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INTRODUCTION |
PDZ1 domains are modular
protein-protein interaction domains that are specialized for binding to
specific C-terminal peptide sequences (1-4) and to other PDZ domains
(5). Many proteins contain multiple PDZ domains, thereby allowing them
to function as multivalent scaffolds for organizing large protein
complexes (6-9). One major class of PDZ-containing proteins, known as
MAGUKs (membrane-associated
guanylate kinases) (10), is characterized by
an SH3 domain and a guanylate kinase-like domain in their C-terminal region. MAGUK proteins are typically localized in specific membrane domains such as cell junctions in epithelial cells (10) and synaptic
junctions in neurons (11-13). MAGUK proteins are thought to play a
central role in the organization of protein complexes at these
specialized membrane domains.
Much of what is known about PDZ protein function has come from studies
of the PSD-95 family of MAGUK proteins that are predominantly localized
in the postsynaptic density of excitatory synapses in the brain. The
first two PDZ domains, PDZ1 and PDZ2, of PSD-95 bind specifically to a
peptide sequence at the very C terminus of Shaker-type potassium
channels and of N-methyl-D-aspartate receptor
NR2 subunits (14-16). This PDZ-mediated interaction results in the
clustering of the ion channel proteins in heterologous cells (15, 17,
18) and is important for the synaptic localization of the membrane
protein-binding partners in vivo (19-21).
Recent studies have investigated the molecular mechanisms of ion
channel clustering by PSD-95. Channel-clustering activity is dependent
on the multimerization of PSD-95, which is mediated by a stretch of
~64 amino acids in the N-terminal region of the protein (termed the
N-segment) (18). Two members of the PSD-95 family, PSD-95 and
chapsyn-110, can heteromultimerize with each other because they both
contain the N-segment at their N-terminal ends. Moreover, a pair of
cysteine residues, Cys3 and Cys5, is conserved
in the N-terminal regions of PSD-95 and chapsyn-110. Mutation of either
or both of these cysteines abolishes multimerization and the
channel-clustering activity of PSD-95 (18). Based on biochemical
experiments in vitro and in vivo, it was
hypothesized that Cys3 and Cys5 form
intermolecular disulfide bonds that stabilize the
N-segment-mediated multimerization of PSD-95 (18).
More recently, these N-terminal cysteines have also been found to be
sites for fatty acid modification (palmitoylation) of PSD-95 (22).
These investigators reported that mutation of Cys3 and
Cys5 inhibited the association of PSD-95 with membranes and
prevented PSD-95 from binding to the potassium channel Kv1.4 in
heterologous cells. This would provide an alternative explanation of
why Cys3 and Cys5 mutations abolish Kv1.4
clustering by PSD-95. The conclusion reached by Topinka and Bredt (22)
is somewhat surprising because there are other members of the PSD-95
family that lack these N-terminal cysteines (e.g. SAP97 and
the Drosophila homolog Discs large). SAP97 and Discs large
(Dlg) are nevertheless membrane-associated proteins (17, 23), and Dlg
is known to play a critical role in the binding and clustering of
Shaker ion channels and FasII at the synapse in vivo
(19-21). In re-examining the role of the N-segment and of
Cys3 and Cys5 in more detail, we report here
that mutation of these N-terminal cysteines has no detectable effect on
the PSD-95 binding of Kv1.4, although it abolishes the ability of
PSD-95 to self-associate and to cluster Kv1.4 at the cell surface. Thus
Kv1.4 binding and homomultimerization are separable functions of
PSD-95, and the former activity is not dependent on the N-terminal
cysteines. Moreover, we demonstrate that cysteine mutants of PSD-95
lose their ability to form a ternary complex with Kv1.4 and the cell adhesion molecule FasII, implying that multimerization is required for
PSD-95 to bind simultaneously to two different PDZ ligands.
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MATERIALS AND METHODS |
DNA Constructs--
All PSD-95 constructs used here contain a
Myc epitope tag inserted between residue 9 and 10 of PSD-95. Expression
constructs GW1-PSD-95, GW1-PSD-95 (C3S/C5S), GW1-N-GFP, GW1-N-PDZ12,
and GW1-N-PDZ12(C3S/C5S) have been described previously (18). To construct HA-tagged PSD-95, the Myc cassette in Myc-tagged PSD-95 was
replaced by an HA epitope. For the GFP-N construct, the Myc-tagged N-segment (corresponding to residues 1-64 of PSD-95) was polymerase chain reaction amplified from Myc-tagged PSD-95 and subcloned into
plasmid EGFP-C1 (CLONTECH). To construct Myc-tagged
SAP97, an AscI restriction site was introduced between
residues 435 and 436 of SAP97 in GW1-CMV by inverse polymerase chain
reaction, and a duplex oligonucleotide encoding the Myc epitope tag was inserted into the AscI site. GW1 expression constructs of WT
Kv1.4, Kv1.4 C-terminal mutant (Kv1.4VA), WT FasII, and FasII
C-terminal mutant (FasIIVA) have been described previously (15,
20).
Antibodies, Transfection, Immunoprecipitation, and
Immunocytochemisry--
Rabbit anti-PSD-95 ("CSK"), guinea pig
anti-PSD-95 ("HM319") antibodies (15, 17), and Kv1.4 antibodies
(24) have been described previously. The sites in the PSD-95 protein
recognized by CSK and HM319 antibodies are indicated in Fig. 1. Myc
monoclonal antibody 9E10 and HA monoclonal antibody 12CA5 were
purchased from Santa Cruz Biotechnology and Boehringer Mannheim,
respectively. FasII monoclonal antibodies 1D4 were kindly provided by
Dr. Corey S. Goodman.
Transfection was performed using the LipofectAMINE reagent according to
the manufacturer's directions (Life Technologies, Inc.). For
immunoprecipitation, COS-7 cells in 35-mm plates at 50-70% confluency
were incubated with a 1-ml Opti-MEM (Life Technologies, Inc.) mixture
containing 1.6 µg of DNA and 6 µl of LipofectAMINE (Life
Technologies, Inc.) for 5 h followed by incubation in Dulbecco's modified Eagle's medium. 48 h later, cells were harvested and lysed in 0.45 ml of radioimmune precipitation buffer. One-third of the
cell lysate was incubated with CSK antibodies or Kv1.4 antibodies at a
final concentration of 2 µg/ml at 4 °C for 2 h. 20 µl of
1:1 slurry of Protein A-Sepharose (Amersham Pharmacia Biotech) was
added, and the mixture was rotated at 4 °C for 1-2 h. The
immunoprecipitates were washed with a series of buffers: first,
radioimmune precipitation buffer; second, 10 mM Tris (pH 7.5), 0.5% Nonidet P-40, 0.5 M LiCl; third, 10 mM Tris (pH 7.5), 0.5 M LiCl; and finally, 10 mM Tris (pH 7.5). The immunoprecipitates were eluted in
SDS-loading buffer, separated by SDS-polyacrylamide gel
electrophoresis, and analyzed by immunoblotting using 0.2 µg/ml Myc
9E10 or Kv1.4 antibodies.
For immunocytochemistry, transfected COS-7 cells were fixed by 2%
formaldehyde in phosphate-buffered saline for 15 min, permeabilized with 0.1% Triton X-100 in Tris-buffered saline for 1 min, and blocked
with 3% horse serum and 0.1% bovine serum albumin in Tris-buffered saline for 30 min at room temperature. The cells were incubated with
primary antibodies at 1 µg/ml at room temperature for 1 h, followed by Cy3- and fluorescein isothiocyanate-conjugated secondary antibodies (Jackson ImmunoResearch) for 1 h. Results were viewed with a Zeiss Axioskop microscope, and images were prepared for publication using Adobe Photoshop.
Membrane Fractionation and Extraction--
Transfected COS-7
cells were suspended in 800 µl of homogenization buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
dithiothreitol, and protease inhibitors) and broken by Dounce
homogenization. Total cell extracts were centrifuged at 1,000 × g for 10 min twice. The supernatants were centrifuged at
100,000 × g for 1 h at 4 °C to separate crude
membrane pellet and S100 soluble fraction. Crude membrane pellets were
further washed with either homogenization buffer, 1 M NaCl,
or 100 mM sodium carbonate-buffered homogenization buffer
and centrifuged at 100,000 × g for 1 h. Equal
fractions of crude membrane pellet, S100 cytosolic fraction, final
washed pellet, and soluble fractions were analyzed by immunoblotting
using the HA antibody for WT PSD-95 and the Myc antibody for N-terminal
cysteine mutant, PSD-95-CS.
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RESULTS |
N-segment Functions as Multimerization Domain Only When Situated at
the N Terminus--
In an earlier study, we showed that deletion
mutants of PSD-95 lacking a short N-terminal region of the protein
(residues 1-64, termed the N-segment) (Fig.
1) lost the ability to form homomultimers
(18). Thus the N-segment is required for self-association of PSD-95.
Moreover, the N-segment is sufficient to confer multimerization on a
heterologous protein. A chimeric protein containing the N-segment from
PSD-95 linked to the green fluorescent protein (Fig. 1,
N-GFP) could be coimmunoprecipitated with wild type
full-length PSD-95 using CSK antibodies directed against the SH3 domain
of PSD-95 (Fig. 2A) (18). In
the absence of PSD-95, N-GFP was not precipitated by CSK antibodies;
furthermore, GFP itself (lacking the N-segment) cannot be
coimmunoprecipitated with PSD-95 (data not shown) (18). These results
indicate that the N-segment alone can confer on a heterologous protein
(GFP) the ability to associate with PSD-95.

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Fig. 1.
Schematic diagram of PSD-95 constructs used
in this paper, and summary of their multimerization, Kv1.4 binding, and
Kv1.4-clustering activities. Multimerization activity was assayed
by coimmunoprecipitation with wild type full-length PSD-95 (see Fig.
2). Kv1.4 binding activity was assayed by coimmunoprecipitation with
Kv1.4 (see Fig. 3). Kv1.4-clustering activity was defined by the
ability to form plaque-like clusters (>2 µm in diameter) in
PSD-95/Kv1.4 double transfected COS-7 cells. The sites in PSD-95
recognized by CSK and HM 319 antibodies are indicated. Double cysteine
C3S/C5S mutants are referred to as N-PDZ12-CS and PSD-95-CS. A Myc
epitope tag (black line) was inserted in the N-segment
(hatched boxes). The numbered boxes refer to PDZ
domains. SH, SH3 domain; GK, guanylate
kinase-like domain; ND, not done.
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Fig. 2.
N-segment and Cys3 and
Cys5 are required for PSD-95 multimerization.
A, N-segment confers PSD-95 association only when fused to
the N-terminal side of GFP. COS-7 cells were co-transfected with PSD-95
and N-GFP or PSD-95 and GFP-N, as indicated. Cell lysates were
immunoprecipitated using CSK anti-PSD-95 antibodies, and the
precipitates were immunoblotted with anti-Myc antibodies (both PSD-95
and GFP constructs are Myc-tagged). B, N-terminal cysteines
(Cys3 and Cys5) are essential for PSD-95
multimerization. COS-7 cell lysates transfected with different
combinations of PSD-95, N-PDZ12, and N-PDZ12-CS (all Myc-tagged) were
immunoprecipitated with CSK antibodies. The precipitates were
immunoblotted with anti-Myc antibodies. C, NEM has no effect
on PSD-95 multimerization. This is a similar experiment as in
B, but NEM (10 mM) was included in the
radioimmune precipitation lysis buffer to prevent oxidation during
extraction and immunoprecipitation. Coimmunoprecipitation of PSD-95 and
N-PDZ12 was not affected by NEM. All input lanes were loaded with 10%
of the extract used for the immunoprecipitation reaction.
IP, immunoprecipitate.
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Interestingly, a chimeric protein in which the PSD-95 N-segment is
fused to the C-terminal end of GFP (diagrammed in Fig. 1,
GFP-N) was unable to associate with full-length PSD-95 by
the co-immunoprecipitation assay, despite being efficiently expressed (Fig. 2A). This suggests that the N-segment has to be placed
at an N-terminal location to self-associate. The GFP moiety, when placed at the N-terminal end but not when placed at the C-terminal end
of the N-segment, may sterically interfere with self-association mediated by the N-segment.
Cys3 and Cys5 Are Critical for PSD-95
Multimerization--
The N-terminal cysteine residues Cys3
and Cys5 are conserved between PSD-95 and chapsyn-110;
these members of the PSD-95 family can form homomultimers with
themselves and heteromultimers with each other (17, 18). To confirm
that Cys3 and Cys5 are important for
multimerization of PSD-95, both these cysteines were substituted with
serines in the context of two different constructs of PSD-95
(generating N-PDZ12-CS and PSD-95-CS) (see Fig. 1). These double
cysteine mutants were assayed for multimerization in
coimmunoprecipitation experiments. Unlike wild type N-PDZ12, the
N-PDZ12-CS mutant could not be precipitated by CSK antibodies in the
presence of full-length PSD-95 (Fig. 2B), suggesting that the Cys3 and Cys5 residues are essential for
PSD-95 self-association. In previous studies, we presented evidence
that PSD-95 can form disulfide-linked multimeric complexes through
residues Cys3 and Cys5 (18). To make sure that
the association of full-length PSD-95 and N-PDZ12 is not because of
artifactual disulfide bonding that occurred during cell lysis,
N-ethylmaleimide (NEM) was included in the lysis buffer at
10 mM to inactivate free sulfhydryl groups. Even in the
presence of NEM, N-PDZ12 was still as efficiently associated with
full-length PSD-95 as in standard lysis conditions (Fig.
2C). The same result was obtained with 20 mM NEM
added to the cell culture for 20 min prior to cell harvesting (data not shown). Taken together, these results indicate that PSD-95
multimerization requires Cys3 and Cys5;
however, PSD-95 multimerization is not because of artifactual oxidation
of these residues during or after cell extract preparation.
N-terminal Cysteine Mutants of PSD-95 Can Bind the Kv1.4 Potassium
Channel--
The double cysteine mutants N-PDZ12-CS and PSD-95-CS lack
multimerization activity (Figs. 1 and 2). In addition, these mutants are incapable of forming cell surface-associated clusters with Shaker-type potassium channel Kv1.4 in heterologous cells (Fig. 3A) (data not shown for
N-PDZ12-CS). Wild type PSD-95 formed plaque-like coclusters with Kv1.4
in doubly transfected COS-7 cells (Fig. 3A, a), as has been
shown previously (15, 17, 18). These PSD-95-Kv1.4 coclusters are on or
closely associated with the cell surface (25). However, in COS cells
coexpressing Kv1.4 and double cysteine PSD-95 mutants, the typical
plaque-like coclusters did not form (Fig. 3A, b)
(data for N-PDZ12-CS not shown). Instead, both Kv1.4 and PSD-95
cysteine mutants were often concentrated in brightly staining
intracellular structures that colocalized with each other. These
intracellular structures were typically spherical and had brightly
immunoreactive perimeters, suggesting that they were membrane-bound.
The membranous nature of these stained structures is consistent with
the fact that the Kv1.4 ion channel is an integral membrane protein.
The appearance of the intracellular staining seen with Kv1.4 and
PSD-95-CS is strikingly reminiscent of that seen upon coexpression of
Kv1.4 and SAP97 in COS-7 cells (25). This may reflect a similarity
between SAP97 and PSD-95-CS in that SAP97 naturally lacks any
N-terminal cysteines equivalent to Cys3 and
Cys5.

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Fig. 3.
N-terminal cysteine mutant of PSD-95 still
binds to Kv1.4 but cannot cluster Kv1.4 at the cell surface.
A, different Kv1.4-clustering behaviors of wild type PSD-95
(a) and cysteine mutant PSD-95-CS (b) shown by
double label immunostaining of transfected COS cells (a1,
a2). The cotransfected cell double-stained for Kv1.4
(a1) and WT PSD-95 (a2). PSD-95 and Kv1.4
immunoreactivities colocalize in plaque-like clusters associated with
the cell surface (b1, b2). PSD-95-CS and Kv1.4
fail to form plaque-like coclusters at the cell surface but instead
colocalize in round intracellular structures with a perinuclear
distribution. In addition, some PSD-95-CS is distributed diffusely in
the cell. B, cysteine mutant PSD-95-CS associates with
Kv1.4. COS-7 cells transfected with a different combination of Kv1.4,
WT PSD-95, and PSD-95-CS mutant, as indicated, were immunoprecipitated
with CSK PSD-95 antibodies. Precipitates were immunoblotted with Kv1.4
antibodies, and then the nitrocellulose membrane was stripped and
re-probed with anti-Myc antibody to reveal PSD-95. C, the
PSD-95 relative SAP97 associates with Kv1.4. COS cells transfected with
Myc-tagged SAP97 ± Kv1.4 were immunoprecipitated with
Kv1.4-specific antibodies. The immunoprecipitate was immunoblotted with
Myc antibody to visualize SAP97; the nitrocellulose membrane was then
stripped and re-probed for Kv1.4. IP, immunoprecipate.
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A recent report suggested that mutations of Cys3 and
Cys5 prevent PSD-95 from binding to Kv1.4 in COS cells, as
measured by coimmunoprecipitation assays (22). However, we find that
double cysteine mutants of PSD-95 coimmunoprecipitate as efficiently with Kv1.4 as does wild type PSD-95 (Fig. 3B). Thus, the
inability of PSD-95 cysteine mutants to form surface clusters with
Kv1.4 is not because of their inability to bind to the potassium
channel. The ability of double cysteine mutants of PSD-95 to bind Kv1.4 is further supported by the colocalization of these proteins in the
brightly staining intracellular structures.
Another member of the PSD-95 family, SAP97, has an additional 94 amino
acids preceding its N-segment-like region, and it does not contain a
pair of cysteine residues at its N terminus. We tested for an
interaction between SAP97 and Kv1.4 by the coimmunoprecipitation assay.
When coexpressed in COS-7 cells, SAP97 was readily co-precipitated by
anti-Kv1.4 antibodies in the presence of Kv1.4 (Fig. 3C),
suggesting that SAP97 and Kv1.4 can form a stable complex. This result
reinforces the conclusion that N-terminal cysteines are not required
for biochemical association of PSD-95 family proteins with Kv1.4 and perhaps other membrane protein-binding partners.
Taken together, the above data indicate that residues Cys3
and Cys5 of PSD-95 are required for PSD-95 multimerization
and for formation of surface-associated clusters with Kv1.4. However,
unlike Topinka and Bredt (22), we find that these N-terminal cysteines
are not important for Kv1.4 channel binding by PSD-95 or its relatives. Because Cys3 and Cys5 are the defined sites of
palmitoylation in PSD-95 (22), our data imply that palmitoylation is
not essential for the interaction of PSD-95 with membrane ion channels.
Effect of Cys3 and Cys5 Mutation on PSD-95
Association with Membranes--
To examine whether Cys3
and Cys5 play a role in the membrane association of PSD-95,
we compared the biochemical fractionation of HA-tagged wild type PSD-95
and Myc-tagged PSD-95-CS mutant coexpressed in COS-7 cells. The
relative distribution of wild type and cysteine mutant PSD-95 could
then be followed in the same extracts by immunoblotting with HA and Myc
antibodies, respectively. In COS cells, the majority of both the wild
type PSD-95 and the PSD-95-CS mutant were found in the cytosolic (S100)
fraction, although substantial amounts were associated with the crude
membrane fraction (Fig. 4). The
association of PSD-95 with membranes was investigated further by
extracting with high salt or high pH buffers. Sodium carbonate buffer
(PH11) was slightly more efficient than 1 M NaCl
in extracting PSD-95 from the crude membrane fraction; however, there
was no major difference between wild type PSD-95 and PSD-95-CS with
regard to sensitivity to these conditions (Fig. 4). Although PSD-95-CS
was perhaps more efficiently extracted than wild type PSD-95, the
difference was slight in contrast to the qualitative difference
observed by Topinka and Bredt (22). The amount of PSD-95-CS that
remained associated with membranes after extraction with high salt or
PH11 buffer was similar to that of wild type PSD-95 (Fig. 4).

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Fig. 4.
Effect of N-terminal cysteine mutations on
membrane association of PSD-95. COS-7 cells were co-transfected
with wild type PSD-95 (HA-tagged) and cysteine mutant PSD-95-CS
(Myc-tagged). Membrane fraction (Memb) and cytosolic S100
fractions (S100) of COS-7 cells were prepared as described
under "Materials and Methods." The membranes were further extracted
with isotonic homogenate buffer (HB), 1 M sodium
chloride (NaCl), or alkaline sodium carbonate buffer
(PH11) to yield pellet (P) and soluble fractions
(S). All fractions were simultaneously analyzed by
immunoblotting with HA antibody to reveal wild type PSD-95 and Myc
antibody to reveal PSD-95-CS.
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Cys3 and Cys5 Are Required for Formation of
a Ternary Complex Containing PSD-95, Kv1.4, and Fasciclin
II--
PSD-95 and its Drosophila homolog Dlg have recently
been shown to bind via their PDZ domains to the cell adhesion molecule FasII as well as to the Shaker potassium channel (20, 21). Indeed, Dlg
can form a ternary complex with Shaker and FasII proteins in
heterologous cells, although Shaker and FasII have the same binding
specificity for PDZ2 of Dlg/PSD-95 (20). We asked whether PSD-95 can
form a ternary complex with Kv1.4 and FasII and whether this ability
depended on multimerization of PSD-95. That is, can a cysteine mutant
of PSD-95 that cannot multimerize bind simultaneously to Kv1.4 and FasII?
COS-7 cells were triply transfected with Kv1.4, FasII, and either WT
PSD-95 or PSD-95-CS mutant, and the cell extracts were immunoprecipitated with Kv1.4 antibodies. The presence of FasII in the
Kv1.4 immunoprecipitate was taken to imply existence of the
Kv1.4-PSD-95-FasII ternary complex (Fig.
5). FasII was precipitated by Kv1.4
antibodies in the presence of wild type PSD-95, but not PSD-95-CS
mutant (Fig. 5, lanes 7 and 10), indicating that
residues Cys3 and Cys5 of PSD-95 are required
for ternary complex formation. The C-terminal FasII(VA) mutant,
which cannot interact with the PDZ2 domain of PSD-95, could not be
coprecipitated with Kv1.4 and PSD-95 (Fig. 5, lane 11).
Moreover, the C-terminal Kv1.4(VA) mutant, which cannot interact with
PSD-95, could not coprecipitate either PSD-95 or FasII (Fig. 5,
lane 12). These controls confirm that the ternary complex is
mediated by C-terminal tail interactions with PDZ domains and that the
immunoprecipitating antibodies (anti-Kv1.4) do not cross-react with
PSD-95 or FasII. The requirement for N-terminal residues
Cys3 and Cys5 in formation of the ternary
complex suggests that a single monomer of PSD-95 cannot bind
simultaneously to FasII and Kv1.4.

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Fig. 5.
Wild type PSD-95, but not N-terminal cysteine
mutant of PSD-95, can associate with two distinct PDZ2-binding proteins
simultaneously. COS-7 cells were triply transfected with different
combinations of wild type or mutant PSD-95, FasII, and Kv1.4. For
PSD-95, C indicates wild type, and S indicates
double C3S/C5S cysteine mutant. For FasII and Kv1.4, V
indicates wild type, and A indicates the mutant in which the
C-terminal valine has been mutated to alanine. Cell lysates were
immunoprecipitated with Kv1.4 antibodies; the precipitates were
immunoblotted with FasII, Kv1.4, and Myc (for Myc-tagged PSD-95)
antibodies. Kv1.4 antibodies were able to coprecipitate both WT PSD-95
and PSD-95-CS mutant in the presence of wild type Kv1.4, but not in the
presence of Kv1.4(VA) mutant. Wild type FasII was coprecipitated by
Kv1.4 antibodies in the presence of wild type PSD-95, but not PSD-95-CS
mutant. Kv1.4(VA) and FasII(VA) mutants were controls to show that the
biochemical associations with PSD-95 were mediated by the C-terminal
PDZ binding motif of these membrane proteins. IP,
immunoprecipitate.
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DISCUSSION |
Two properties appear to be essential for ion channel clustering
by PSD-95 in heterologous cells: 1) the ability of PSD-95 to bind
directly to the ion channel by a PDZ-mediated interaction (17, 18, 25);
and 2) the ability of PSD-95 to self-associate into multimers (18).
Deletion mutants of PSD-95 that lack the Kv1.4-binding domains PDZ1 and
PDZ2 lose Kv1.4-clustering activity (18). Deletion mutants of PSD-95
that lack the N-segment responsible for multimerization are also unable
to cluster Kv1.4 (18).
Multimerization of PSD-95 and surface clustering of Kv1.4 are dependent
on a pair of cysteines in the N-segment of PSD-95 (Cys3 and
Cys5). These cysteines are potential sites for disulfide
bonding and/or palmitoylation. Palmitoylation of these cysteine
residues has been proposed to mediate membrane association of PSD-95
and to allow its binding to Kv1.4, a membrane ion channel (22).
However, we find that these palmitoylation sites are not required for
Kv1.4 binding. Nor did the presence or absence of these cysteines have a major effect on the biochemical association of PSD-95 with membranes, at least when tested in heterologous cells. Perhaps most significantly, natural homologs of PSD-95 that lack these cysteines (such as SAP97 and
Dlg) interact efficiently with Shaker-type potassium channels in
heterologous cells and in native tissues (19, 25). Taken together, it
seems unlikely that palmitoylation of Cys3 and
Cys5 is essential for membrane association of PSD-95 or for
PSD-95 interaction with Kv1.4 or other binding partners in the
membrane. Although palmitoylation is not critical for membrane
interactions of PSD-95 in general, it may facilitate the targeting of
PSD-95 specifically to the plasma membrane. We noted that cysteine
mutants of PSD-95 tended to be trapped in intracellular membranous
organelles (Fig. 3A). Our results are still consistent with
the possibility that disulfide bond formation involving
Cys3 and Cys5 is important in the ability of
PSD-95 to organize large two-dimensional clusters of ion channels at
the cell surface. Given the potential significance of these two
cysteines for PSD-95 and chapsyn-110 function, it would be valuable to
examine in greater detail the relative roles played by palmitoylation
and disulfide bonding of these two residues.
Whatever the chemical modifications of Cys3 and
Cys5, it is clear that these cysteine residues are required
for the N-segment-mediated multimerization of PSD-95. Mutations of
these residues abolish PSD-95 self-association (Fig. 2B).
The coimmunoprecipitation experiments in the presence of NEM are
consistent with PSD-95 being multimerized prior to cell lysis via
disulfide bonds involving Cys3 and Cys5.
Alternatively, N-segment multimerization may be mediated by noncovalent
bonds, and the Cys3 and Cys5 residues could be
important in some other way for the structure and function of the
N-segment. It is also possible that palmitoylation is required for the
multimerization mediated by the N-segment, although the mechanism of
such involvement is unclear.
The other major conclusion of this study is that the N-terminal
cysteines are required for PSD-95 to coordinate a ternary complex of
two distinct membrane proteins, FasII and Kv1.4. Mutation of
Cys3 and Cys5 does not affect Kv1.4 binding by
PSD-95, but it prevents association of Kv1.4 and FasII together in a
PSD-95-based complex (Fig. 5). The simplest explanation of these
results is that a single monomer of PSD-95 cannot bind simultaneously
to both FasII and Kv1.4. Rather, the ternary complex is constructed on
a multimer of PSD-95, each monomer of PSD-95 binding individually to
either Kv1.4 or FasII. Such a mechanism neatly explains how PSD-95 can
form a ternary complex with two distinct membrane proteins, both of
which have the same binding preference for PDZ2 of PSD-95. In
vivo, there are multiple proteins that have overlapping
specificities for the various PDZ domains of PSD-95. A multimeric
scaffold of PSD-95 based on N-segment-mediated association would
provide a simple mechanism for several different proteins with the same PDZ specificity to be brought together in a physical complex.