large part on the
correct subcellular distribution of effector proteins. At specialized sites of cell-cell contact,
membrane proteins are clustered with specific adaptor
proteins linked to the cytoskeleton, enabling cellular adhesion, motility, and intercellular as well as intracellular signaling events to occur (Kofron et al., 1997; Tsukita et al., 1996
, 1997
). Some proteins, which are localized to specific
cell junctions and presumably influence their function,
have been identified. In epithelial cells, the specialized site
of cell contact known as the tight junction (zonula occludens) has been shown to be composed of transmembrane proteins such as occludin (Furuse et al., 1993
) and
juxtamembrane proteins such as ZO-1 (Stevenson et al.,
1986
), ZO-2 (Jesaitis and Goodenough, 1994
), ZO-3 (Haskins et al., 1998
), cingulin (Citi et al., 1988
), 7H6 (Zhong et
al., 1993
), and AF-6 (Mandai et al., 1997
; Yamamoto et al.,
1997
).
In the nervous system specialized sites of cell-cell contact are vital for ensuring growth cone pathfinding during
embryogenesis and neuronal signaling and information
processing at developed synapses. Voltage-gated and ligand-gated ion channels are clustered at specific membrane sites at synapses in neuronal cells facilitating the
coordinated transmission of electrical signals (Froehner, 1993). Shaker-type K+ channel and N-methyl-D-aspartate
(NMDA)1 receptor 2 (NR2) subunits are localized specifically at presynaptic and postsynaptic sites, respectively
(Kim et al., 1995
; Kornau et al., 1995
). Recently, a subfamily of the membrane-associated putative guanylate kinases
(MAGUKs) comprised of PSD-95/SAP90 has been shown
to bind directly to these ion channels and colocalize with
them at synapses in the brain (Hunt et al., 1996
; Kim et al.,
1995
; Kornau et al., 1995
; Niethammer et al., 1996
). The interaction is mediated by the direct association of the COOH terminus of the ion channel receptors with the NH2-terminal two PDZ domains present in PSD-95/SAP90. PDZ domain-containing proteins, such as that exemplified by the
PSD-95 subfamily of MAGUKs, are thought to function as
clustering proteins at specific synaptic membranes on the
surfaces of neurons (Gomperts, 1996
). In addition, these
proteins may organize overall synaptic structures, since perturbations in the Drosophila homologue of PSD-95,
discs large (DlgA), lead to aberrant synaptic structures in
the fly nervous system (Lahey et al., 1994
).
A number of the juxtamembrane proteins identified as components of specialized junctions in epithelial and endothelial cells such as ZO-1, ZO-2, ZO-3, and AF-6 also have PDZ domains and therefore may also function as clustering agents for as yet unidentified receptor proteins. To identify potential receptor targets of one of these proteins, AF-6, we screened the database for receptors having potential PDZ interacting termini and tested whether these termini could interact with the PDZ domain of AF-6.
We observed that AF-6 is able to interact with a specific subset of the Eph RTK proteins. Two-hybrid and in vitro binding assays confirmed that the AF-6 PDZ domain interacts specifically with the COOH terminus of the Eph receptors. Cotransfection of AF-6 with the Eph receptors in heterologous cells induces a clustering of AF-6 with the Eph receptors at sites of cell-cell contact. AF-6 is phosphorylated when cotransfected with Eph receptors but not with a kinase-deficient mutant, demonstrating that AF-6 is a substrate of Eph receptors.
Eph receptor tyrosine kinases (RTKs) are highly expressed in the nervous system (Orioli and Klein, 1997) and
some can be localized to specific membrane regions of
cell-cell contact on neurons (Henkemeyer et al., 1994
). It
is demonstrated by electron microscopy that both, AF-6
and the receptors, tightly colocalize at postsynaptic membranes of excitatory synapses in the hippocampus and at
other sites of membrane specialization on neurons. This
observation is further corroborated by showing that endogenous AF-6 physically interacts with Eph receptors in
whole rat brain extracts.
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Materials and Methods |
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Mammalian Two-hybrid Analysis
A partial clone of mouse AF-6 (amino acids [aa] 850-1,129) encompassing
the PDZ domain was subcloned into the VP16 activation domain expression vector pSNATCH (Buchert et al., 1997). Oligonucleotide adaptors
encoding the last 10 amino acids of the COOH termini of the various
RTKs as listed in Fig. 1 were then cloned into the Gal4 DNA-binding domain mammalian expression vector pSNAG (Buchert et al., 1997
). Human 293T cells from a 12-well tissue culture plate were transfected with
20 ng of the expression plasmids along with 50 ng of a chloramphenicol acetyltransferase (CAT) reporter gene construct and the cells were
harvested 36-48 h later. A CAT enzyme-linked immunosorbent assay
(ELISA) was performed according to the manufacturer's instructions
().
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In Vitro and Coimmunoprecipitation Assays
293T cells were transiently transfected separately with expression vectors
encoding the full-length receptors, EphA7 (MDK1), EphB3 (Hek2),
EphB2 (Nuk), and Ephrin-B1 (Xlerk2) along with a partial, FLAG-tagged clone of the mouse AF-6 (aa 850-1,129) encompassing the PDZ
domain. 48 h after transfection cells were lysed in buffer A (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 mM DTT, 1 mM
PMSF, 1 mM benzamidine, 2.8 µg/ml aprotinin) and placed on ice for 30 min with occasional shaking. Samples were pelleted for 10 min at 4°C at
10,000 g. The supernatant was then precleared with protein A-Sepharose
beads and the precleared supernatant incubated with 1 µl (2.7 µg) anti-FLAG M2 antibody () and incubated with rotation at 4°C
for 1 h. Protein A-Sepharose beads (50 µl) were then added to the cell extracts and incubated with rotation at 4°C for a further 45 min. Cell extracts
were then pelleted and washed four times with 1 ml of buffer A and resuspended in loading buffer, run on 10% SDS-PAGE gels, and then blotted
as described (Harlow and Lane, 1988). Blots were probed with the
anti-receptor-specific antibodies. For glutathione-S-transferase (GST)-
pull down experiments the partial clone of the mouse AF-6 (aa 850-1,129)
encompassing the PDZ domain was fused COOH-terminal to GST and
produced and coupled to glutathionine beads as described (Ridley and
Hall, 1992
), except after binding, beads were washed five times in lysis
buffer; PBS, 1 mM DTT, 1 mM PMSF, 1 mM benzamidine, 2.8 µg/ml
aprotinin. The green flourescent protein (GFP)-peptide (EphB3pep) was
visualized on the blots with a monoclonal anti-GFP antibody ()
as recommended by the manufacturer.
Phosphorylation of AF-6
293T cells were transiently transfected separately with expression vectors
encoding the full-length receptors EphB2 (Nuk), EphB3 (Hek2), a kinase-deficient mutant of EphB3 (EphB3K665R), and Ephrin-B1 (Xlerk2)
along with a FLAG-tagged full-length AF-6 cDNA. 48 h after transfection
cells were lysed in a 10-cm culture dish with 1 ml of NETN-buffer (20 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40 supplemented
with 1 mM PMSF, 1 mM Na3VO4, 2.8 µg/ml aprotinin, 50 mM NaF, and
1 mM benzamidine) and placed on ice for 15 min. Lysates were collected,
transferred to precooled Eppendorf tubes, and then incubated in a shaker
for another 15 min at 4°C. Samples were then pelleted for 10 min at 4°C at
10,000 g. The supernatant was then precleared with protein A-Sepharose
beads and the precleared supernatant incubated with 4 µl (2.7 µg/µl) anti-FLAG M2 antibody () and incubated with rotation at 4°C
for 1 h. Protein A-Sepharose beads (40 µl) were then added to the cell extracts and incubated with rotation at 4°C for a further 45 min. Cell extracts
were then pelleted and washed four times with 1 ml of NETN-buffer, resuspended in loading buffer, run on a 7.5% SDS-PAGE gel, and then
blotted as described (Harlow and Lane, 1988). Western blots were probed
either with the monoclonal anti-FLAG M2 antibody, or the monoclonal
anti-pTyr antibody (PY99; Santa Cruz).
Phosphorylation of AF-6 in the Cellular Lysate
293T cells were transiently transfected separately with expression vectors
encoding the full-length receptors EphB2 (Nuk), EphB3 (Hek2), and a kinase-deficient mutant of EphB3 (EphB3K665R) along with a FLAG-tagged full-length AF-6 cDNA. 48 h after transfection, cells were lysed in
a 10-cm culture dish with 1 ml of NETN-buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40 supplemented with 1 mM
PMSF, 1 mM Na3VO4, 2.8 µg/ml aprotinin, 50 mM NaF, and 1 mM benzamidine) and then placed on ice for 15 min. Lysates were collected, transferred to precooled Eppendorf tubes, and then incubated in a shaker for
another 15 min at 4°C. Samples were then pelleted for 10 min at 4°C at
10,000 g. Equal amounts of lysate were resuspended in loading buffer,
boiled for 5 min at 95°C, run on a 7.5% SDS-PAGE gel, and then blotted
as described (Harlow and Lane, 1988). Western blots were probed either
with the monoclonal anti-pTyr antibody (PY99; Santa Cruz), with the monoclonal anti-FLAG M2 antibody (), or with the anti-
receptor-specific antibodies.
Coimmunoprecipitation from Whole Rat Brain Lysates
Brain coimmunoprecipitation with EphB3 was done essentially as described (Irie et al., 1997). In brief, three fresh rat brains were homogenized in a glass teflon homogenizer in 15 ml of NL buffer: 20 mM Hepes-NaOH, pH 8.0, 1% NP-40, 0.2 M NaCl, 2 mM EGTA and inhibitors (1 mM
benzamidine, 1 mM PMSF, 50 mM NaF, 2.8 µg/ml aprotinin, 1 mM
Na3VO4. This homogenate was then vigorously shaken for 15 min at 4°C
and centrifuged for 60 min at 4°C at 100,000 g to give a supernatant and a
pellet fraction. The supernatant was precleared with 30 µl of protein
A-Sepharose beads and then incubated for 90 min with 50 µl polyclonal
rabbit anti-EphB3 antibody, 12.5 µg monoclonal anti-AF-6 antibody
(Transduction Laboratories), or 12.5 µg monoclonal anti-FLAG M2 antibody (). 60 µl of protein A-Sepharose was added for another 90 min at 4°C. Then, beads were washed five times with NL buffer and
30 µl of SDS-loading buffer were added. The pellet received after the centrifugation was extracted a second time, this time using 15 ml of the more stringent radioimmunoprecipitation assay (RIPA) buffer (which disrupts
the interaction between the interacting partners): 20 mM Tris-HCl, pH
7.5, 0.1% SDS, 0.5% deoxycholic acid, 1% Triton X-100, 137 mM NaCl,
10% glycerine, 2 mM EDTA, and inhibitors as described above. This homogenate was then processed identically as described for the homogenate
and subsequently washed five times with RIPA buffer and taken up in 30 µl of SDS-loading buffer. Fractions were analyzed by SDS-polyacrylamide gel (7.5%) electrophoresis and by immunoblotting with antibodies
to AF-6 and EphB3.
Brain coimmunoprecipitation with EphB2 was done as follows. Four fresh rat brains were homogenized using the FastPrep FP120 machine (BIO 101). Brains were cut in small pieces and put into FastDNA Kit tubes (BIO 101). The tubes were filled with HO buffer (50 mM Hepes-NaOH, pH 7.5, 1% Triton X-100, 0.15 M NaCl, 1 mM EGTA, 1.5 mM MgCl2, 10% glycerol) and inhibitors (1 mM benzamidine, 1 mM PMSF, 50 mM NaF, 2.8 µg/ml aprotinin, 1 mM Na3VO4) and the machine run at speed 4.5 for 15 s. To this homogenate HO buffer was added up to 20 ml and vigorously shaken for 15 min at 4°C. Subsequently, supernatant and pellet was obtained through centrifugation twice for 1 h at 13,000 g. The supernatant was precleared with 30 µl of protein A-Sepharose beads and then incubated for 90 min with 10 µg polyclonal rabbit anti-EphB2 antibody (Santa Cruz), 12.5 µg monoclonal anti-AF-6 antibody (Transduction Laboratories), or 12.5 µg monoclonal anti-FLAG M2 antibody (). 60 µl of protein A-Sepharose was added for another 90 min at 4°C. Then beads were washed five times with HO buffer and 30 µl of SDS-loading buffer were added. The pellet received after the centrifugation was extracted a second time exactly as described for the second extraction in the EphB3 coimmunoprecipitation (see above). Fractions were analyzed by SDS-polyacrylamide gel (7.5%) electrophoresis and by immunoblotting against AF-6.
Antibodies and Immunofluorescence Microscopy
Monoclonal anti-AF-6 (Transduction Laboratories), rabbit polyclonal anti-ZO-1 (, monoclonal anti-FLAG M2 antibody (), monoclonal anti-GFP (), and rabbit polyclonal anti-Lerk2 (Ephrin-B1; Santa Cruz) antibodies were purchased from commercial sources. Rabbit polyclonal antibodies against EphB3, EphB2, and EphA7 were kindly provided by B. Hock and K. Strebhardt (Georg-Speyer-Haus, Frankfurt, Germany [anti-EphB3]), S. Holland (Samuel Lunefeld Research Institute, Ontario, Canada [anti-EphB2]), and A. Ullrich (Max-Planck-Institute for Biochemistry, Martinsried, Germany [anti-EphA7]). All antibodies directed against the different Eph receptors and the Ephrin-B1 ligand recognize the cytoplasmic domain of their protein targets.
Immunofluorescence microscopy of 293T cells was performed as follows. Cells were cultured on a coverglass and fixed with 3% paraformaldehyde in PBS for 20 min at room temperature (RT). The fixed sample
was then treated with 0.5% Triton X-100 in PBS for 90 s at RT and
washed three times with PBS. Samples were then incubated with the primary antibodies for 1 h at 4°C in 5% newborn goat serum/PBS. Samples
were then washed and incubated with either rhodamine-conjugated anti-rabbit or anti-mouse IgG or FITC-conjugated anti-mouse IgG antibodies
for 1 h at 4°C. Samples were then washed again and embedded in Mowiol
(Hoechst) and examined with a confocal laser scanning microscope
equipped with an Ar/Kr laser (model TCS4D; Leica). Bleed-through between the green and the red channel was completely blocked by adjusting
the intensity of the laser beam with an acousto-optical (Leica). Images
were further processed and merged with the Imaris software (Bitplane)
and finally printed with a Fudjix Pictrography 3000 (Fuji). For MDCK
cells, samples were fixed and permeabilized in 70% methanol/30% acetone at 20°C for 20 min. Samples were washed three times with PBS and
then treated as described for 293T cells.
Animals and Tissue Preparation
Oncins France strain A (OFA) line rats (180-200 g; RCC) were used in this study. Environmental conditions for housing of the rats and all procedures that were performed on them were in accordance with animal research licences granted by the Cantonal Veterinary Authority of Zürich and followed the Codes of Practice established by the University Veterinary officers. 10 rats were deeply anesthetized with halothane and then perfused transcardially with 30 ml of 0.9% NaCl, followed by 500 ml of fixative containing 3-4% paraformaldehyde and 0.2% picric in 0.1 M phosphate buffer (PB), pH 7.4, at a rate of ~18 ml/min. The brain from eight rats was removed into cold (4°C) PB. Coronal sections throughout brain were cut on a vibrating microtome at 60-70 µm. To enhance the penetration of the immunoreagents, the sections were equilibrated in 30% sucrose in PB, rapidly frozen in liquid nitrogen, and then thawed in PB. The brain from two rats was postfixed in 0.1% glutaraldehyde containing fixative for 2 h and blocked for cryo-ultramicrotomy. Tissue blocks, 0.8-mm thick, were transferred into 2 M sucrose containing 15% polyvinyl pyrrolidone solution for cryoprotection.
Immunohistochemistry
The sections were preincubated in 10% normal goat serum (NGS) in 0.05 M TBS, pH 7.4, for 45 min and then incubated in primary antibody solutions in TBS that was supplemented with 2% NGS and 2% BSA for 36-48 h at 4°C with constant gentle shaking. They were washed four times for 20 min in 1% NGS in TBS and then incubated in biotinylated goat anti-mouse or anti-rabbit IgG (1:200, Vector Labs) for 12 h at 4°C followed by 3 h of incubation in an avidin-biotin-peroxidase complex (1:100, Elite ABC; Vector Labs) at RT. Antigenic sites were visualized by incubation in 3,3'-diaminobenzidine (0.05% in TBS, pH 7.6; ) in the presence of 0.0048% H2O2. The reaction was stopped by several washes in TBS. Immunotreated sections were postfixed in a solution of 1% osmium tetroxide (Oxchem) in phosphate buffer, pH 7.4, for 45 min, stained with 2% uranyl acetate for 2 h, dehydrated, and flat embedded into Durcupan ACM (Fluka). The sections were examined first in Leica DMR light microscope. Areas of interest were cut from the slide and reembedded. Serial thin sections were collected on pioloform-coated copper grids, and examined in a Philips CM100 electron microscope. The specificity of the immunolabeling was proven by the absence of staining when the primary antibodies were omitted.
Immunogold Staining on Cryo-ultrathin Sections
Cryo-ultrathin sections, 90-nm thick, were cut at 120°C, picked up on pioloform-coated nickel grids, and then processed according to Tokuyasu
(1986)
(for review see Liou et al., 1996
). In brief, sections were preincubated on drops of 5% NGS in PBS for 30 min at RT, incubated either in
one of the primary antibodies or in the mixture of anti-AF-6 and one of
the anti-Eph receptor antibodies (1:200) in PBS containing 1% BSA overnight at 4°C. Gold-conjugated secondary antibodies (5-, 10-, and 15-nm
gold-conjugated anti-mouse and anti-rabbit IgG [Nanoprobes]; 8-nm anti-rabbit IgG provided by B. Guhl [University of Zürich, Zürich, Switzerland]; 1:50) or mixture of anti-mouse and anti-rabbit IgGs, conjugated to
different size gold particles, for double labeling were applied for 2 h at
RT. Then sections were postfixed in 1% glutaraldehyde in PBS and embedded in 2% methylcellulose and 3% uranyl acetate mixture (9:1).
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Results |
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AF-6 Interacts with Specific Eph Receptors
PDZ domains specifically recognize the COOH-terminal
ends of target proteins, commonly XS/TXV-COOH (Saras
and Heldin, 1996). The PDZ domain has been shown to
link diverse membrane proteins with effector molecules
inducing receptor clustering and to serve as a scaffold
to assemble different components of a signaling cascade
(Tsunoda et al., 1997
). Different PDZ domains have been demonstrated to have distinct COOH-terminal peptide
binding specificities (Saras and Heldin, 1996
; Songyang et al.,
1997
). We surmized that the PDZ domain of AF-6 might
be able to recognize the COOH-terminal regions of
membrane-associated proteins. Therefore, the GenBank/ EMBL/DDBJ database was searched for membrane-spanning molecules having potential PDZ domain-binding
COOH termini. More than 20 different receptors broadly
classified among the large RTK family had COOH-terminal ends potentially able to interact with PDZ domains. Fusion proteins consisting of the DNA-binding domain
(DBD) and COOH-terminal peptides, encompassing the
last 10 amino acids of these various receptors, were constructed and tested in a mammalian two-hybrid interaction
assay for their ability to interact with the PDZ domain of
AF-6. The results are depicted in Fig. 1, a and b. Of all
the different receptors analyzed, only a specific subset of
members of the Eph subfamily of RTKs were able to
interact with the AF-6 PDZ domain. EphA6 (Ehk2), (Ciossek et al., 1995
), EphA7 (Mdk1), (Ciossek et al., 1995
),
EphB2 (Nuk) (Henkemeyer et al., 1994
), and EphB3 (Hek2)
(Bohme et al., 1993
) all activated the reporter gene between 10- and 60-fold over background levels and EphB6
(Hep) (Matsuoka et al., 1997
) activated sixfold over background levels.
Interaction of the AF-6 PDZ Domain with Recombinant Eph Receptors
The binding specificities observed in the mammalian cell
two-hybrid assays with the PDZ domain of AF-6 and the
peptide ligands were verified with recombinant receptors
in vitro. cDNAs encoding full-length EphB2, EphA7, and
EphB3 were expressed in 293T cells and their ability to
immunoprecipitate with a FLAG epitope-tagged AF-6
PDZ domain construct (FLAG AF-6 PDZ) was analyzed.
Fig. 2 a indicates that both the full-length EphB2 and
EphA7 receptors, when cotransfected into 293T cells with
FLAG AF-6 PDZ, specifically coimmunoprecipitate with
the FLAG-tagged AF-6 PDZ domain fusion protein
whereas no receptor immunoprecipitates with the FLAG-tagged vector alone (Fig. 2 a, FLAG, compare lanes 3 and
4 and lanes 7 and 8). As a further control the Ephrin-B1
(Xlerk2) (Jones et al., 1997) receptor ligand, which has an
intracellular COOH terminus characteristic of PDZ-binding peptides but which our mammalian two-hybrid analysis had indicated did not interact with the PDZ domain of
AF-6, was transfected. When the Ephrin-B1 cDNA expression vector was cotransfected with either FLAG AF-6
PDZ or the empty vector (FLAG), no Ephrin-B1 protein
coimmunoprecipitated with the FLAG-tagged fusion proteins, indicating the specificity of the interaction of the
AF-6 PDZ domain for only certain Eph receptors (Fig. 2
a, compare lanes 10-12).
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In further experiments, a GST AF-6 PDZ domain fusion protein was coupled to glutathione beads and mixed with 293T cellular extracts expressing GFP-tagged receptor COOH termini peptides or the full-length EphB3 receptor to determine whether specific binding to the AF-6 PDZ domain was observed. Similarly a GFP-EphB3 COOH-terminal peptide containing the last ten amino acids (designated as EphB3pep) as well as the full-length EphB3 receptor were specifically retained by GST AF-6 PDZ-coupled beads but not by GST-coupled control beads (Fig. 2 b, compare lanes 1-3 and lanes 4-6). Mutation of the very COOH-terminal valine residue in the full-length EphB3 receptor to an alanine residue abolished binding of the EphB3 receptor (EphB3ala) to FLAG AF-6 PDZ (Fig. 2 b, compare lanes 7-9).
Colocalization of AF-6 and Eph Receptors at the Membrane in Transfected Cells
To observe a possible direct interaction between AF-6 and these RTK proteins, a plasmid was constructed coding for a full-length AF-6 fused to GFP at its NH2 terminus, and cotransfected with various full-length receptors in 293T cells. The GFP-tagged AF-6 protein gave a generally diffuse fluorescent signal throughout the cytoplasm and nucleus when transfected alone (data not shown). However, when cotransfected with full-length EphB2, EphA7, or EphB3 RTKs (Fig. 3, a1-d1), a dramatic shift in the subcellular localization of the GFP-tagged AF-6 protein occurred with a strong signal enriched at the cellular membrane, often at sites of cell-cell contact (Fig. 3, a2-d2, compare with e2). Costaining for the transfected receptors in these cells with a rhodamine-coupled antibody specific for the primary anti-receptor antibodies revealed that the GFP AF-6 signal colocalized with the membrane-associated receptor signals. As a control Ephrin-B1 and GFP AF-6 were transfected. Even though the Ephrin-B1 signal was enriched at the plasma membrane there was no colocalization of the GFP AF-6 signal and Ephrin-B1, with the GFP AF-6 signal diffusely spread throughout the cotransfected cells (Fig. 3, e1 and e2). This is consistent with the result that AF-6 is unable to associate with the COOH-terminal peptide of Ephrin-B1 in a mammalian two-hybrid experiment (Fig. 1).
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Transfected Eph Receptors Colocalize with Endogenous AF-6 in MDCK Cells
It has recently been reported that AF-6 is localized at the
plasma membrane at specialized sites of cell-cell contact.
In the polarized epithelial cell line MDCKII, AF-6 has
been shown to colocalize with the tight junction-associated
protein ZO-1 at apical lateral membrane sites of cell-cell
contact (Yamamoto et al., 1997). In fibroblasts, AF-6 has
also been shown to colocalize with ZO-1 at cadherin-based, spot-like cell-cell adherens junction (Mandai et al.,
1997
). To determine the effect of exogenous Eph receptors on the subcellular localization of endogenous AF-6, EphA7 was transfected into MDCK cells. This caused an
enrichment of the endogenous AF-6 protein at sites of
cell-cell contact (Fig. 4, a-f), indicating that Eph receptors
can be a target for the endogenous AF-6 protein. The
colocalization and enrichment of endogenous AF-6 with
EphA7 seems to occur only at their extreme apical positions (Fig. 4, compare e with f). This could mean that endogenous AF-6 can not freely diffuse along the lateral
membrane, whereas the transfected and overexpressed receptor can do so. Alternatively, the interaction may require additional factors only present at extreme apical positions.
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AF-6 and Eph Receptors Cocluster at Postsynaptic Specializations in the Rat Brain Hippocampus
Immunocytochemical techniques with light and electron
microscopic analyses were combined in order to determine whether AF-6 colocalizes with Eph receptors in rat
brain. In addition, the localization of ZO-1, a protein
which is found together with AF-6 at tight junctions in polarized epithelial cells and at cadherin-based cell-cell junctions in fibroblasts (Mandai et al., 1997; Yamamoto et al.,
1997
), was examined. At low magnification, we observed overlapping spatial distributions in the immunolabeling
patterns of AF-6, ZO-1, EphB2, EphA7, and EphB3.
Strong immunoreactivity occurred in a variety of regions
with high synaptic density, specifically the hippocampus,
caudate putamen, cerebellum, superficial gray layer of superior colliculus, external cortex of inferior colliculus, medial mammillary nucleus, sensory nuclei of thalamus, and
neocortex (data not shown). Because the architecture and
function of hippocampal circuitry is well known (Johnston
and Amaral, 1998
), we focused our examination on the
subcellular localization of AF-6, ZO-1, and Eph receptors
in hippocampal regions. The strong immunolabeling of
AF-6, ZO-1, and Eph receptors was found in all dendritic
layers, strata oriens and radiatum of CA1 and CA3 regions, and the dentate moleculare of the hippocampus
(data not shown). Electron microscopic analysis revealed
the presence of immunoperoxidase reaction products at
the majority of asymmetric axo-spinous and axo-dendritic
synapses in stratum radiatum of the CA1 region (Fig. 5 a,
c-e). Immunogold labeling of cryo-ultrathin sections of
CA1 have shown that AF-6 (Fig. 6 a) and Eph receptors
(Fig. 6, b and c) are localized at postsynaptic densities.
Further examination of double-immunogold staining revealed colocalization of AF-6 and Eph receptors over postsynaptic specializations of excitatory synapses in CA1
(Fig. 6, d-f).
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Immunoreaction products for ZO-1, in contrast, accumulated mainly in presynaptic axonal terminals that
formed asymmetric synapses with either dendritic spines
of pyramidal neurons (Fig. 5 b) or dendritic shafts of inhibitory interneurons. ZO-1 immunoreactivity over postsynaptic densities was rarely observed. The different localization of AF-6 and ZO-1 in the rat hippocampus contrasts with the reported interaction of these two proteins at tight
junctions in polarized epithelial cells (Yamamoto et al.,
1997). This implies that at least in the case of a specialized
cell-cell contact, such as the synapse, AF-6 and ZO-1 do
not interact with each other and might fulfill different roles.
AF-6 Is an In Vivo Substrate of Eph Receptors
To obtain some information on the biological significance of the interaction between AF-6 and Eph receptors, especially whether AF-6 is a substrate of the Eph receptor tyrosine kinases, we transiently transfected 293T cells with AF-6 along with either wild-type EphB3, EphB2, or a kinase-deficient mutant EphB3 receptor (EphB3K665R). The result shown in Fig. 7 a demonstrates that AF-6 is tyrosine phosphorylated only when coexpressed with wild-type EphB2 or EphB3 receptors. No phosphorylation occurred with a kinase-negative mutant receptor. Phosphorylation of AF-6 is dependent on previous activation of the receptor which is achieved by overexpression in 293T cells (Fig. 7 a and data not shown). When transfected AF-6 is immunoprecipitated from 293T cells, strong phosphorylation of the immunoprecipitated AF-6 is detected when it is cotransfected with the wild-type EphB2 and EphB3 receptors, whereas AF-6 is not phosphorylated when transfected alone or with the kinase-negative mutant EphB3K665R (Fig. 7 e, top). Taken together, these results demonstrate that AF-6 is phosphorylated by the EphB2 and EphB3 receptors in a kinase-dependent fashion and demonstrates that AF-6 is a substrate for a subgroup of Eph receptors.
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EphB2 and EphB3 Coimmunoprecipitate AF-6 from Whole Rat Brain Lysates
Finally, to confirm that these findings are also biologically relevant in the living organism, we tested whether complexes of endogenous AF-6 with Eph receptors occur in total rat brain lysates. Indeed, endogenous AF-6 coimmunoprecipitated with both EphB2 and EphB3, but not when the total rat brain extract was immunoprecipitated with an anti-FLAG M2 antibody (Fig. 8, a and b, left). The pellet, obtained after the first extraction, was reextracted with the stronger RIPA buffer, known to disrupt protein-protein interactions. This treatment destroyed the interaction even though endogenous AF-6 was still precipitable (Fig. 8, a and b, right). This further demonstrates the specificity of the interaction and suggest a physiological role for the interaction between AF-6 and Eph receptors in the brain.
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Discussion |
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At the sites of cell-cell contact of epithelial or endothelial
cells, known as the tight junction or zonula occludens,
transmembrane proteins have been identified to form
complexes with ZO-1, ZO-2, ZO-3, and AF-6. A characteristic feature of these proteins is the presence of at least
one and more often several PDZ domains. The name PDZ
is based on the three proteins with such domains, PSD-95,
DlgA, and ZO-1. PDZ domains can mediate specific interactions to the COOH-terminal regions of target proteins
and many appear to be involved in the clustering or docking of integral membrane proteins to particular sites of
membrane specialization (Fanning and Anderson, 1996).
The clustering function of PDZ domain-containing proteins has been most clearly demonstrated for a family of
synapse-associated proteins (SAPs) which are thought to
play a central role in the molecular organization of synapses (Garner and Kindler, 1996
; Sheng, 1996
).
Since potential PDZ-containing receptor targets of tight
junction-associated transmembrane proteins have not been
identified, we screened the GenBank/EMBL/DDBJ database for receptor proteins having potential PDZ domain-binding COOH-terminal ends. We noticed that a significant number of receptors in the RTK family have potential
PDZ domain-binding COOH-termini (XS/TXV-COOH),
suggesting that they might interact with as yet unidentified
PDZ domain-containing juxtamembrane proteins. These
termini were analyzed for their ability to bind to the PDZ
domain of AF-6. Using a mammalian cell-based two-hybrid assay we determined that the AF-6 PDZ domain
was able to interact with specific members of the Eph subfamily of RTKs but not with any of the other receptors
tested. Of the 14 different members of the Eph RTK subfamily (the largest subfamily of RTKs) eight of them have
COOH termini, suggesting they might interact with a PDZ
domain-containing protein. Of the eight receptors tested here, five appear to interact with AF-6. Comparing the
peptide sequences of receptors able to bind to the PDZ
domain of AF-6 (Fig. 1 b, boldface) with each other, indicated that there seems to be a preference for a hydrophobic residue at the 2 position of the binding peptide as
well as for a valine at the 0 position. Thus, the potential
consensus sequence of COOH termini able to bind to AF-6
PDZ domain is XV/IXV-COOH. However, the EphA3
receptor peptide exhibited no binding to the AF-6 PDZ
domain even though it conformed to this consensus
(PVPV-COOH), showing that residues at positions other
than
2 and 0 are also important for binding. In a recent
publication, Songyang et al. (1997)
selected artificial peptides on the basis of binding to an immobilized GST-AF-6
PDZ domain fusion protein. However, their selected peptide sequences bear little relationship to the Eph receptor
COOH termini able to bind to AF-6. This may indicates
that the PDZ domain of AF-6 is a very flexible interaction
domain, with the potential to bind to different target sequences.
Our transient transfection studies demonstrated that
AF-6 is specifically recruited to sites of cell-cell contact
via interaction with its cognate Eph receptor binding partners in heterologous 293T and MDCK cells. If AF-6 has a
role in clustering Eph receptors at sites of membrane specialization, the endogenous proteins should colocalize in
normal tissues. Additionally, the findings that Eph family
receptors can be clustered at sites of cell-cell contact in
the brain (Henkemeyer et al., 1994), lead us to analyze the expression patterns of the three AF-6 interacting Eph receptors and that of AF-6 in brain sections from adult rats.
A very tight colocalization was observed. This was particularly the case at postsynaptic membranes of excitatory synapses in the hippocampus, indicating that the observed interaction between these receptors and AF-6 may have
physiological consequences. Interestingly, ZO-1 which in
polarized epithelial cells tightly colocalizes with AF-6 at
the lateral apical membrane, exhibited an expression pattern with marked staining of presynaptic membrane specializations in the brain. In contrast, we found AF-6 and
the Eph receptors to colocalize at the postsynaptic membrane, as demonstrated by double-immunogold labeling
experiments, reinforcing the view that AF-6 may be involved in the clustering of these receptors.
The clustering of AF-6 and certain Eph receptors at
postsynaptic densities may be analogous to that observed
with a family of SAPs and ionotropic receptors at the same
membrane location. SAPs such as SAP97, SAP90/PSD-95,
SAP102, and Chapsyn110/PSD-93 contain three PDZ domains which mediate the interactions with the COOH-terminal ends of the voltage-gated K+ channel and the NR2
subunits of the NMDA receptor (Sheng, 1996). These interactions appear to mediate the clustering of these ion channels at synaptic membranes. AF-6 may also be involved in mediating the clustering of the Eph receptors at
postsynaptic sites. In addition, like AF-6, SAP97 has also
been localized to the lateral membrane between epithelial
cells (Mueller et al., 1995
), hence, these two proteins may
also be involved in the clustering of receptors at other sites
of cell-cell contact than just at synaptic membranes.
Our demonstration that AF-6 is an in vivo substrate for
the kinase activity of a subgroup of the family of Eph receptors is in agreement with recent data (Hock et al.,
1998) and supports a functional role of the Ras-binding
protein AF-6 in the signal transduction mediated by Eph
receptors. Moreover, endogenous AF-6 physically interacts with the two Eph receptors EphB2 and EphB3 in the
adult rat brain. This is especially intriguing, considering the fact that little is known about the function of the Eph
receptors in an adult organism. Most of the data on the
role of Eph receptors stems from observations made during embryonic development of the nervous system where
these receptors are involved in axonal pathway selection,
guidance of cell migration, and establishment of regional patterns. Therefore, our data would complement the distinct function of certain Eph receptors during embryogenesis and suggest an additional role of this class of RTKs in
the developed nervous system.
It is of interest to note that the postsynaptic localization
of the Eph receptors overlaps substantially with that observed for glutamate NMDA receptors. Of the three major classes of ionotropic glutamate receptors, the AMPA,
kainate, and NMDA receptors, it is the latter which has
been found to have a role in long-term potentiation (LTP),
excitotoxicity, and synaptic development (Choi and Rothman, 1990; Bliss and Collingridge, 1993
). The properties of
the NMDA receptor are modulated by serine/threonine
phosphorylation (Chen and Huang, 1992
; Wang and
Salter, 1994
). Recently it has also been demonstrated that
tyrosine phosphorylation of the NR2B and NR2A subunits of the NMDA receptor may regulate the properties of the NMDA receptor channel (Wang and Salter, 1994
;
Lau and Huganir, 1995
; Wang et al., 1996
; Yu et al., 1997
).
The tyrosine phosphorylation status of NMDA receptor
subunits changes upon induction of LTP (Rosenblum et
al., 1996
), suggesting that regulation of the tyrosine phosphorylation of the NMDA receptor may play an important
role in neuronal modulation. The identity of the kinase(s)
which phosphorylate ionotropic receptor channels such as
the NMDA receptor at the postsynaptic density have yet
to be identified. The colocalization of some of the Eph receptors with that of NMDA receptors at the postsynaptic density suggests these Eph receptors may be candidates for kinases able to influence the phosphorylation
status of other postsynaptic density associated proteins,
such as the NMDA receptor. Such a role would provide a
rationale for the observed clustering of these receptors
and AF-6 at synaptic sites in the adult.
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Footnotes |
---|
Address correspondence to K. Moelling, Institut für Medizinische Virologie, Universität Zürich, Gloriastrasse 30/32, CH-8028 Zürich, Switzerland. Tel.: (41) 1 634 2652. Fax: (41) 1 634 4967. E-mail: moelling{at}immv.unizh.ch
Received for publication 24 June 1998 and in revised form 4 November 1998.
C.M. Hovens's present address is Dept. of Surgery, Royal Melbourne
Hospital, Clinical Sciences Building, Parkville VIC. 3050 Australia.
We are grateful to I. Daar at the National Cancer Institute (Frederick, MD) for providing the Ephrin-B1 clone. We thank B. Hock and K. Strebhardt (both from the Georg-Speyer-Haus, Frankfurt, Germany) for the EphB3 cDNA and EphB3 antibody; S. Holland (Samuel Lunenfeld Research Institute, Ontario, Canada) for the EphB2 cDNA and EphB2 antibody; A. Ullrich (Max-Planck-Institute for Biochemistry, Martinsried, Germany) for the EphA7 cDNA and EphA7 antibody; A. Hajnal (University of Zürich, Zürich, Switzerland) for MDCK cells and stimulating discussions; B. Guhl for advice with cryomicrotomy and tissue processing; and T. Hoechli (Elektronenmikroskopisches Zentrallabor, Zürich, Switzerland) for expert computer assistance.
This work was supported by grants from the Swiss National Science Foundation (31-43779.95 and 24.6317) and the Swiss Cancer League.
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Abbreviations used in this paper |
---|
aa, amino acid; FLAG AF-6 PDZ, FLAG epitope-tagged AF-6 PDZ domain construct; GFP, green fluorescent protein; GST, glutathione-S-transferase; MAGUK, membrane-associated guanylate kinase; NGS, normal goat serum; NMDA, N-methyl-D-aspartate; NR2, NMDA receptor 2; PB, phosphate buffer; RIPA, radioimmunoprecipitation assay; RT, room temperature; RTK, receptor tyrosine kinase; SAP, synapse-associated protein.
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