Selectivity and Promiscuity of the First and Second PDZ
Domains of PSD-95 and Synapse-associated Protein 102*
Indra Adi
Lim
,
Duane D.
Hall§, and
Johannes W.
Hell¶
From the Department of Pharmacology, University of Wisconsin,
Madison, Wisconsin 53706-1532 and the Department of Pharmacology,
University of Iowa, Iowa City, Iowa 52242-1109
Received for publication, December 22, 2001, and in revised form, March 12, 2002
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ABSTRACT |
PDZ domains typically interact with the
very carboxyl terminus of their binding partners. Type 1 PDZ
domains usually require valine, leucine, or isoleucine at the very
COOH-terminal (P0) position, and serine or threonine
2 residues upstream at P
2. We quantitatively defined the
contributions of carboxyl-terminal residues to binding selectivity of
the prototypic interactions of the PDZ domains of postsynaptic density
protein 95 (PSD-95) and its homolog synapse-associated protein
90 (SAP102) with the NR2b subunit of the
N-methyl-D-aspartate-type glutamate receptor. Our studies indicate that all of the last five residues of NR2b contribute to the binding selectivity. Prominent were a requirement for
glutamate or glutamine at P3 and for valine at
P0 for high affinity binding and a preference for threonine
over serine at P2, in the context of the last 11 residues
of the NR2b COOH terminus. This analysis predicts a COOH-terminal
(E/Q)(S/T)XV consensus sequence for the strongest binding
to the first two PDZ domains of PSD-95 and SAP102. A search of the
human genome sequences for proteins with a COOH-terminal
(E/Q)(S/T)XV motif yielded 50 proteins, many of which have
not been previously identified as PSD-95 or SAP102 binding partners.
Two of these proteins, brain-specific angiogenesis inhibitor 1 and
protein kinase C
, co-immunoprecipitated with PSD-95 and SAP102 from
rat brain extracts.
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INTRODUCTION |
Postsynaptic density protein 95 (PSD-95)1 (1), also known as
synapse-associated protein 90 (SAP90) (2), is a neuronal protein that
is specifically enriched at the postsynaptic densities of dendritic
spines (3). PSD-95 is a member of a family of proteins that includes
SAP102 (4), SAP97 (hDlg) (5, 6), and PSD-93 (Chapsyn-110) (7, 8). All
members of this family share the same general structure: three
PSD-95/Dlg/Zo-1 (PDZ) domains, one Src homology 3 domain, and one
guanylate kinase homology domain. All of these domains mediate
protein-protein interactions. Therefore, PSD-95 and related proteins
are thought to function as structural scaffolds that assemble protein
complexes, thereby facilitating signal transduction. NR2 subunits of
the NMDA-type glutamate receptor (9) and Shaker-type
K+ channels (10) were the first proteins described to bind
to PSD-95. These interactions occur via the first two PDZ domains of
PSD-95. Additional neuronal proteins that have subsequently been
characterized as interacting with the PDZ domains of PSD-95 include
neuronal nitric-oxide synthase (7, 11), CRIPT (12), SynGAP (13, 14),
Citron (15), and two isoforms of the plasma membrane Ca2+
pump (PMCA2b and -4b) (16) (see Table
I). In addition, a number of novel
PDZ domain-containing proteins have been described that are also
localized to the synapse, including MAGI-1 (17), S-SCAM (MAGI-2) (18),
GRIP (19), ABP (GRIP2) (20), CASK (LIN-2) (21), and PICK1 (22).
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Table I
Known PSD-95-associated proteins
Proteins that have been observed to associate with the PDZ domains of
PSD-95 (as well as related proteins that do not associate with PSD-95
PDZ domains) are listed along with their COOH-terminal PDZ-domain
binding consensus sequence. Residues highlighted in boldface indicate
optimal residues for that position for binding to PDZ1 and -2 of
PSD-95. Relative affinities (for binding different PDZ domains or
relative to similar proteins) are also listed. These are denoted by
(+), +, or ++ in order from weakly to strongly binding. See Discussion
for full details and citations. ND, not determined. Nedasin was not
tested for PSD-95 binding; given are interactions with SAP102 (last
line).
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PDZ domains are unique among protein-protein interaction domains,
because the proteins that bind to PDZ domains generally do so by their
very COOH-terminal residues. The subunits of the Shaker K+
channel and NMDA receptor that bind to the first two PDZ domains of
PSD-95, SAP102, and PSD-93 have the sequence E(S/T)DV-COOH. The
co-crystal structure of the third PDZ domain of PSD-95 and a peptide
ligand derived from the very COOH terminus of CRIPT (TKNYKQTSV-COOH)
has been solved (23). This work showed that the very COOH-terminal
valine and the threonine two positions upstream (the 0- and
2-positions P0 and P2, respectively) form
crucial interactions in the PDZ domain binding pocket. Thus, the
minimal consensus sequence for binding to the PSD-95 PDZ domains has
been defined as an (S/T)XV motif, where X can
represent any residue. However, it is clear that other residues must
contribute to specificity for a given PDZ domain, because various
proteins and ion channels that have an (S/T)XV motif do not
bind any of the PDZ domains of PSD-95 under conditions under which the
other ligands do. Examples include the neuronal inwardly rectifying
K+ channels Kir3.2 and Kir3.3 (COOH-terminal sequence is in
both cases ESKV (24)); the Na+ channel Nav1.5
(ESIV (25)), which is present not only in muscle but also brain (26);
and diacylglycerol kinase 
(ETAV (27)). Furthermore, the
1 adrenergic receptor does not interact with the first
two PDZ domains of PSD-95 although it does conform to the
(S/T)XV motif (SKV (28)). This receptor does, however, bind to the third PDZ domain of PSD-95 (28). This PDZ domain possesses a
binding preference that is quite different from the first two PDZ
domains (e.g. see Ref. 12) of PSD-95 and does not interact with NR2 subunits (9, 29) or Shaker-type K+ channels (10).
Similarly, Neuroligin carries the sequence TRV at its COOH terminus but
only interacts with the third and not the first two PDZ domains of
PSD-95 (30).
PDZ domains can be broadly divided into several categories, based on
their general ligand specificity. Type I PDZ domains, found on PSD-95
and its homologs, bind (S/T)X(V/I/L) COOH termini, whereas
type II PDZ domains (e.g. the fourth and fifth PDZ domain of
GRIP and the PDZ domain of CASK), bind an 
-X- motif, where represents a hydrophobic residue (preferably tyrosine or phenylalanine at P2) (31, 32). A third type of PDZ domain present in
neuronal nitric-oxide synthase shows a preference for aspartate at
P2 (DXV motif) (33, 34), although it also
accepts other residues at P2 (e.g. isoleucine
(35)). Additionally, another kind of binding has been described and is
exemplified by an internal (non COOH-terminal) sequence in neuronal
nitric-oxide synthase that binds to syntrophin's PDZ domain and the
second PDZ domain of PSD-95 (7, 36).
The interaction between the NMDA receptor NR2b subunits and PSD-95 is
one of the best defined PDZ domain interactions. The NMDA receptor is
critical for a number of neuronal functions such as synaptic
transmission and synaptic plasticity. PSD-95 and its homologs may be
crucial for the assembly of postsynaptic proteins and signal
transduction involving the NMDA receptor (8, 13, 37, 38). Therefore, we
evaluated the role of the various residues at the very COOH terminus of
the NMDA receptor in determining the affinity and specificity of its
interaction with PSD-95 and its relative, SAP102.
Various combinatorial methods have been employed to study the
specificity of PDZ domains. The yeast two-hybrid method has been used
extensively in the characterization of PDZ domains and their
selectivity. But although it is a powerful and sensitive assay, it is
prone to give a high background of false positives and is only useful
for a qualitative assessment of binding. Precise affinity
determinations are not possible. Oriented peptide libraries have been
used to determine the consensus binding sequence for a variety of PDZ
domains (32). Different PDZ domains were immobilized and incubated with
batches of solubilized peptide libraries. Peptides retained by the PDZ
domains were sequenced, and the frequency of residues at each position
was quantified. An increase in the probability for a given residue at a
certain position by a factor of 1.5 was considered to be significant.
This method can reveal preferences of the various PDZ domains for
certain residues at a given position but does not allow quantitative
comparisons of affinities of defined peptides. Because this method does
not determine actual sequences of any of the PDZ domain-binding
peptides, it does not permit judgment of whether a preference for a
residue at one position is linked to the presence of another residue at another position. Furthermore, the method used by Songyang et al. (32) is best suited for detecting preferences critical at a
given position for high affinity binding. However, it would be
difficult if not impossible to determine with this method whether certain residues are forbidden at a given position if this position accepts most of the other residues. Most recently, a COOH-terminal phage display has been used to explore the specificity of the seven PDZ
domains of the human homologue of InaD (39). This approach identified
putative consensus binding sequences for each PDZ domain, although many
interactions may have been of low affinity, perhaps with
KD values above 10 µM, because the
high display density on the phage surface may have resulted in avidity effects (39). As for the other methods, it is not possible to determine
affinities of the phage-displayed peptides and compare the precise
contribution of each residue.
To understand how each position in the PDZ binding motif may contribute
to the affinity for the PDZ domain, we used several different libraries
of selected peptides based on the COOH terminus of the NR2b subunit of
the NMDA receptor to identify the important positions and residues
involved in the binding of NR2b to the PDZ domains of PSD-95 and
SAP102. The use of solid-phase colorimetric and in-solution
fluorescence polarimetric peptide binding assays allowed us to
quantitatively determine the effects of amino acid substitutions at
various positions along the peptide sequence on PDZ domain binding. We
were surprised by a strong, often severalfold preference of the first
two PDZ domains of PSD-95 and of SAP102 for valine at the 0-position
over the related amino acids leucine and isoleucine, for threonine at
the 2-position over serine, and for glutamate and glutamine at the
3-position over aspartate. Analysis of the human genome indicated
that besides NMDA receptor NR2 subunits and K+ channels of
the Shaker-type Kv1 family, only a limited number of other neuronal
proteins possess COOH termini that would predict a strong interaction
with the first two PDZ domains of PSD-95 or SAP102. Despite the large
number of proteins with COOH-terminal (S/T)XV motifs that
can potentially bind to PDZ domains, our analysis indicates a clear
selectivity of specific PDZ domains for a limited number of proteins.
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EXPERIMENTAL PROCEDURES |
Production of GST Fusion Proteins--
NH2-terminal
GST fusion proteins of the second PDZ domain of rat PSD-95 (residues
154-248) and those carrying residues 142-235 (PDZ1), 237-333 (PDZ2),
and 399-485 (PDZ3) of rat SAP102 were expressed from a modified pGEX2T
vector named pGHEB and kindly provided by Dr. Craig C. Garner
(University of Alabama, Birmingham, AL). To create vectors for the
expression of GST fusion proteins of the first and third PDZ domain of
PSD-95, cDNA sequences encoding human PSD-95 residues 82-202
(PDZ1) and 344-443 (PDZ3; residue numbers refer to full-length human
PSD-95 sequence as given in Ref. 40) were excised with EcoRI
from corresponding GAD10 vectors, which contained the respective
sequences that had originally been inserted into the GAD10
EcoRI cloning sites (10) (kindly provided by Morgan Sheng,
MIT, Cambridge, MA). The latter two protein sequences are completely
identical to residues 39-159 and 301-400 encoding the first and third
PDZ domain of rat PSD-95, respectively. DNA fragments were purified by
agarose gel electrophoresis and ligated into EcoRI-digested
pGEX 4T-1 (Amersham Biosciences) before screening the resulting clones
for correct orientation of the inserts. All vectors were confirmed by
DNA sequencing with the AmpliTaq system (PerkinElmer Life Sciences).
Fusion proteins were expressed and purified as described (41, 42) with
the following modifications. The vector DNA was electroporated into the
E. coli strain BL21 
-DE3 cells. 50-ml cultures were grown
overnight, diluted 1:10 with LB medium, and incubated until the
cultures reached an A600 of 1.0 (2-4 h). Fusion
protein expression was induced with
isopropyl--D-thiogalactopyranoside (100 µM, 2 h). Cells were harvested; resuspended in TBS
(10 mM Tris-Cl, pH 7.4, 150 mM NaCl); digested
with lysozyme; supplemented with 10 mM EDTA, 15 mM dithiothreitol, and protease inhibitors (0.2 mM phenylmethanesulfonyl fluoride, 1 µg/ml pepstatin A,
0.1 µg/ml leupeptin, 0.1 µg/ml aprotinin); and lysed by adding
1.5% (final concentration) sarkosyl (43), followed by sonication. Lysates were clarified by ultracentrifugation (45-Ti rotor, 40,000 rpm,
186,000 × g, 1 h). Sarkosyl was neutralized by
adding 2-4% (final concentration) Triton X-100 from a 20% stock
solution. Lysates were incubated with glutathione-Sepharose (Amersham
Biosciences) overnight at 4 °C. Resins were washed with TBS and
eluted by adding 15 mM glutathione in 150 mM
NaCl, 50 mM Tris-Cl, pH 8. The fusion proteins were
dialyzed against TBS and subsequently quantified by determining
A280 and, in parallel, by the bicinchoninic acid assay (Pierce) and a Coomassie assay (Pierce) in microtiter plate format. The purity and quality of all fusion proteins was evaluated by
SDS-polyacrylamide gel electrophoresis and detection by staining with
Coomassie Brilliant Blue and by immunoblotting with anti-GST antibodies
to ensure minimal degradation. Purified GST fusion proteins usually did
not show contamination with other proteins by Coomassie Brilliant Blue
staining and were discarded otherwise.
Peptide Synthesis--
Peptides based on the NR2b COOH terminus
and the COOH termini of other potential PDZ ligands were synthesized
manually using a Multipin Peptide Synthesis kit according to the
manufacturer's instructions (Chiron-Mimotopes) (44). This method
utilizes standard solid phase peptide synthesis with Fmoc-protected
amino acids, followed by deprotection and cleavage of the peptide from
the solid-phase support with trifluoroacetic acid. We typically
synthesized peptides using a 96-well plate array of polystyrene pins
serving as the solid phase support. After cleavage, peptides were
cleaned with two ether/petroleum ether extractions to remove leftover protecting groups. The remaining trifluoroacetic acid was eliminated by
dissolving the peptide in water/acetic acid/acetonitrile followed by
lyophilization. Peptides were dissolved in water and stored frozen
until used. All peptides contained at their NH2 termini a
lysine, followed by a serine-glycine spacer. The
NH2-terminal lysine residue was modified with a biotin or
fluorescein tag (Fmoc-biotinyl-lysine and Fmoc-fluoresceinyl-lysine
were from Anaspec). The NH2-terminal tag-KSG sequences were
followed by 11 residues corresponding to the wild type (WT) or
modified COOH termini of NR2b and of other PDZ-binding proteins.
Selected peptides were analyzed by mass spectrometry, and all of the
peptides were quantified with a BCA assay.
Fluorescence Anisotropy Plate Assays--
Saturation binding
curves for PDZ domain-peptide interactions were determined by examining
the change in fluorescence polarization (FP) (45) of a fixed
concentration of the various fluorescein-labeled peptides (100 nM) with increasing amounts of a GST-PDZ domain fusion
protein. Peptides and fusion proteins were diluted using TBS with 1%
bovine serum albumin to prevent absorption of the peptide to the plate.
After combining the fluorescent peptide and PDZ domain in a black
384-well plate and allowing the system to come to equilibrium (1-2 h),
the plate was read on a Victor2 V (PerkinElmer Life
Sciences) plate reader in the fluorescence polarimeter mode to obtain
polarization values (P). P is calculated according to the equation P = (Iv g *
Ih)/(Iv + g *
Ih), with Iv and
Ih corresponding to the vertical and horizontal
fluorescence intensities, respectively; g is a calibration
correction, obtained from reading the polarization value of a 100 nM fluorescein solution, which possesses a P
value of 0.027 under our conditions at room temperature. The
g value is calculated from the equation g = (Iv0/Ih0) * (1 0.027/1 + 0.027), where Iv0 and
Ih0 are the vertical and horizontal fluorescent
intensities of the fluorescein solution in TBS plus 1% bovine serum
albumin. The P values of fluorescent peptide solutions
without PDZ domains were subtracted from the P values
obtained by titrations with PDZ domains. The resulting P
values were plotted against the concentration of the PDZ domain. To
determine the KD value, a curve was fitted by the equation Y = B *
X/(KD + X), with B
being the maximum P value that would be reached at
saturation as indicated by the extrapolation of the fitted curve.
Solid-phase Colorimetric Plate Assays--
Streptavidin-coated
96-well microtiter plates (Pierce) were incubated with 1% bovine serum
albumin in TBS and subsequently with saturating amounts of biotinylated
peptides. After two washing steps with TBS to remove any unbound
peptide, GST-PDZ domain fusion proteins were added in TBS at varying
concentrations and allowed to incubate for 1-2 h. Following two quick
washing steps with TBS, bound GST fusion proteins were detected by
incubation with anti-GST antibodies (41) for 1/2 h, washed twice
with 0.1% Triton X-100 in TBS, and incubated with Protein A coupled to
horseradish peroxidase (HRP; 1:10,000 in TBS) for 1/2 h. After
washing with 0.1% Triton X-100 in TBS, 100 µl of the colorimetric
HRP substrate solution "Slow-TMB" (Pierce) was added to each well.
After 5 min, the reaction was stopped with 100 µl of 1 M
H2SO4, and the plate was read at 650 nm.
Immunoprecipitation and Immunoblotting--
The protease
inhibitors pepstatin A (1 µg/ml), leupeptin (10 µg/ml), aprotinin
(20 µg/ml), phenylmethanesulfonyl fluoride (200 nM), and
calpain inhibitor I and II (8 µg/ml each) were present in all
extracts. Membrane fractions were prepared from whole rat brain,
extracted with 1% deoxycholate at pH 8.5 (and, in some cases, 1%
Triton X-100 or 1% SDS), and cleared by ultracentrifugation as
described (41, 46, 47). Extracts (0.5 ml) were incubated on ice with
antibodies (all produced in rabbits) against PKC (Invitrogen),
frizzled 2, BAI1 (48), or control rabbit IgG. After 1.5 h, 4 mg of
protein A-Sepharose were added, samples were tilted for 2.5 h, and
the resins were washed and extracted with 20 µl of SDS sample buffer
before immunoblotting (46, 49) with antibodies against PSD-95, SAP102
(50), or BAI1. All experiments were performed at least three times with
comparable results.
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RESULTS |
Affinity Determinations of Fluorescein-labeled Peptides for the
First Two PDZ Domains of PSD-95 and SAP102--
Type I PDZ domains
typically bind to SXV consensus sequences located at the
very COOH termini of their interaction partners. Bulk sequencing of
peptides that bound to PDZ domains during incubation with peptide
libraries indicated some preference (in the range of 1.5-2-fold) for
certain amino acids over others at most positions within the
COOH-terminal 10 residues (32). However, residues of the COOH-terminal
(S/T)XV motif for a given PDZ domain can be quite flexible.
For example, serine at the 2-position (P2) can usually
be substituted by threonine as is the case for Kv1.4 (10), and
isoleucine or leucine can often replace valine at P0
(51-55). Our goal was to quantify sequence selectivity of the first
two PDZ domains of PSD-95 and SAP102 for target binding using defined
peptides. We determined binding constants by measuring FP of
fixed concentrations of fluorescein-labeled peptides with increasing
concentrations of PDZ domains. Sequence selectivity indicated by these
in-solution measurements was corroborated by an ELISA-styled assay with
biotinylated peptides, which were attached to streptavidin-coated
plates and incubated with increasing concentrations of PDZ domains.
Constant concentrations of the NR2b WT peptide were incubated with
increasing concentrations of purified GST-PDZ fusion proteins, and FP
was determined with a Victor2 V plate reader (Fig.
1). The peptide contained the 11 COOH-terminal residues of NR2b and the linker sequence KSG at the
NH2 terminus with the NH2-terminal lysine
fluorescein tagged. The rotational mobility of this fluorescent peptide
decreased when bound to a PDZ fusion protein. Therefore, as the
proportion of fluorescent peptide bound to PDZ fusion proteins
increased, the aggregate FP of the solution increased. The measured
values could be fitted to saturation curves from which the
KD values were determined. We found that the second
PDZ domain of PSD-95 and SAP102 bound with higher affinity than the
first PDZ domain (0.91 versus 2.3 µM for
PSD-95; 0.64 versus 1.41 µM for SAP102). These
values are in good agreement with those obtained earlier for
in-solution interaction assays for PDZ domain binding to their
respective ligands (12, 56). It is also interesting to note that SAP102 had slightly higher affinity for the NR2b WT peptide than PSD-95 for
corresponding PDZ domains.
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Fig. 1.
KD determination of
peptide-PDZ domain interactions by FP. 100 nM
solutions of fluorescein-labeled peptides containing the COOH-terminal
11 residues of NR2b were incubated with increasing amounts of GST
fusion proteins of PDZ1 or -2 domains of PSD-95 or SAP102, and changes
in FP were determined. Shown are plots of these P value
changes against the concentration of the PDZ domains
(symbols) and curves fitted by the equation
Y = B * X/(KD + X). To allow for direct visual comparison of required PDZ
domain concentrations in the 50% saturation range, data were
normalized to B = 1, with B being the
maximal P value change that would be reached upon saturation
as extrapolated by the curve fittings. KD values are
given following the symbol key.
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Substantially lower KD values have been observed by
solid phase-based assays including surface plasmon resonance measurements (30, 57). However, these solid phase measurements were
based on interactions of immobilized peptides carrying respective COOH-terminal SXV motifs with recombinant proteins that
contained more than one PDZ domain. Accordingly, these solid phase
assays may not reflect the true value for a single PDZ domain-target interaction because one protein may simultaneously bind two or more of
the immobilized peptides, thereby dramatically reducing its off-rate
and causing rebinding and avidity effects (58, 59). Similar
considerations apply for experiments with single PDZ domains of SAP102
expressed as GST fusion proteins (4) because the GST moieties can
dimerize and thereby result in avidity effects in solid phase assays
(58). To avoid these problems, we primarily worked with peptides in
solution. In our solid phase assays, we used GST fusions with a single
PDZ domain, and the wells were coated with our peptides at low density.
We tested peptides derived from proteins that were initially identified
as binding partners for one or more of the three PDZ domains of PSD-95
and were either strong or weak interactors (Fig. 2A). Highest affinities were
observed for binding of the second PDZ domains of PSD-95 and SAP102 to
NR2a, NR2b, NR2c, and the K+ channel Kv1.4. Binding to the
Kv1.1 K+ channel and the COOH terminus of the NR1 C2'
domain, which constitutes one of the two alternatively spliced COOH
termini of the NR1, was much weaker. The first PDZ domains of PSD-95
and SAP102 exhibited substantially lower affinities for these peptides
than the second PDZ domains but showed a similar ranking order, with
NR2a, NR2b, NR2c, and Kv1.4 being much stronger interactors than Kv1.1
and NR1 C2'. The relative ranking of all of these affinities agrees with earlier semiquantitative data. For example, yeast two-hybrid assays suggested much stronger interactions of the second than of the
first PDZ domain of PSD-95 with NR2a, NR2b, and NR2c (29), and PSD-95
interacts much better with Kv1.4 than with Kv1.1 in this assay (10).
NR1 is generally not considered to bind to PSD-95 (60). However, the
C2' COOH terminus does conform to the (S/T)XV motif. Several
pieces of evidence suggest that the NR1 C2' region can associate with
PSD-95 and SAP102, although it is unknown whether this interaction is
mediated by the first two or rather the third PDZ domains of these two
proteins (9, 61, 62). The low affinities observed for the Kv1.1 and NR1 C2'-derived peptides may therefore reflect that binding of C2' to the
first two PDZ domains is not as firm as for the other interactions described above and may require addition interactions for
stabilization. PSD-95 and SAP102 may form interactions to NR1 in
parallel to those with NR2 subunits in the NMDA receptor complex,
thereby increasing the stability of the complex. However, we cannot
rule out the possibility that Kv1.1 and NR1 does not at all associate with one of the first two PDZ domains of PSD-95 or SAP102 in
vivo. Removal of the last 4 residues of NR2b by shifting the
peptide sequence 4 positions toward the NH2 terminus
exposes another sequence at the very COOH terminus that constitutes an
(S/T)XV motif with isoleucine substituting for valine at the
COOH terminus. However, this peptide did not show detectable binding at
all.
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Fig. 2.
KD values for binding of
PDZ1 and PDZ2 domains of PSD-95 and SAP102 to peptides derived from
established binding partners determined by FP. Fluorescein-tagged
peptides with the COOH-terminal sequences of different binding partners
of PSD-95 and SAP102 (A) and chimeric peptides carrying
various COOH-terminal portions of GluR1 and NR2b (B;
residues derived from NR2b are underlined) or GluR2 were
titrated with purified GST fusion proteins of PSD-95 and SAP102 PDZ1 or
-2 domains (the identity of the PDZ domain is given at the
top of each column). NR2b 4 refers to a peptide
that was NH2-terminally shifted by 4 positions away from
the COOH terminus, eliminating the COOH-terminal ESDV sequence. FP
measurements were performed, and KD values were
calculated as described under "Experimental Procedures." To
facilitate reading of the relative binding affinities,
KD values were normalized to the WT NR2b
KD, which was set to equal 1, and the inverses of
the normalized values were plotted as bars.
Numbers beside the bars give the
actual KD values (in µM;
N.D., not determined). The peptide sequence is listed in the
far right column.
KD values greater than the 10-fold
KD for WT NR2b are generally listed in the chart as
0, because accurate values for peptides with weak affinity were
difficult to determine. Each assay was usually done in
quadruplicate.
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We also analyzed the interactions to the first two PDZ domains of
PSD-95 and SAP102 with the COOH termini of Neuroligin and CRIPT, two
proteins that bind to the third PDZ domain of PSD-95. As expected (12,
30), the Neuroligin peptide did not show any detectable binding;
however, the CRIPT peptide exhibited relatively strong binding to the
second, although not the first, PDZ domains of PSD-95 and SAP102. In
fact, the COOH-terminal CRIPT sequence comes close to the optimal
binding sequence for the first two PDZ domains of PDS-95 and SAP102
(see below), and the original work on CRIPT described a weak
interaction of this protein with the second, although not the first,
PDZ domain of PSD-95 in the yeast two-hybrid system (12). In summary,
the results described in this and the previous paragraphs correspond
well to earlier qualitative and semiquantitative observations and
afford a reliable survey of binding constants. Accordingly, our assay
is well suited to determine the contribution of certain residues at the
COOH terminus of NR2b for PDZ domain binding by comparing the resulting affinity values of the various peptides with alterations in their sequences.
The COOH termini of GluR1 (TGL) and GluR2 (VKI) subunits of the
AMPA-type glutamate receptors constitute consensus sequences for
binding type I and type II PDZ domains, respectively. GluR1 has been
shown to be associated with SAP97, which, like PSD-95, contains type I
PDZ domains, but no interaction with PSD-95 or SAP102 was detectable
(41, 50). GluR2 binds to type II PDZ domains present in GRIP1 and -2 (19, 20). Peptides derived from these COOH termini did not show
detectable binding (Fig. 2B), further confirming the
specificity of our assays. We produced chimeric peptides based on the
GluR1 COOH-terminal sequence. Starting at the very COOH-terminal
position P0, we replaced an increasing number of GluR1
residues with those of NR2b. Little specific binding was detectable
until at least the last 4 GluR1 residues (i.e.
P3-P0) were replaced with those from NR2b
(Fig. 2B). Substituting positions P4 and
P5 further improved binding of the peptides, especially
to the PDZ1 domains, which required these substitutions for detectable
peptide interactions. Of note, binding by PDZ2 of PSD-95 and SAP102 was not very sensitive to substituting isoleucine for glycine at
P4 in these experiments, in contrast to those with single
point mutations described below. This difference may reflect that
contributions of each position may be influenced by the context of the
other positions, including those upstream of P4, although
these latter positions appear to have little influence on binding when
probed by single point mutations (see below).
Contribution of the Residues of the Very COOH Terminus of NR2b to
Binding to the First Two PDZ Domains of PSD-95 and SAP102--
To
evaluate the role of each position at the COOH terminus of NR2b in PDZ
binding, we systematically introduced single point mutations in this
sequence and determined the KD values of the
resulting peptides by FP (Fig. 3). We
also evaluated the relative contributions of each position in solid
phase assays with the corresponding biotinylated peptides anchored at
streptavidin plates. The density of streptavidin in the plate wells was
low, a characteristic of the plates we used that helped to avoid
avidity and rebinding effects. For most biotinylated peptides, the
solid phase binding assays were performed at single concentrations of the PDZ fusion proteins (Fig. 4), but for
selected peptides, titration curves with increasing amounts of the PDZ
fusion proteins were obtained (Fig. 5).
These solid phase assays corroborated the relative contribution of each
residue to PDZ binding (compare Fig. 3 with Figs. 4 and 5). The
apparent KD values calculated from the solid phase
titration assays were generally higher than those for the FP
measurements, usually by a factor of 2-4. This increase in the
apparent binding constant is due to unbinding of ligand during the
incubation and washing steps subsequent to the initial binding reaction
of the purified PDZ fusion proteins with the immobilized peptides.
Those steps are performed after the removal of unbound PDZ domains and
take a minimum of 1.5 h. If longer washing periods are applied,
the apparent KD values increase further (data not
shown). Therefore, the solid phase titration assays provide not true
but only "apparent" KD values. However, the
ranking order from these solid-phase titration assays agrees very well
with that obtained by FP and therefore corroborates the latter.
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Fig. 3.
Survey of KD values of PDZ domain
binding to NR2b-derived peptides with single amino acid substitutions
determined by FP. KD values were determined by FP
with fluorescein-tagged peptides derived from the WT NR2b COOH
terminus. Titrations were performed with purified GST fusion proteins
containing PDZ1 or -2 domains of PSD-95 or SAP102 (the identity of the
PDZ domain is indicated at the top of each
column). Each peptide carried a single substitution at one
of the 11 COOH-terminal positions designated P0 (the very
COOH-terminal position) to P10. The column on
the left indicates the residue in the WT NR2b sequence,
followed by the position number and the amino acid residue substituted.
Numbers following each bar indicate measured
KD values, whereas bars represent inverse
KD values normalized to WT NR2b, which equals 1, to
facilitate survey of relative binding affinities. KD
values greater than the 10-fold KD for WT NR2b are
generally listed in the chart as 0, because accurate values for
peptides with weak affinity were difficult to determine. Each assay was
typically performed in quadruplicate.
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Fig. 4.
Solid phase survey of relative affinities of
NR2b-related peptides to the first and second PDZ domain of PSD-95 and
SAP102. NH2-terminally biotinylated peptides as
indicated on the left were bound to streptavidin-coated
wells, and a fixed, nonsaturating concentration of GST-PDZ domain
fusion protein was added. Bound GST-PDZ was detected after washing with
anti-GST antibodies, followed by Protein A-HRP and a colorimetric
reaction with HRP substrate. OD values at 650 nm were normalized to WT
NR2b peptide values and plotted. Numbers beside
the bars are the averaged OD values from quadruplicate
determinations. NR2b V0A S2A refers to the NR2b WT sequence
with alanine substituted for valine at position P0 and
alanine substituted for serine at P2. These substitutions
effectively eliminated the binding of this peptide to PSD-95 PDZ
domains.
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Fig. 5.
Apparent KD
values of interactions between selected NR2b-related peptides and
the second PDZ domain of PSD-95 and SAP102 by the solid phase
assay. Selected NH2-terminally biotinylated peptides
as given on the left were attached to streptavidin-coated
wells and titrated with increasing concentrations of GST-PDZ domain
fusion protein. GST-PDZ retained on the plate was measured after
incubation with anti-GST antibodies and subsequently with protein A-HRP
by reading the A650 after a colorimetric
reaction with HRP substrate. Curves were fitted by the equation
Y = B * X/(KD + X), and subsequent KD calculations were
performed. Bars shown in the graph are the inverse of the
normalized KD values. KD values
are normalized to the KD of the WT NR2b peptide,
which is set to 1. Numbers beside the
bars show actual KD values
(µM).
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Initial experiments on the interaction of PSD-95 with NR2 and Kv1.4
COOH termini, which were the first studies identifying PDZ domain
interactions with target proteins, indicated that the serine (NR2
subunits) or threonine (Kv1.4) at P2 and the valine at
P0 are the most crucial residues and cannot effectively be
replaced by alanine, hence the reference to the
"(S/T)XV" motif. Later studies on other type I and also
type II PDZ domains indicated that valine at P0 can be
replaced by other hydrophobic residues including leucine and isoleucine
(51-55). We observed that valine at P0 was by far superior
for binding to the first two PDZ domains of PSD-95 and SAP102 as
compared with other hydrophobic residues, including leucine,
isoleucine, and phenylalanine. The presence of valine versus
leucine or isoleucine at P0 may therefore be crucial in
determining the specificity of a COOH-terminal interaction with a PDZ
domain, with valine increasing the selectivity for PSD-95 and SAP102.
NR2 subunits, Kv1.1, and Kv1.4, carry either aspartate or glutamate at
P-1. Substituting glutamate for aspartate in the NR2b peptide sequence
did not cause big changes in affinities for PDZ1 or -2 of PSD-95 and
SAP102. Glutamine-substituted peptides bound equally well, and so did
asparagine-substituted ones, at least for PDZ domain 95/1 in the
solid phase single concentration survey assay (Fig. 4). However,
asparagine substitution substantially reduced binding to the other
three PDZ domains in that assay (we did not perform FP assays for this
substitution). Serine showed a profile similar to that of asparagine,
with nearly unaltered binding affinity for PDZ domain 95/1 but
reduced affinity for the other three PDZ domains. Alanine-substituted
peptides showed good binding, although somewhat weaker than that
of WT, with affinities in the range around 2-4 µM. A
change to glycine or lysine strongly reduces binding affinities with
KD values above 10 µM, a concentration
range at which the precise KD values often proved
hard to determine with our survey assays. An exception is PDZ domain
102/2, which exhibited a KD value around 4 µM for the glycine-substituted peptide, suggesting that
glycine can be tolerated at this position to some degree, at least by certain PDZ domains. In summary, P1 is quite flexible,
and our PDZ domain interactions tolerate very well various residues
including aspartate, glutamate, glutamine, and alanine; asparagine and
serine work well for 95/1 binding, and glycine works to some extent for
102/2 interaction, although binding to other PDZ domains appears
compromised for peptides with the last three substitutions. The
positively charged residue lysine is generally not acceptable at
P1 for binding to these PDZ domains.
Structural studies indicate that either a serine or a threonine is
required at P2 for type I PDZ domain interactions; serine
and threonine at this position form a hydrogen bridge with the
histidine residue at the first position of the B helix (see Fig. 8),
which is conserved in type I PDZ domains (see Table III). The
2-position of NR2b and of other NR2 subunits has a serine. Replacing
this serine with threonine as present in Kv1.4 and Kv1.1 resulted in a
substantial increase in binding affinities for all four PDZ domains
(Figs. 3 and 5). As expected, an alanine replacement caused a strong decrease in binding to most PDZ domains, although some binding in the
low micromolar range was observed for 102/2 (Fig. 3; see also Fig. 4).
Peptides with the bulky tryptophan at this position did not show
binding (Fig. 4). Accordingly, serine at P2 can be
replaced by threonine, which increases binding, but other residues are
not well tolerated.
Glutamate is conserved at the 3-position in NR2 subunits and Kv1.4,
but this position was initially considered less critical for PDZ
binding, in part because Kv1.1, although a weaker interactor than Kv1.4
(10) (see also Fig. 2), carries a hydrophobic residue at this position
and because substituting glutamine for glutamate in Kv1.4 did not alter
its interaction with PSD-95 (9, 10). We found that only glutamate or
glutamine at P3 allows for high affinity interaction with
PDZ1 and -2 of PSD-95 and SAP102. Other substitutions including those
with aspartate, serine, threonine, alanine, leucine, phenylalanine, or
lysine substantially reduced or eliminated binding. The fact that
aspartate but not glutamine substitutions decreased binding indicates
that the length of the side chain but not the negative charge is
critical at this position for interaction with our PDZ domains. Both
the 
-carboxyl group of the glutamate residue and the amide group at
the -position of the glutamine residue are capable of forming hydrogen bonds, and this ability seems to be important (see below).
Like the 1-position, the 4-position appears to tolerate a variety
of residues. NR2a and -2b, NR2c and Kv1.1, and Kv1.4 and NR1 have
isoleucine, leucine or valine, respectively, at this position, and
replacing the valine in Kv1.4 with either arginine or tryptophan did
not alter PSD-95 binding in yeast two-hybrid interaction assays (10).
In addition, more recently identified binding partners for PDZ1 and -2 of PSD-95 include Kir2.1 and -2.3 (24, 51), and ErbB4 (63),
which possess an arginine at that position. Our peptide binding assays
confirm that P4 of NR2b is quite flexible and accepts not
only hydrophobic residues (e.g. leucine; Figs. 4 and 5) but
also lysine, which carries a positive charge similar to arginine.
However, glycine substitution resulted in a strong loss of binding,
indicating that the removal of a side chain does disturb binding. This
position is, therefore, not completely neutral. In fact, alanine,
methionine, and aspartate substitutions also strongly reduce binding.
Collectively, these data indicate that P4 accepts various
hydrophobic and positively charged residues, but the presence of other
residues including glycine, alanine, methionine, or aspartate selects
against binding to PDZ1 and -2 of PSD-95 and SAP102.
Positions 5 through 10 generally accepted most substitutions,
suggesting that they are less critical in determining PDZ domain
interactions. However, aspartate was not well tolerated at
P5 (Figs. 3 and 4). Replacing tyrosine with an aspartate
at P10 resulted in a strong increase in binding affinity
for the second PDZ domains of PSD-95 and SAP102 in the FP assay (Fig.
3) but not the solid phase assay (Fig. 4). It is quite possible that this position is too close to the plate surface to be fully accessible in the solid phase assay for interaction with the PDZ domain and may,
therefore, only show an effect in the FP assay. These results suggest
that selected positions past the P4 may make some,
although quite limited, contributions to PDZ domain interactions.
Affinities of Peptides for the Third PDZ Domains of PSD-95 and
SAP102--
We also tested the CRIPT- and Neuroligin-derived peptides
along with those corresponding to the COOH termini of the various glutamate receptor subunits and Kv1.4 in binding assays with the third
PDZ domains of PSD-95 and SAP102. The CRIPT peptide exhibited by far
the highest affinity for both PDZ domains with a KD in the range of 3 µM for PDZ3 of PSD-95 as determined by
titration of FP (Fig. 6A).
Neuroligin binding was also saturable but showed a much higher
KD of 28 µM in this assay (Fig.
6A), whereas the NR2b wild-type peptide does not show
specific binding for PDZ3 (Fig. 6B). For nearly all of the
peptides based on the NR2b COOH-terminal sequence, FP and the solid
phase assay indicated little to no specific binding (data not shown).
However, the NR2c peptide consistently showed distinguishable binding
above background (Fig. 6B). In fact, FP titration indicated
that association of this peptide with PDZ3 is saturable with a
KD around 25 µM (Fig. 6A).
This finding opens up the possibility that the COOH terminus of NR2c
may not only interact with the first two but also the third PDZ domains
of PSD-95 and SAP102. Although the affinity is relatively low for the
latter interactions, they may help stabilize association of NR2c with
the first two PDZ domains of PSD-95 and SAP102. In a similar way, CRIPT
may not only interact with the third but also with the second and
perhaps first PDZ domains of PSD-95 and SAP102. The idea of combining weak and strong COOH-terminal interactions with PDZ domains also receives support by early findings that NR2 subunits and Kv1.4 interact
most robustly with the second PDZ domains of PSD-95 and SAP102 and more
weakly with the first PDZ domains (10, 29). Because each NMDA receptor
and each Kv1.4 channel possesses several subunits with appropriate
COOH-terminal SXV motifs, simultaneous interactions of one
receptor or channel complex with several PDZ domains appears
likely.
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Fig. 6.
Affinity determination of interactions
between selected peptides and the third PDZ domains of PSD-95 and
SAP102 by FP and colorimetric assays. A, 100 nM
solutions of fluorescein-labeled peptides containing the COOH-terminal
11 residues of NR2c, CRIPT, or Neuroligin were incubated with
increasing amounts of GST fusion proteins containing the third PDZ
domain of PSD-95, and the change in fluorescence polarization was
determined. Curves were fitted by the equation Y = B * X/(KD + X), and
the KD value was determined. Shown are plots of the
binding curves against concentration of the GST-PSD-95 third PDZ
domain. FP value changes were normalized to the maximal calculated FP
value change for each curve. KD values are given on
the right. B, select peptides were incubated with
a fixed concentration of a GST fusion protein with the third PDZ domain
of either PSD-95 (95/3) or SAP102 (102/3). The
concentration of the fusion protein was nonsaturating with respect to
CRIPT peptide binding. The change in FP (indicated in P values × 103) of fluorescein-conjugated peptides after
GST-PDZ domain addition (first and third
columns) and the OD at 650 nm for biotinylated peptides
after GST-PDZ domain addition and detection with anti-GST/Protein
A-HRP/colorimetric HRP substrate Slow-TMB (second and
fourth columns) are shown. Peptide sequences are
given in the far right column. Each
peptide was evaluated in quadruplicate.
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Survey of the Available Human Genome Sequences for Potential
Binding Partners for the First Two PDZ Domains of PSD-95 and
SAP102--
To obtain an impression of how many potential binding
partners with a COOH-terminal (E/Q)(S/T)XV motif exist, we
screened proteins as predicted by the currently available draft version of the human genome for this motif. We did not use any restrictions at
the 1-position and 4-position. We identified 54 proteins and
divided them into four categories (Table
II). Group 1 contains all proteins
with a COOH-terminal (E/Q)(S/T)(D/E/Q/N)V sequence. Of note, nearly all
proteins in that group carry a hydrophobic or positively charged
residue at P4, although this had not been a criterion for
grouping those proteins together. We predict that these proteins are
likely to bind strongly to the first two PDZ domains of PSD-95 and
SAP102. In fact, five of the 14 members of this group had been
identified as binding partners (in boldface type at the top of the list
in Table II), and we present evidence that at least one member of the
brain-specific angiogenesis inhibitor family (BAI1) does also interact
with PSD-95 (see below). Group 2 proteins differ from group 1 by having
a residue different from the binding-promoting aspartate, glutamate, asparagine, or glutamine at P1 but carrying an
advantageous aliphatic or positively charged residue at
P4 (i.e. isoleucine, leucine, valine,
arginine, or lysine). Only two of the 16 proteins in this category are
actually known to interact with the first two PDZ domains of PSD-95 or
SAP102 (PMCA4b and CRIPT). Group 3 proteins have no favorable residue
at either P1 or P4, and only two of 20 proteins have been shown to bind to PSD-95 or SAP102. Finally, group 4 contains proteins with a lysine or an arginine at P1,
which inhibit binding to our PDZ domains. Three of the four proteins,
Kir3.2, Kir3.3 (24), and the 1-adrenergic receptor (28),
have actually been tested for binding to PDZ1 or -2 of PSD-95, with
negative results. In the same studies, parallel pull-down experiments
resulted in interactions that serve as positive controls (the
inward-rectifying K+ channels Kir2.1 and Kir2.3 bound to
PSD-95 in Ref. 24, presumably via the second PDZ domain (51), and the
1 adrenergic receptor bound to the third, although not
the first or second, PDZ domain of PSD-95 in Ref. 28). These results
confirm our finding that a lysine and perhaps an arginine are
inhibiting binding to the first two PDZ domains of PSD-95 and
SAP102.
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Table II
Human proteomic search for proteins containing PSD-95 PDZ binding
consensus sequence (E/Q)(S/T)XV
We performed a BLAST search for COOH-terminal
(E/Q)(S/T)XV-containing proteins. The search was restricted
to the working draft sequence of the human genome protein products. For
each match, the GenBankTM accession number is given, along with
the COOH-terminal consensus sequence (residues that potentially
increase affinity for PSD-95 PDZ binding are in boldface type) and if
there is evidence whether (+) or not () the protein (or its mRNA)
is expressed in the brain (? indicates that there is no evidence for
brain expression, +/ indicates that expression in the brain is low
and spatially narrow). The entries are grouped (1 through 4) in order
from the highest predicted affinity group to the lowest. Protein names
in boldface type indicate confirmed association with PSD-95 PDZ
domains.
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A comparison with Table I, which lists all known interaction partners
of the first two PDZ domains of PSD-95 and SAP102 with COOH-terminal
SXV motifs, indicates that Table II identifies most proteins
with COOH-terminal (E/Q)(S/T)XV sequences (NR2a to -d, Kv1.4, Kir3.2, 3.3, 1 adrenergic receptor, PMCA4b,
CRIPT, Citron, and Sema4c in Table I). Maguin and SynGAP also possess
COOH-terminal (E/Q)(S/T)XV sequences (Table I) but were not
identified, because the sequences for these two proteins predicted from
the human genome projects only show versions with truncated COOH
termini, perhaps due to inaccuracies in the draft sequence (64). Kv1.1, Kv1.2, Kv1.3, Kir2.1, Kir2.3, 2-adrenergic receptor,
PMCA2b, ErbB4, Nedasin, and Neuroligin (Table I) do not conform to the (E/Q)(S/T)XV motif and were not detected in our screen,
which is, therefore, not complete in terms of binding partners for the first two PDZ domains of PSD-95 and SAP102. However, most of the latter
proteins are known or predicted to interact with those PDZ domains more
weakly than proteins with a COOH terminus that matches the
(E/Q)(S/T)XV sequence (e.g. Kv1.4 and PMCA4b bind much stronger than Kv1.1-3 and PMCA2b, respectively; see
"Discussion").
Not all of the proteins in Table II are actually potential binding
partners of PSD-95 and SAP102 because their expression patterns do not
overlap. For example, PSD-95 and SAP102 are only detectable in brain,
whereas the PDZ binding kinase is abundant in testis and placenta and
weakly expressed in heart but not brain (65, 66). Tyrosinase-related
protein 1 is involved in melanin syntheses and restricted to
melanocytes. It binds to the PDZ domain of GIPC (67), but due to its
restriction to melanocytes it would not constitute an interaction
partner for PSD-95 or SAP102 in neurons. The Na+ channel
Nav1.5 was originally described as muscle-specific and interacts in these tissues with the PDZ domains of syntrophins (68).
Recently, limited expression of Nav1.5 in the brain has been described (26), but PDZ-domain interaction assays for a Nav1.5-derived peptide were negative (25), suggesting that
Nav1.5 does not interact with PSD-95 or SAP102 even if
co-expressed in some neurons. Another example of a protein that has a
COOH-terminal (E/Q)(S/T)XV motif and is present in neurons
but does not appear to interact with PSD-95 is diacylglycerol kinase
, which binds to the PDZ domains of syntrophins but not of PSD-95
(27).
Interaction of Brain-specific Angiogenesis Inhibitor BAI1 with
PSD-95--
Many proteins revealed by our proteomic search have not
been considered in terms of association with PSD-95. We were able to
obtain antibodies for three of these proteins (BAI1, PKC, and
frizzled 2) and evaluated those proteins for co-immunoprecipitation with PSD-95 and SAP102 from rat brain extracts. Antibodies against BAI1
(48) recognize three bands in brain extract, all of which migrate in
the range around 150 kDa; the lower two bands form a doublet, which is
often not resolved (Fig. 7A
and data not shown). These bands are enriched after immunoprecipitation
with the same antibodies (Fig. 7A), suggesting that all
three polypeptides are specifically recognized by the antibody and
represent various isoforms of BAI1. BAI1 immunoprecipitates also
contained PSD-95, but SAP102 was never detectable in the BAI1 complex
(Fig. 7A). To ensure that the BAI1-PSD-95 complex was
present in the intact brain and did not form during homogenization, we
homogenized parallel samples in a 50-fold larger buffer volume than
under our standard conditions before collecting crude membranes by
ultracentrifugation from both conditions. Subsequent
immunoprecipitation resulted in comparable immunoreactivity for
BAI1-associated PSD-95 (Fig. 7A). We did observe
coprecipitation of BAI1 and PSD-95 and a lack of coprecipitation of
BAI1 and SAP102 not only after extraction with 1% deoxycholate but
also after extraction with either 1% Triton X-100 or 1% SDS (data not
shown). Together with an analogous finding for PMCA2b, which also
selectively binds to PSD-95 but not SAP102 (52), our observations that
PSD-95, but not SAP102, is associated with BAI1 in the brain indicate
that PSD-95 and SAP102 may play different roles in their cellular
context by interacting with distinct, although partially overlapping
protein pools. BAI1-related BAI2 and BAI3 also have a COOH-terminal
SXV motif (YQTEV) with a hydrophobic residue at
P4 and a glutamate at P1 and carry an
acidic residue at P10. Because these residues at these
positions increase binding of our NR2b-derived peptides to the first
two PDZ domains of PSD-95, it is likely that BAI2 and -3 are also
interaction partners for PSD-95.
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Fig. 7.
Co-immunoprecipitation of PSD-95 and SAP102
with BAI1 and PKC but not frizzled 2. Rat
forebrain (0.8 g) was homogenized either in 8 ml (1× in A
and C) or 400 ml (B, 50× in A and
C) sucrose buffer before collection of a crude membrane
fraction by ultracentrifugation and solubilization with deoxycholate.
A, proteins were immunoprecipitated with anti-BAI1 or
anti-frizzled 2 antibodies or with nonimmune control rabbit IgG.
Immunoblotting for PSD-95 and SAP102 revealed that PSD-95, but not
SAP102, is present in the same immunocomplex as BAI1.
Immunoprecipitation of frizzled 2 did not result in
co-immunoprecipitation of either of the PDZ domain-containing proteins.
The Load lane represents the deoxycholate
solubilized membrane fraction that was used for immunoprecipitation.
B, immunoblot for frizzled 2 showing that frizzled 2 was
effectively immunoprecipitated from the solubilized membrane fraction
used in A. C, immunoprecipitations of PKC and
immunoblotting for PSD-95 and SAP102 were performed in a fashion
similar to that in A. Mouse IgG was used as control. Both
PSD-95 and SAP102 are found in the same immunocomplex as PKC. Each
experiment was repeated at least three times with similar
results.
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In parallel with BAI1, we immunoprecipitated the Wnt receptor
frizzled 2 (69-71). The frizzled 2 antibody recognized one major band
in brain extract of the expected molecular mass by immunoblotting (Fig.
7B). Immunoprecipitation with this antibody, but not with a
control antibody, resulted in the same band, indicating that this
antibody effectively immunoprecipitated frizzled 2 under our
conditions. However, we never observed coprecipitation of either PSD-95
or SAP102 with frizzled 2, whether membrane extracts were prepared with
1% deoxycholate, Triton X-100, or SDS (Fig. 7A and data not
shown). The COOH-terminal sequence of frizzled 2 matches that of
frizzled 1 (GETTV) and is very similar to the COOH termini of frizzled
4 (SETVV) and frizzled 7 (GETAV). All of these sequences predict at
best weak interactions with the first two PDZ domains of PSD-95 or
SAP102 (group 3 in Table II). Our negative results suggest that
frizzled 2 and its homologs do not bind to PSD-95 or SAP102 in
vivo.
Interaction of PKC with PSD-95 and SAP102--
We performed
similar experiments looking for co-immunoprecipitation of PKC with
PSD-95 and SAP102. PKC is listed in group 2 (Table II) with an
unfavorable alanine at P1 but a beneficial leucine at
P4 in its COOH-terminal sequence (LQSAV), suggesting an
intermediate affinity for the first two PDZ domains of PSD-95 and
SAP102. PKC has been shown earlier to bind with its COOH terminus to
the PDZ domain of PICK1 (22). Both PSD-95 and SAP102 coprecipitated with PKC (Fig. 7C). As in the previous experiments (Fig.
7A), immunoprecipitations with appropriate control
antibodies were negative for PSD-95 and SAP102, indicating the
specificity of the immunoprecipitations. Similar results were obtained
when 1% Triton X-100 or 1% SDS instead of the routinely employed
deoxycholate was used for solubilization of the membrane fractions
(data not shown). Comparable amounts of PSD-95 and SAP102
coprecipitated with PKC whether the brain tissue was homogenized in
the standard volume or at a 50-fold higher dilution, indicating that
PKC was associated with PSD-95 and SAP102 in vivo before
homogenization of the brain tissue (Fig. 7C). The novel
finding that BAI1 and PKC are associated with PSD-95 testifies to
the utility of our proteomic approach in defining the potential pool of
interaction partners for various PDZ domains.
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DISCUSSION |
Our results demonstrate proteins with COOH-terminal
(E/Q)(S/T)XV sequences possess the potential for stable
interactions with the first two PDZ domains of PSD-95 and SAP102. This
potential is further increased if P1 is aspartate,
glutamate, asparagine, or glutamine and if P4 is a
hydrophobic residue including leucine, isoleucine, valine, or perhaps
tryptophan, lysine, or arginine (10). Aspartate and perhaps
glutamate at P10 may also foster binding to the first two
PDZ domains of PSD-95 and SAP102. Nevertheless, the 0-, 2-, and
3-positions are highly sensitive to the precise residue present and
are most crucial in determining the selectivity of the NR2b COOH
terminus for binding to the first two PDZ domains of PSD-95 and SAP102.
We found a severalfold preference of these PDZ domains for valine at
P0 over the related amino acids leucine and isoleucine, for
threonine at P2 over serine, and for glutamate and
glutamine at P3 over aspartate. Accordingly, even
conservative substitutions at these positions such as serine for
threonine or aspartate for glutamate affect binding. As discussed in
the following paragraphs, these findings are in good agreement with
published results on proteins that bind to these PDZ domains (see Table
I).
The first identified binding partners for PSD-95 were the NR2 subunits
and Kv1.4. They not only match the (E/Q)(S/T)XV motif; they
also possess an aspartate or glutamate at P1 and an
isoleucine, leucine, or valine at P4. They are therefore
likely to be among the strongest interactors in vivo. Kv1.1,
Kv1.2, and Kv1.3 also have (S/T)XV motifs and hydrophobic
residues at P4 and aspartate at P1.
However, they possess hydrophobic residues at P3, which
should strongly reduce binding to the first two PDZ domains of PSD-95
and SAP102 (Figs. 3-5). In fact, PSD-95 association with Kv1.1, Kv1.2,
or Kv1.3 appears to be much weaker than with Kv1.4 in a yeast
two-hybrid assay and a clustering assay in mammalian cells (10, 72). In
a clustering assay, Shaker, a Drosophila homolog of the Kv1
subfamily, exhibits an interaction with PSD-95 that is as strong as the
interaction between Kv1.4 and PSD-95 (72). In contrast to Kv1.1, Kv1.2,
and Kv1.3, Shaker perfectly matches positions 0 through 3 of the
Kv1.4 COOH terminus, and P4 carries an isoleucine. These
findings corroborate our consensus sequence for binding to the first
two PDZ domains of PSD-95 and SAP102.
The next two proteins in Table I are the inwardly rectifying
K+ channels Kir2.1 and Kir2.3 (24, 51). Both proteins have
an isoleucine rather than a valine at P0, which should
strongly reduce binding affinity for those PDZ domains. However, they
carry an arginine at P4; an aspartate or glutamate,
respectively, at P10; and, in the case of Kir2.1, a
glutamate at P1. According to our analysis, these
residues help to strengthen the interaction with our PDZ domains and
may promote stable binding even if the COOH-terminal position is not
valine but the related isoleucine. Kir3.2 and Kir3.3 do conform to the
generic (S/T)XV motif, but a careful analysis indicates that
they do not bind to PSD-95 or SAP102 (24). The lack of detectable
interaction is probably due to the presence of the positively charged
lysine at P1, which inhibits binding to our NR2b-derived
peptides at this position. Similarly, the 1 adrenergic
receptor carries an ESXV motif with lysine at
P1 and does not appear to interact with either the first
or the second PDZ domain of PSD-95 (28); it does bind to the third PDZ
domain of PSD-95 (28), indicating that the third PDZ domain is quite
different from the first two PDZ domains. The 2
adrenergic receptor with the COOH-terminal DSPL sequence binds to the
PDZ domain of the Na+/H+ exchange regulatory
factor (53) but does not seem to associate with PSD-95 (28), probably
because of the COOH-terminal leucine without compensatory residues at
the 1-, 4-, or 10-position.
The plasma membrane Ca2+ ATPase PMCA4b has a COOH-terminal
interaction sequence that is predicted to allow for high affinity interaction: threonine at P2, the hydrophobic leucine at
P4, and aspartate at P10. It strongly binds
to PDZ1 and -2 of PSD-95 and SAP102 (16, 52). PMCA2b also binds to the
first two PDZ domains of PSD-95, but these interactions are much weaker
than those observed with PMCA4b (16), and no binding could be observed
between PMCA2b and SAP102 (52). This binding behavior would be
predicted from our findings that the leucine at the 0-position of
PMCA2b should strongly reduce binding to PDZ1 and -2 of PSD-95 and
SAP102. Maguin-1 matches the (E/Q)(S/T)XV motif,
co-immunoprecipitates with PSD-95 from rat brain extracts, and binds to
one or more PDZ domains of PSD-95 (73). Although the exact PDZ domains
that interact with Maguin-1 have yet to be defined, based on our
analysis it could bind to the first two domains because it has not only
the (E/Q)(S/T)XV motif at its COOH terminus but also an
isoleucine at P4. However, the histidine at
P1 is partially positively charged and may potentially be
inhibitory for the binding to the first two PDZ domains and perhaps
direct it to the third PDZ domain.
CRIPT also possesses that (E/Q)(S/T)XV motif and a lysine at
P4, which, like isoleucine at this position, increases
binding to the first two PDZ domains of PSD-95 and SAP102. Although it
was originally described mainly as a binding partner for the third PDZ
domain of PSD-95 (12), CRIPT did interact with the second PDZ domain in
the original yeast two-hybrid assays (12). A peptide derived from the
COOH terminus of CRIPT bound in our titration assays with
KD values between 1 and 2 µM to the
second PDZ domains of PSD-95 and SAP102, although binding to the first PDZ domains was either very weak or not detectable (Fig. 2). We propose
that the COOH terminus of CRIPT strongly interacts not only with the
third but also the second PDZ domains of PSD-95 and SAP102. This
binding pattern would explain why application of a membrane-permeable
peptide derived from the CRIPT COOH terminus effectively dispersed
PSD-95 from the postsynaptic site (74).
Similar to CRIPT's QTSV COOH-terminal sequence, Citron has the
sequence QSSV at its COOH terminus and binds preferentially to the
third and possibly also to the second PDZ domain of PSD-95 (15, 75). It
carries aspartate at P4, in contrast to CRIPT's lysine,
and P2 is a serine rather than a threonine; accordingly,
we predict that Citron binds in vivo much more weakly than
CRIPT to the second PDZ domain of PSD-95. Indeed, Citron's interaction
with PDZ2 of PSD-95 appears to be much less favorable than with PDZ3
(15). The Citron-related COOH-terminal sequence EESSV is present in the
semaphorin Sema4C and predicts a modest affinity for the first two PDZ
domains of PSD-95 and SAP102. Sema4C co-immunoprecipitates with PSD-95
from rat brain and binds to a fusion protein containing
TOP
ABSTRACT
INTRODUCTION
