1 Department of Physiology University of California at San Francisco, San
Francisco, CA 94143-2140, USA
* These authors contributed equally to this work
Author for correspondence (e-mail:
bredt{at}itsa.ucsf.edu)
Accepted 15 April 2003
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Summary |
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Key words: Synapse, Clustering, Receptor, Palmitoylation
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Introduction |
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A role for PSD-95 in receptor clustering was first suggested based on the
potent interaction of both N-methyl-D-aspartate (NMDA) receptors and
Shaker-type K+ channels with the PDZ domains of PSD-95
(Kim et al., 1995;
Kornau et al., 1995
).
Furthermore, mutations of discs large (dlg), a
Drosophila homolog of PSD-95, prevent proper post-synaptic clustering
of Shaker-type K+ channels at the larval neuromuscular junction
(Tejedor et al., 1997). In
addition to clustering ion channels, PDZ domains from PSD-95 organize
signaling enzymes (Brenman et al.,
1996
) and cell adhesion molecules
(Irie et al., 1997
) at the
postsynaptic density. PSD-95 also contains SH3 and guanylate kinase (GK)
domains, which both mediate regulatory intramolecular interactions
(McGee et al., 2001
;
Tavares et al., 2001
) and
recruit additional proteins to the macromolecular complex
(Brenman et al., 1998
;
Kim et al., 1997
;
Takeuchi et al., 1997
).
Through this network of interactions, PSD-95 and related MAGUK protein
complexes regulate postsynaptic development
(El-Husseini et al., 2000b
;
Sala et al., 2001
) and
plasticity (Migaud et al.,
1998
).
Biochemical mechanisms for ion channel clustering by PSD-95 remain
uncertain. Some aspects of channel clustering can be reproduced in a
heterologous cell transfection assay in which co-expression of PSD-95 and
Kv1.4 yields formation of plasma membrane patches containing both PSD-95 and
Kv1.4 (Kim et al., 1995). One
model for this clustering posits that the clusters of PSD-95 and Kv1.4 reflect
a molecular lattice, which forms based on the multivalent nature of both
PSD-95 and Kv1.4 (Hsueh et al.,
1997
). Shaker-type K+ channels are multimeric based on
their inherent tetrameric structure. Although the oligomeric structure of
PSD-95 is unclear, previous studies have proposed that PSD-95 occurs as a
tetramer formed through intermolecular disulfide bonds involving cysteine
residues 3 and 5 (Hsueh et al.,
1997
).
Here, we mechanistically analysed the oligomerization and clustering of PSD-95. We find that multimerization of PSD-95 involves only its first 13 amino acids. Remarkably, these 13 amino acids are sufficient for oligomerization of two heterologous proteins. Multimerization of PSD-95 does not involve disulfide bonds but instead requires palmitoylation of two cysteine residues in the 13 amino acid motif. This lipid-dependent multimerization is a unique property of the N-terminus of PSD-95 and is not seen with other palmitoylation domains. Clustering K+ channel Kv1.4 requires interaction of palmitoylated PSD-95 with tetrameric K+ channel subunits but, surprisingly, does not require multimerization of PSD-95. Finally, blocking palmitoylation with 2-bromopalmitate disperses PSD-95/ion-channel clusters, indicating that K+ channel clustering by PSD-95 is a reversible process regulated by protein palmitoylation.
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Materials and Methods |
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Construction of cDNA plasmids
Epitope-tagged constructs of PSD-95 were generated by PCR as described
previously (El-Husseini et al.,
2000a). To generate the Kv1.4 constructs, nucleotides encoding
amino acids 1-350 and 351-655 of Kv1.4 were amplified separately by PCR with a
hemagglutinin (HA) epitope encoded in the 5' primer at amino acid 351. A
three-part ligation reintroduced Kv1.4 into GW1
HindIII/EcoRI with a KpnI site joining the two
separate PCR products. To produce (Kv1.4)2, the coding region of
HA-tagged Kv1.4 was amplified by PCR twice: once with a 5' primer
containing a HindIII site and a 3' primer with a BglII
site and a second time with a 5' primer containing an in-frame
BglII site and a 3' primer with a stop codon and an
EcoRI site. A three-part ligation reintroduced both Kv1.4 channel
subunits into GW1 HindIII/EcoRI with a BglII site
between the two subunits. To link four Kv1.4 channel subunits together to
produce (Kv1.4)4, the entire coding region of HA-tagged Kv1.4 was
amplified twice and subcloned back into GW1 as described above for
(Kv1.4)2 but without the stop codon in the second subunit. A third
subunit was added in-frame at the BglII site and a fourth in-frame
with a stop codon at the EcoRI site. The Tac (interleukin-2 receptor
-subunit) cDNA, modified by the introduction of an XbaI site
at the 5' end, was kindly provided by R. Edwards (University of
California, San Francisco). A binding consensus for PSD-95 from the C-terminal
12 amino acids of the NR2B subunit was subcloned into this cDNA with oligos
using the introduced XbaI site and an ApaI site in the
multi-cloning site of the vector. The C-terminal prenyl motif of paralemmin
(-DMKKHRSKSCSIM) was added to the extreme C-terminus of PSD-95(C3,5S)-GFP with
PCR primers encoding this motif.
Cell transfection and immunoprecipitation
COS7 or HEK293 cells were grown in Dulbecco's modified Eagle's medium
(DMEM) containing 10% fetal bovine serum, penicillin and streptomycin. Cells
were transfected using Lipofectamine reagent according to the manufacturer's
protocol (Gibco). For routine immunoprecipitation studies, transfected cells
were incubated in lysis buffer [25 mM Tris HCl, pH 7.4, plus 1 mM EDTA, 150 mM
NaCl, 1% Triton X-100, 1 mM dithiothreitol (DTT), 1 mM PMSF] at 4°C for 30
minutes. In some experiments, as indicated, 0.1% SDS replaced Triton X-100 in
the cell lysis buffer; in these experiments, 1% Triton X-100 was added after
cell lysis. Lysates were cleared by centrifugation at 17,000 g
for 15 minutes and incubated for 1 hour at 4°C with 2 µg sheep
anti-PSD-95 antibody (Brenman et al.,
1996) or rabbit anti-GFP antibody (Clontech).
Immunofluorescent labeling
COS7 cells were grown on coverslips in 24-well plates and transfected as
described above. Coverslips were removed from culture wells and fixed in 2%
paraformaldehyde for 30 minutes. After washing with PBS containing 0.3% Triton
X-100 (PBST) three times for 5 minutes each, the cells were incubated in PBST
containing 3% normal goat serum for 1 hour at room temperature to block
non-specific antibody interactions. Primary antibodies were added in block
solution for 1 hour at room temperature, followed by donkey anti-mouse or goat
anti-rabbit secondary antibodies conjugated to Cy2 or Cy3 fluorophores
(diluted 1:200 in block solution) for 1 hour at room temperature. Images were
taken under fluorescence microscopy with a 100µ oil-immersion objective
(NA=1.4) affixed to a Leica upright microscope.
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Results |
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To determine whether PSD-95 multimerization involves intermolecular covalent bonds, we denatured the protein lysates with 0.1% SDS in the absence of reducing agents. This procedure disrupts non-covalent protein/protein interactions but does not disrupt disulfide bonds. We found that denaturing cell lysates with SDS disrupted the PSD-95 multimers (Fig. 1), indicating that they are not linked by covalent bonds.
Palmitoylated N-terminal 13 amino acids of PSD-95 mediate
oligomerization
To define the interaction interface between the PSD-95 multimers, we
constructed and analysed a series of progressively larger deletion constructs
encoding PSD-95 fused to GFP. Each of these constructs was transfected into
COS cells and assessed for interaction and coimmunoprecipitation with
full-length PSD-95 (data not shown). These experiments demonstrated that a
construct containing only the first 13 amino acids of PSD-95 associates with
full-length PSD-95 (Fig. 2A).
This interaction between 1-13 and full length PSD-95 is disrupted by SDS,
indicating that it is not covalent (Fig.
2B).
|
Having determined that the first 13 amino acids of PSD-95 are sufficient for interaction with the full-length protein, we next asked whether this 13 amino acids motif alone could mediate oligomerization. To assess this, we co-transfected COS cells with constructs encoding the first 13 amino acids of PSD-95 linked both to GFP and to maltose binding protein. Remarkably, these two fusion proteins efficiently co-immunoprecipitated (Fig. 2C).
The first 13 amino acids of PSD-95, which mediate multimerization, are also
necessary and sufficient for palmitoylation
(El-Husseini et al., 2000a),
raising the possibility that multimerization and palmitoylation are
interrelated. Consistent with this possibility, mutating palmitoylated
cysteines 3 and 5 prevents multimerization of PSD-95
(Fig. 3A)
(Hsueh et al., 1997
). More
importantly, mutating leucine-4 to serine, which disrupts palmitoylation of
PSD-95 (El-Husseini et al.,
2000a
), also prevents multimerization
(Fig. 3B).
|
To assess more directly the role of palmitoylation, we treated transfected
cells with 2-bromopalmitate, which blocks the palmitoyl transferase that
mediates protein palmitoylation (Webb et
al., 2000) and blocks PSD-95 palmitoylation
(El-Husseini et al., 2002
).
Cells co-transfected with PSD-95-GFP and 1-13-GFP were treated with 20 µM
2-bromopalmitate for 4 hours and multimerization was assessed by
co-immunoprecipitation. These experiments showed that 2-bromopalmitate blocks
multimerization of PSD-95 (Fig.
3C). As a control, we treated cells with palmitate and found that
this does not interfere with multimerization
(Fig. 3C).
We next asked whether another palmitoylated motif, from the growth
associated protein GAP-43, would also mediate multimerization. Previous
studies have shown that the N-terminal ten amino acids of GAP-43 determine its
palmitoylation (Liu et al.,
1993) and we previously reported that fusing these ten amino acids
to PSD-95 yields a robustly palmitoylated chimera
(El-Husseini et al., 2000a
). To
test whether the palmitoylation motif of GAP-43 also mediates multimerization,
we tagged such GAP-43/PSD-95 chimeras with either GFP or FLAG at their
C-termini. In co-transfected COS cells, we find that these differently tagged
GAP-43 chimeras do not interact (Fig.
3D), indicating that oligomerization is a specific property of the
PSD-95 palmitoylation motif.
Clustering by PSD-95 requires interaction with a multivalent ion
channel
We next asked whether protein multimerization participates in ion channel
clustering with PSD-95. Certain aspects of channel clustering by PSD-95 can be
reproduced in a co-clustering experiment in heterologous cells
(Kim et al., 1995). In this
assay, co-transfection of PSD-95 with Shaker K+ channel Kv1.4
causes a striking redistribution of both proteins to raft-like clusters on the
cell surface (Fig. 4A,B). To
determine whether the tetrameric structure of Kv1.4 contributes to its
clustering with PSD-95, we constructed expression plasmids containing two or
four Kv1.4 subunits linked in tandem. Previous studies have shown that
Shaker-type K+ channel subunits, which contain six transmembrane
domains, can be expressed as functional channels when fused together in tandem
arrays (Isacoff et al., 1990
).
As was done in these previous studies, our constructs were designed such that
the intracellular C-terminus of one Kv1.4 subunit is linked in-frame to the
N-terminus of another subunit to form a single polypeptide with 12
transmembrane domains and two C-terminal cytoplasmic tails per channel to
interact with PSD-95. We also linked together two (Kv1.4)2 subunits
to form a (Kv1.4)4 channel containing 24 transmembrane domains and
a single C-terminal cytoplasmic tail per channel. These Kv1.4 channels contain
an HA epitope in the extracellular domain so that surface expression can be
monitored by immunofluorescent staining of intact cells.
|
We find that (Kv1.4)2 and (Kv1.4)4 channels are
expressed on the surface of the cells. However, in co-transfections with
PSD-95, they do not efficiently form clusters
(Fig. 4C,D and data not shown).
Co-expression of PSD-95 with wild-type Kv1.4 yields clusters in 60% of
cells, whereas PSD-95 and (Kv1.4)2 form clusters in 3% of cells,
and PSD-95/(Kv1.4)4 did not form clusters in any cells
(n=100 for each). To determine whether monomeric single-pass
transmembrane proteins can be recruited to clusters, we appended the PSD-95
binding consensus (-SDV) from the C-terminus of NMDA receptor 2B (NR2B) to the
short cytoplasmic tail of the interleukin-2 receptor
-subunit (IL-2 or
Tac), a well-characterized plasma membrane protein. When expressed with PSD-95
in COS7 cells, no clusters of Tac-SDV are formed
(Fig. 4E,F). However, when
Kv1.4 is co-expressed with PSD-95 and Tac-SDV, clusters of all three proteins
are formed (Fig. 4G,H). This
co-clustering requires the PDZ binding site on Tac, because the native Tac
construct remains diffuse in cells containing PSD-95/Kv1.4 clusters
(Fig. 4I,J). These results
suggest that the tetrameric nature of the Kv1.4 ion channel is necessary for
efficient formation of clusters.
Ion channel clustering by PSD-95 requires palmitoylation but not
multimerization
One model for protein clustering by PSD-95 suggests that clusters represent
a macromolecular lattice that relies on the multivalent nature of both PSD-95
and Kv1.4. However, this model is inconsistent with experiments showing that
co-transfections of Kv1.4 with the monomeric GAP-43/PSD-95 chimera
(Fig. 3D) shows clusters that
appear identical to those formed with Kv1.4 and wild-type PSD-95
(Fig. 5A-D). Although difficult
to address experimentally, it is possible that binding of this chimera to
Kv1.4 induces multimerization of the chimera. To assess further the role of
palmitoylation in K+ channel clustering, we acutely blocked
palmitoylation with 2-bromopalmitate. Co-transfected COS cells showing patches
of Kv.1.4 and PSD-95 were treated with 20 µM 2-bromopalmitate and, within
4-8 hours, the patches disappeared and were replaced by round perinuclear
blobs that appear to be intracellular protein aggregates
(Fig. 5E,F). These perinuclear
blobs resemble those seen when palmitoylation-deficient mutants of PSD-95 are
co-transfected with Kv1.4 (Fig.
5G,H). Cells treated with palmitate as a control showed normal
patches of PSD-95 and Kv.1.4 (Fig.
5I,J).
|
To help verify that the effects of 2-bromopalmitate on K+
channel clustering are specific for palmitoylation of PSD-95, we used a
non-palmitoylated construct of PSD-95 containing a C-terminal CAAX domain from
paralemmin, which is isoprenylated
(El-Husseini et al., 2000a).
This PSD-95 construct forms clusters with Kv1.4 that are somewhat smaller but
generally resemble those formed with wild-type PSD-95
(Fig. 5K,L). As expected,
2-bromopalmitate does not affect clustering mediated by isoprenylated PSD-95
(Fig. 5M,N). Finally, we asked
whether the prenylated PSD-95-CAAX can multimerize and found that
PSD-95-prenyl multimerizes with neither itself nor palmitoylated PSD-95
(Fig. 6).
|
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Discussion |
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A remarkable aspect of this study is that only 13 amino acids of PSD-95 are needed for oligomerization. Although the mechanism for this multimerization remains uncertain, it is lipid dependent and requires protein palmitoylation. The interaction is not disrupted by solubilization with Triton X-100 but is disrupted by SDS. We sought to determine the stoichiometry of the PSD-95 oligomers by performing gel filtration chromatography studies on PSD-95 extracted from both transfected cells and brain homogenates. In both cases, we found that PSD-95 migrates in the excluded volume, suggesting a molecular mass of greater than 1000 kDa (data not shown). This behavior is consistent with PSD-95 being present in a very high molecular weight complex or perhaps in lipid rafts.
Many palmitoylated proteins partition in specialized non-ionic detergent
resistant membrane (DRM) domains (Lisanti
et al., 1995; Moffett et al.,
2000
; Shaul et al.,
1996
) and a small proportion of PSD-95 occurs in these raft-like
domains (Perez and Bredt,
1998
). However, this partitioning cannot by itself explain the
multimerization of PSD-95, because the palmitoylated protein GAP-43
efficiently sorts to DRMs (Arni et al.,
1998
), but we find that GAP-43 does not form multimers. Although
both PSD-95 and GAP-43 are doubly palmitoylated, the palmitoylation motif of
GAP-43 differs from that of PSD-95 in that it contains two adjacent cysteines
as well as nearby basic residues. It is possible that the structural
differences between these palmitoylation motifs favors, in the case of PSD-95,
interactions between palmitoyl groups.
Whatever the mechanism for multimerization, the 13 amino acid motif of PSD-95 is one of the smallest domains found in any protein capable of mediating specific homomultimerization. Appending this short motif to heterologous proteins provides a novel mechanism for artificially forming protein multimers at the cell surface. This facile tool for constructing multimeric protein networks is likely to have general utility for both cell biological and pharmaceutical research.
Another surprise from this work is that multimerization of PSD-95 is not
essential for ion channel clustering. This finding questions the model that a
multivalent lattice of both PSD-95 and an interacting ion channel mediates ion
channel clustering by PSD-95. However, we do find that N-terminal
palmitoylation of PSD-95 is essential for channel clustering. Because
palmitoylation also targets PSD-95 to cellular endomembranes
(El-Husseini et al., 2000a), it
has been proposed that PSD-95 actively recruits ion channels from endosomes to
the plasma membrane. Indeed, this model has recently been supported by similar
studies showing that rapsyn, a myristoylated peripheral membrane protein,
actively recruits nAChR from endosomes to mediate synaptic clustering
(Marchand et al., 2000
).
If multimerization of PSD-95 is not needed for channel clustering, what
might be the function for this unique property of the N-terminus? One
possibility is that multimerization of PSD-95 might be crucial for its roles
in early synaptic development. Studies in cultured cells indicate that PSD-95
clusters at earlier developmental stages than do other postsynaptic proteins
(Rao et al., 1998).
Oligomerization of PSD-95 through a lipid-dependent mechanism could provide an
autonomous mechanism for formation of these antesynaptic complexes. An
alternative possibility is that multimerization of PSD-95 is important for its
assembly of synaptic protein networks. Previous studies have shown that
coupling of NMDA receptors to downstream effectors such as neuronal nitric
oxide synthase (nNOS) and to physiological responses such as synaptic
plasticity and learning requires PSD-95
(Migaud et al., 1998
). Through
protein multimerization, PDS-95 might help to recruit nNOS and other
intracellular proteins to the NMDA receptor complex
(Craven and Bredt, 1998
;
Kennedy, 1998
;
O'Brien et al., 1998
;
Sheng and Wyszynski,
1997
).
Because protein palmitoylation is reversible
(Dunphy and Linder, 1998;
Milligan et al., 1995
), it
provides a mechanism for regulating multimerization and ion channel clustering
of PSD-95. Indeed, blocking palmitoylation with 2-bromopalmitate disperses
preformed PSD-95/K+ channel clusters, indicating that maintenance
of these clusters requires continued palmitoylation of PSD-95. It will now be
important to identify the palmitoyl transferase and palmitoyl thioesterase
enzymes that regulate palmitoylation, multimerization and clustering of
PSD-95.
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Acknowledgments |
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Footnotes |
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3 Present address: Gladstone Institute of Neurological Disease, San
Francisco, CA 94141, USA
4 Present address: Department of Neuroscience, Genentech, South San
Francisco, CA 94080, USA
5 Present address: Department of Psychiatry, University of British Columbia,
Vancouver, BC V6T 1Z3, Canada
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