From the Department of Neuroplasticity, Research
Center on Aging and Adaptation, and the § Second Department
of Anatomy, Shinshu University School of Medicine, 3-1-1 Asahi,
Matsumoto 390-8621, Japan and the ¶ Department of Molecular
Neurobiology, Brain Research Institute, Niigata University School of
Medicine, Niigata 951-8585, Japan
Received for publication, November 29, 2000, and in revised form, December 26, 2000
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ABSTRACT |
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We cloned a cDNA encoding a novel
synGAP, synGAP-d (GenBankTM accession number
AB016962), from a rat brain cDNA library. The clone consisted of
4801 nucleotides with a coding sequence of 3501 nucleotides, encoded a
protein consisting of 1166 amino acids with >99% homology with 1092 amino acid overlaps to synGAP, and contained a 13-nucleotide insertion
to the previously reported synGAP mRNAs, which suggested that the
clone was a splice variant of synGAP. We also found that there are at
least seven variants in the 3' portion of the synGAP mRNA and that
they encoded five different protein isoforms. The coding sequence of
these C-terminal variants were classified into The synaptic Ras-GTPase activating protein synGAP (or
p135 synGAP) is a brain-specific protein of about 130 kDa, and the
synGAP cDNA was cloned independently by two groups (1, 2). There are three isoforms of synGAP proteins: synGAP-a or p135 synGAP, synGAP-b, and synGAP-c (GenBankTM accession numbers
AF058789, AF048976, AF058790, and AF050183, respectively). They are
different at the N termini, and p135synGAP has N-terminal and
C-terminal variants (GenBankTM accession numbers AF053938
and AF055883, respectively) (1). The synGAP protein is condensed in
postsynaptic density (PSD),1
probably through interaction with the PDZ domains of
PSD-95/SAP90 via its C-terminal amino acids. The protein negatively
regulates Ras activity by stimulating GTPase activity of Ras. Activity
of the synGAP is regulated by the phosphorylation by
Ca2+/calmodulin-dependent protein kinase II
(CaMKII); phosphorylation by CaMKII reduces the synGAP activity,
leading to Ras activation by increasing the GTP-bound form of Ras (1).
In this respect, synGAP is a key molecule that enables
cross-talking between Ca2+/calmodulin and Ras/MAPK
signaling pathways. Both signaling pathways are believed to function at
the postsynaptic sites in vivo (3, 4). A recent study
implicated Ras signaling and MAPK in the modulation of synaptic
transmission (5-8), and certain neurotrophins, such as brain-derived
neurotrophic factor, of which signals are processed by the Ras/MAPK
pathway system, are involved in synaptic plasticity (9-11). Thus,
similar to the Ras guanine nucleotide-releasing factor (or Ras guanine
nucleotide exchange factor) (Ras-GRF or CDC25) (12), synGAP may
contribute to synaptic plasticity. Neurofibromin 1 protein, a
Drosophila-specific RasGAP, is involved in the learning and
memory of the fruit fly (13).
In this study we cloned a novel isoform of synGAP, synGAP-d, and
further identified various isoforms of synGAP. We also identified the
nonphosphorylated form of CaMKII Materials--
cDNA RT-PCR--
Total RNA was isolated from the rat tissues
using ISOGEN according to the manufacture's instructions. The RNA
was incubated with DNase I (Qiagen) at 37 °C for 30 min and treated
by Isogen again. RT-PCR was essentially followed by the protocol of
the RNA PCR kit (Avian Myeloblastosis Virus) version 2.1. One
microgram of total RNA was used for the RT-PCR reaction. cDNA was
produced at 42 °C for 30 min, and the following PCR reaction was 28 cycles, unless stated otherwise, of 30 s of 94 °C, 30 s of
60 °C, and 1 min of 72 °C. We tested 7-35 cycles of the PCR and
confirmed that the quantity of amplified synGAP gene products did not
reach the saturating level at the end of 28 cycles of the reaction. PCR
products were separated by electrophoresis using 6% polyacrylamide gel
and visualized by ethidium bromide. The ethidium bromide-stained images
were printed by a CCD camera system (ATTO printograph, ATTO Bioscience
& Technology, Tokyo, Japan).
The two sets of PCR primers spanning the alternatively spliced
junction in the 3' tail portion of the synGAP were synthesized. Primer
set 1 corresponded to nucleotides 4500-4519 (5' primer, 5'-AGAAGCGCTTGAGGCAGCAG-3') and 4658-4639 (3'-primer,
5'-GTCGAGCAGCCTCTTCTTGG-3') of the synGAP-d cDNA, and primer set 2 corresponded to nucleotides 4319-4339 (5' primer,
5'-TACTCGAAGTCCATGGACGAG-3') and 4707-4687 (3' primer,
5'-TCACACGCGGGTTTGTTGGAC-3') of the synGAP-d cDNA. Primers for
glyceraldehyde-3-phosphate dehydrogenase were 5'-GCTGTGGGCAAGGTCATC-3' and 5'-CCTGGTCCTCAGTGTAGC-3' (sense and antisense, respectively); those
for rat ninjurin2 (GenBankTM accession number AF250322)
were 5'-ATGGAGTCAGACCGAGAAATC-3' and 5'-AGCCGCATGGCATTGGAC-3' (sense
and antisense, respectively).
Construction and Expression of the GST Fusion
Protein--
cDNAs corresponding to amino acid residues 729-1166
of synGAP-d protein, amino acid residues 787-1249 of synGAP-b protein, the C-terminal 21-amino acid portion of synGAP- Generation of Antibodies--
Antiserum specific to PSD protein
was produced by immunizing a rabbit using whole PSD proteins as
immunogen after sonicating the protein to maximally unfold the PSD structures.
Peptide Immunoprecipitation and Western Blotting--
PSD proteins were
solubilized at 4 °C for 2 h in a low stringent buffer (16) or
at 4 °C for 30 min in 50 mM Tris-HCl buffer (pH 7.5)
containing 137 mM NaCl, 2.7 mM KCl, 5 mM EDTA, 5 mM EGTA, 2% SDS, 50 mM
NaF, 100 µM sodium vanadate, and protease inhibitor mixture (solubilization buffer). In the latter case, five volumes of
dilution buffer containing 2% Triton X-100 (17) were further added and
incubated at 4 °C for an additional 2 h. The supernatant was
obtained after centrifugation at 10,000 rpm for 10 min, incubated at
4 °C for 3 h or overnight with antibodies, and then mixed for 2 h with protein G plus/protein A-agarose. Alternatively, the supernatant was incubated with antibody immobilized chemically to
protein G plus/protein A-agarose. The gel was pelleted and washed three
times, and immunoprecipitated proteins were separated by SDS-PAGE.
For Western blotting, proteins separated by SDS-PAGE were
electroblotted onto polyvinylidine difluoride membranes. The membranes were blocked with 5% skimmed milk, incubated with antibodies at room
temperature for 1 h, and immunostained using the chemiluminescent substrate. Chemiluminescence was captured and visualized with a CCD
video camera system (Atto Densitograph Lumino CCD AE-6930, ATTO
Bioscience & Technology, Tokyo, Japan).
Pull-down Assay--
PSD proteins (20 µg) were solubilized by
boiling for 5 min in the solubilization buffer (see above) containing 1 mM dithiothreitol, five volumes of Triton X-100-containing
dilution buffer were added, and the mixture incubated at 4 °C for
2 h. Supernatant was obtained by centrifugation at 10,000 rpm for
15 min and incubated at 4 °C for 2 h with peptide Cell Culture and Cell Staining--
Whole cerebral neocortices
of embryonic day 18 rats (Sprague-Dawley) were mechanically dissociated
and plated onto poly-D-lysine-coated dishes at a lower
density (500-800 cells/mm2). Cortical neurons were grown
with Dulbecco's modified Eagle's medium containing 0.5 mM
pure glutamine (Ajinomoto, Tokyo, Japan), 2% fetal bovine serum,
nutrient mixture N2, and 10 mM Hepes (pH 7.3) (18). The
following day the original medium was replaced by the medium lacking
serum (serum-free N2 medium). Cells (15 days on culture) were fixed
with 4% paraformaldehyde as described previously (18) and stained with
rabbit anti- Subcellular Fractionation--
Synaptic plasma membrane fraction
of the forebrain was prepared from Wistar rats (6 weeks old, male) by
the method of Cohen et al. (19) with modification by Wu
et al. (20), and the PSD fraction was successively prepared
as described previously (14, 21). The postsynaptic raft (or dendritic
raft) fraction (22) was obtained as a band between 0.32 and 1.0 M sucrose after sucrose gradient centrifugation to obtain
the PSD fraction. P1 (fraction containing nuclei and cell debris), P2
(crude mitochondrial fraction), and Syn (synaptosomal fraction) were
obtained during PSD isolation. Protein from these fractions was assayed
using the method of Lowry et al. (23) with bovine serum
albumin as the standard. All preparations were stored at Molecular Cloning of synGAP cDNA--
Antiserum specific to
PSD protein was produced by immunizing a rabbit using whole PSD protein
as the immunogen. The antibody recognized a number of proteins in the
PSD fraction and relatively few proteins in the other subcellular
fractions prepared from the brain, and it did not cross-react with
Triton X-100-insoluble cytoskeletal proteins of other tissues such as
the liver (data not shown). We used this antibody for the initial
screening of the cDNA library. About 500,000 plaques of the
601f DNA was 4801 bases long and contained a 3501-base open reading
frame (1124-4624). The nucleotide sequence of the coding region had a
>99% identity in >1092 amino acid overlaps with those of various
types of synGAPs. Therefore, we named the clone synGAP-d (GenBankTM accession number AB016962). The synGAP-d protein
most resembled synGAP-b (GenBankTM accession number
AF058790), and the differences between the two proteins are illustrated
in Fig. 1 (b and c). SynGAP-b protein was 58 amino acids longer than synGAP-d in the N terminus (Fig. 1c). Ala61 and Ala62 in the
synGAP-b were replaced by Tyr and Phe, respectively, in the synGAP-d.
SynGAP-d had deletions of two amino acids, Val1136 and
Lys1137, shown in synGAP-b. SynGAP-d mRNA had an
insertion of 13 nucleotides near the 3' tail of the coding sequence,
which made a frameshift, producing a C-terminal tail completely
different from that of synGAP-b (Fig. 1b). A splice variant
with this 13-base nucleotide had also been reported in p135synGAP by
Chen et al. (1) (GenBankTM accession number
AF055883). The C-terminal of the synGAP-d lacked the (S/T)XV
motif, an association motif with PSD-95. The synGAP-d had a pleckstrin
homology domain, a C2 domain, a GAP domain, and a Pro-rich
domain as found in other synGAP proteins.
Identification of synGAP mRNA by RT-PCR and
Classification of synGAP Isoforms--
The presence of synGAP-b
and synGAP-d mRNAs was confirmed by RT-PCR in the total RNA
prepared from the rat forebrain. Two primer sets, sets 1 and 2, used
for the RT-PCR were located surrounding the 13-oligonucleotide insert
in the synGAP-d (Fig. 2a).
RT-PCR using primer sets 1 and 2 produced two and three major bands, respectively (Fig. 3a). We
purified these bands by extracting the DNA from the cut bands,
subcloned them into pGEM-T easy vector (Fig. 3b), and
determined their nucleotide sequences. Unexpectedly, we obtained seven
different sequences with minor differences. These seven synGAP clones
of 3' tail coding sequences were classified based on sequence
similarity and named systematically as synGAP-
These seven synGAP mRNAs encoded five different synGAP protein
isoforms (Fig. 2b). There are at least four synGAP proteins in which the C-terminal tails were different. The C-terminal
(S/T)XV motif, a PDZ-binding motif, was present only in the
synGAP- Tissue Distribution and Developmental Changes in the Expression of
synGAP mRNA Variants--
Expression of synGAP variants was
examined in various tissues by RT-PCR using two different primer sets
(Fig. 4). All of the synGAP variants were
specifically expressed in the brain. The expression of the
Developmental changes in the expression of synGAP variants were
examined in the rat forebrain by RT-PCR (Fig.
5). The level of synGAP mRNAs was as
high as the adult level 1 day and 1 week after birth. The assay
condition was judged to be appropriate because expression of the nerve
injury-induced protein (ninjurin) (24, 25) assayed under the same
conditions showed developmental change with a gradual increase,
saturating at 6 weeks after birth. Amplified synGAP gene products did
not reach the saturating level at the end of 28 cycles of PCR.
Cellular Distribution of synGAP-
Then we examined the subcellular distribution of synGAP-
Next we examined the intracellular distributions of the synGAP- Identification of synGAP-
We first tested the interaction of the synGAP-
Next we surveyed the binding protein(s) by a co-immunoprecipitation
study (Fig. 10a). In this
case, we adopted the solubilization method of Lau et al.
(17), because this method enabled the efficient solubilization of PSD
proteins and also the maintenance, in part, of the undissociated
functional protein complexes. In fact, this method solubilized PSD
proteins nearly completely (data not shown). Thus, this solubilization
method was judged to be the most appropriate for the
immunoprecipitation of PSD proteins. We used antibody immobilized
chemically to agarose gel, because otherwise immunoglobulin heavy and
light chains hindered detection of some proteins in the polyacrylamide
gel. We tested some cytoskeletal proteins and PSD-enriched proteins by
Western blotting. The CaMKII
Finally we searched the direct binding partner using the pull-down
method (Fig. 10b). When solubilizing, PSD proteins were completely dissociated to monomeric conditions by boiling in the presence of SDS and dithiothreitol (17). This method enabled the
detection of a direct binding partner alone. We first detected the
pull-down materials separated in a polyacrylamide gel by silver staining and found that only one 50-kDa protein, comigrating with the
CaMKII
We next examined the effect of CaMKII autophosphorylation on the CaMKII
We have cloned a novel gene, synGAP-d, that encodes a synGAP
isoform with an unique C-terminal 21-amino acid sequence. The cloned
synGAP-d was most similar to synGAP-b except for the N-terminal and
C-terminal tail sequences. SynGAP-d protein is 58 amino acids shorter than synGAP-b in the N terminus and has a replacement near the
N terminus, and synGAP-d DNA has a deletion of six nucleotides near the
C-terminal portion of the protein and an insertion of 13 nucleotides in
the 3' portion of its coding sequence; this causes a frameshift and
produces a 21-amino acid-long C-terminal sequence unique to the
synGAP-d.
We also found that there are various synGAP isoforms of which the
C-terminal portions are unique. Three isoforms of the synGAP protein
with different N-terminal sequences have been reported: synGAP-a
(identical to p135synGAP), -b, and -c (1, 2) (Fig. 1c).
p135synGAP possesses putative splice variants with insertions in the
N-terminal or C-terminal region (1). The present finding increases the
repertoire of the synGAP protein isoforms.
We classified and named the synGAP isoforms identified in this study
based on the types of differences (Fig. 2). We omit "a," "b,"
"c," or "d" from the names. All these a, b, and c isoforms are
N-terminal variants as shown in Fig. 1c, whereas the
C-terminal sequences are common to all of these variants. Variation in
the C terminus found in synGAP-d may exist in combination with these N-terminal variations, because this C-terminal variation has also been
reported in p135synGAP (1) and synGAP-d, and anti- Furthermore we identified a specific binding protein for the synGAP- The present findings, together with previous observations (1, 2),
suggest that both the (S/T)XV motif-containing synGAP and
the 1,
2,
1,
2,
3,
4, and
, and synGAP-d was classified as the
1 form. The
previously reported synGAPs (synGAP-a, -b, and -c and p135synGAP) can
be classified as the
1 isoform. All isoforms were expressed
specifically in the brain. Unexpectedly, the
isoform, which lacks a
C-terminal PSD-95-binding motif ((S/T)XV), was more
restricted to the postsynaptic density fraction than the
motif-containing
1 isoform. The
isoform did not interact with
PSD-95 but specifically interacted with a nonphosphorylated
subunit of Ca2+/calmodulin-dependent protein
kinase II through its unique C-terminal tail.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit as a specific binding protein of synGAP-
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAP II library from rat
brain cortex and Bluescript plasmid were purchased from Stratagene (La
Jolla, CA); Isogen was purchased from Nippon Gene KK (Tokyo, Japan);
DNase I was from Qiagen (Tokyo, Japan); RNA PCR kit (Avian
Myeloblastosis Virus) version 2.1 was from Takara Co. Ltd.
(Ohtsu, Japan); pGEM-T easy vector was from Promega Corporation
(Madison, WI); keyhole limpet hemocyanine, protease inhibitor mixture,
and anti-actin were from Sigma; Affi-Gel 10 resin was from
Bio-Rad; anti-synGAP (anti-
1) (number 06-900) was from Upstate
Biotechnology, Inc. (Lake Placid, NY); anti-CaMKII
subunit antibody
was from Life Technologies, Inc.; anti-PSD-95 (P43520) was from
Transduction Laboratories (Lexington, KY); anti-neurofilament (70 kDa)
and anti-
-internexin were from Chemicon (Temecula, CA);
anti-
-tubulin, pGEX-4T-1 vector, and BL21 bacteria were from
Amersham Pharmacia Biotech; horseradish peroxidase-coupled goat
anti-rabbit immunoglobulin IgG (H&L) was from Calbiochem (San Diego,
CA); horseradish peroxidase-coupled goat anti-mouse Ig (G + A + M) was
from Cappel (West Chester, PA); SuperSignal, SuperSignal ULTRA, and
SuperSignal BLAZE chemiluminescent detection reagents for Western
blotting were from Pierce; polyvinylidine difluoride membrane was from
Millipore; and protein G plus/protein A-agarose was from Oncogene
Research Products (Cambridge, MA). Peptide
(KGAAPGPPRHG) was
chemically synthesized by Iwaki Glass (Funabashi, Japan). All other
chemicals were of reagent grade.
, the C-terminal 48-amino acid portion of synGAP-
2, and the C-terminal 35-amino acid
portion of synGAP-
were subcloned into pGEX-4T-1 vector. The
constructs were transfected into BL21 cells, and transcription was
induced by isopropyl-1-thio-
-D-galactopyranoside. The
whole cell lysates were analyzed by SDS-PAGE followed by staining with Coomassie Brilliant Blue R-250 or Western blotting.
, the C-terminal peptide of synGAP-
with
N-terminally added lysine (KGAAPGPPRHG), was immobilized to keyhole
limpet hemocyanine and used to immunize a rabbit to generate antiserum. Anti-synGAP-
antibody (anti-
) was affinity-purified by the
peptide
immobilized to Affi-Gel 10. Anti-CaMKII antibody and
anti-phosphorylated CaMKII peptide antibody were produced as reported
previously (14, 15).
coupled
to Affi-Gel 10 (bed volume, 5 µl). The gel was washed four times by
brief centrifugation and aspiration. The pull-down proteins separated
by SDS-PAGE were analyzed by silver staining or Western blotting.
, anti-PSD-95, or anti-synaptophysin antibody combined
with fluorescently labeled secondary antibodies. They were examined and
photomicrographed with a Zeiss Axioskop microscope.
80 °C
until use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAP
cDNA library were plated and screened at first, and we obtained
five different clones, of which three DNA sequences were identical to
the fodrin
subunit, plectin, and neurofilament H subunit, and one
of which, clone 601, had not previously been reported in the initial
data base. We proceeded with the cloning using clone 601 cDNA as a probe and obtained three independent cDNAs, 6011, 6012, and 6013. Finally, we obtained the sequence of 601f by compiling the 601, 6011, 6012, and 6013 clones (Fig.
1a).
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Fig. 1.
Cloning of a synGAP-d gene. a,
isolated clones with a partial sequence of synGAP-d. b,
comparison of synGAP-b and synGAP-d. Structures of synGAP-b and
synGAP-d DNAs are illustrated, and the differences in the nucleotide
sequences and amino acid sequences are indicated. c,
N-terminal amino acid sequences of various synGAP isoforms are shown.
The sequences common to all the isoforms and those common to the a, b,
and d isoforms are double-underlined and
underlined, respectively. ORF, open reading
frame.
(
1 and
2),
(
1,
2,
3, and
4), and
, as shown in Fig. 2a. We omitted "d" from the names, because this classification might also be used in other synGAP isoforms in addition to synGAP-d. The
presence of synGAP-
2, which was longer than the end point of the 3'
primer, was confirmed by RT-PCR using a different 3' primer, which
hybridized to the far 3' portion (data not shown), and was supported by
the finding that synGAP-
2 produced a C-terminal tail with 11 amino
acid sequences extremely similar to those of human RasGAP and nGAP
(GenBankTM accession number AL035702 and AF047711,
respectively). The single base g insertion seen in the
2,
1,
2, and
4 may not be an artificial by-product produced by PCR,
because this insertion was found in the 6013 clone. SynGAP-a, -b, and
-c, with common C-terminal sequences, can also be named as
synGAP-a
1, synGAP-b
1, and synGAP-c
1, respectively, when
adopting the present classification.
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Fig. 2.
A schematic diagram showing the
different nucleotide and amino acid sequences of synGAP C-terminal
variants. a, comparison of nucleotide sequences of the
synGAP mRNA isoforms. Only the 3' portions are shown (compare with
Fig. 1b). Vertical arrows indicate the positions
of stop codons. #1 and #2 refer to position of
primer sets 1 and 2. b, comparison of amino acid sequences
of synGAP C-terminal isoforms. Only the C-terminal portions are shown.
The C-terminal sequence of the isoform is the same as that in the
p135synGAP C-terminal variant (1). Peptide
and
Peptide
indicate the sequences that were used to produce
anti-
1 and anti-
, respectively. Peptide
was also used for the
pull-down assay.
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Fig. 3.
Separation of alternatively spliced variants
of synGAP cDNA by polyacrylamide gel electrophoresis. a,
RT-PCR using RNA isolated from the rat forebrain as a template. The
lengths (base number) of the isoforms are shown in
parentheses. b, various synGAP cDNA clones
subcloned into pGEM-easy T vector were digested with EcoRI
and separated in polyacrylamide gel. The lane marked FB
shows the RT-PCR products from forebrain mRNA using primer set 2. An arrowhead indicates the band in the forebrain
sample.
1 protein. All of the
isoform proteins showed the same
C-terminal tail sequence, which was the same as that found in the C
terminus of p135synGAP (1).
form was
extremely low compared with those of
and
, and bands close to
but slightly larger than the
band were detected in the PCR of
non-neuronal tissues. Expression profiles of synGAP-
and
were
similar between the forebrain and cerebellum, whereas the expression
levels of both genes in the cerebellum were lower than those in the
forebrain.
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Fig. 4.
Tissue distribution of synGAP isoforms
revealed by RT-PCR. RT-PCR was performed using primers sets 1 (a) and 2 (b). The total RNA was isolated from
forebrain (FB), cerebellum (CBL), heart
(He), lung (Lu), liver (Li), skeletal
muscle (Mu), and testis (Te). An
arrowhead indicates the position of the band.
G3PDH, glyceraldehyde-3-phosphate dehydrogenase.
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Fig. 5.
Developmental changes in the synGAP isoform
expression revealed by RT-PCR. RT-PCR was performed using primer
set 1 (a), primer set set 2 (b), and
primers for rat ninjurin (rNinj2, GenBankTM
accession number AB016962) (c), followed by acrylamide
(a and b) or agarose (c) gel
electrophoresis. Total RNA was prepared from the rat forebrain at
postnatal day 1 (1d) through 20 weeks (1w,
2w, 3w, 4w, 6w, and
20w). The numbers of PCR cycles are shown on the
right in a, and 28 cycles were used for
b and c. G3PDH,
glyceraldehyde-3-phosphate dehydrogenase.
--
Before using anti-
in
immunochemical and immunocytochemical studies, we first tested the
specificity of the antibody by Western blotting (Fig.
6). Anti-
reacted specifically with
the expressed GST-synGAP-
1 C-terminal half protein but not with the
expressed GST-C-terminal half synGAP-
protein (Fig. 6a).
The antibody also reacted with the GST 21-amino acid C-terminal peptide
of synGAP-
but not with GST itself, the GST-
2 C-terminal peptide,
nor the GST-
C-terminal peptide (Fig. 6b). Thus, the
anti-
specifically reacted with the C-terminal tail portion of
synGAP-
. Anti-synGAP antibody purchased from Upstate Biotechnology,
Inc. (termed here as anti-
1) reacted with the GST C-terminal half
synGAP-
1 protein but not with the GST C-terminal half synGAP-
protein, the GST-
2-C-terminal peptide, nor the GST-
C-terminal
peptide (Fig. 6). Thus, these antibodies reacted mutually exclusively
and specifically to synGAP-
1 and -
, respectively.
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Fig. 6.
Specificity of the
anti- 1 and anti-
.
a, GST fusion proteins were expressed in BL21 cells by
induction with isopropyl-1-thio-
-D-galactopyranoside
(IPTG), and whole lysates were Western blotted by anti-
1
or anti-
. GST-
-C and GST-
1-C contain amino acid residues
729-1166 of synGAP-d
1 and amino acid residues 787-1249 of synGAP-b
(
1 form), respectively. b, GST fusion proteins containing
various isoforms were expressed, and whole lysates were Western blotted
by anti-
or anti-
1. GST-
, GST-
2, and GST-
contain the
C-terminal 21 amino acids of the
isoform, the C-terminal 48 amino
acids of the
2 isoform, and the C-terminal 35 amino acids of
the
isoform, respectively. Molecular masses are shown on the
left in kDa.
1
and -
(Fig. 7). Both antibodies
detected bands in the 130 kDa region that were considered to be
synGAP proteins. The distribution of synGAP-
was highly restricted
in the PSD fraction, which was confirmed by the picture taken at higher
sensitivity (Fig. 7b, lower panel). SynGAP-
1
was also highly enriched in the PSD but was also detected, although the
content was low, in the other subcellular fractions: postsynaptic (or
dendritic) raft, synaptic plasma membrane, synaptosome, and P2 and P1
fractions. The distribution patterns of PSD-95 and the CaMKII
subunit verified the subcellular fractionation, with both proteins
being enriched in the PSD fraction.
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Fig. 7.
Localization of the
1 and
isoforms in the
various subcellular fractions. Proteins (20 µg) of various
fractions prepared from the rat forebrain were separated on 10%
polyacrylamide gel and detected with anti-
1 (a), anti-
(b), anti-PSD-95 (c), or anti-CaMKII
(d). The lower panel of b shows a
picture taken at higher sensitivity. S, Syn, and
PSR refer to soluble, synaptosome, and postsynaptic raft
(22) fractions, respectively. Molecular masses are shown on the
left in kDa. Only the regions close to PSD-95 or CaMKII band
are shown in the lower two gels because of space
limits.
1 and
-
in cultured neurons (Fig. 8).
Immunoreactivity for synGAP-
was localized in the dendrites
including spines and co-localized with PSD-95, a postsynaptic marker in
the forebrain (26). Their co-localization in the spines was shown
typically in Fig. 8 (insets in a and
b). The distribution of synGAP-
and synaptophysin, a presynaptic marker (27), was compared by double immunofluorescent staining (Fig. 8c). Synaptophysin immunoreactivity was
distributed in a patch-like pattern and localized in close proximity to
synGAP-
. Patchy stainings of synaptophysin are believed to be
localization of presynaptic terminals. Thus, the postsynaptic
localization of synGAP-
was indicated by immunostaining of cultured
cortical neurons as well.
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Fig. 8.
Localization of the isoform in cultured cortical neurons. Subcellular
localization of the novel synGAP isoform was examined in the cultured
cerebral neurons. PSD-95 (a) and the
isoform
(c) were finally labeled with Texas red (red),
and synGAP-
(b) and synaptophysin (c) were
labeled with fluorescein isothiocyanate (green).
Insets in a and b show the area around
a typical spine indicated by arrows.
-binding Protein--
SynGAP-
1
possessed the C-terminal (S/T)XV motif, which was suggested
to bind to the first or second PDZ domain of the PSD-95 protein (28).
Therefore, synGAP-
1 is suggested to be anchored to PSD with
intervention by PSD-95. Unexpectedly, synGAP-
was more
restricted to the PSD fraction than synGAP-
1, although synGAP-
lacked the PSD-95-binding motif (Fig. 7). This finding suggested searching the synGAP-
-binding protein(s) in the PSD proteins.
with PSD-95 in the
PSD by co-immunoprecipitation assay (Fig.
9). SynGAP-
1, but not synGAP-
, was
co-precipitated when PSD proteins were immunoprecipitated with
anti-PSD-95 antibody. In reverse, PSD-95 was co-precipitated when PSD
proteins were immunoprecipitated with anti-
1 but not when they were
immunoprecipitated with anti-
.
View larger version (18K):
[in a new window]
Fig. 9.
Co-immunoprecipitation of synGAP isoforms
with PSD-95. PSD proteins (300 µg) were solubilized at 4 °C
for 2 h with low stringent buffer and then immunoprecipitated with
either anti-PSD-95 ( -PSD-95) antibody,
anti-
1, or anti-
. Western blotting was carried out using various
antibodies shown on the right. The target protein bands for
Western blotting are indicated by asterisks. IP
and WB refer to immunoprecipitation and Western blotting,
respectively.
subunit,
-actin, and
-internexin
were co-immunoprecipitated with synGAP-
. The amounts of these
co-immunoprecipitated proteins were low compared with synGAP-
,
probably because of dissociation of the functional complexes, to some
extent, after solubilization of PSD proteins with SDS. These proteins
were not precipitated when using only protein G plus/protein A-agarose
gel that was treated in a similar way to immobilize the antibody to the
gel. Thus, these precipitations were judged to be specific. The
-tubulin and CaMKII
subunit were not co-immunoprecipitated with
synGAP-
.
View larger version (32K):
[in a new window]
Fig. 10.
Survey of the isoform-binding proteins by co-immunoprecipitation and pull-down
assays. a, PSD proteins (20 µg) were solubilized in a
solubilization buffer containing 2% SDS at 4 °C for 30 min, five
volumes of 2% Triton X-100 were added, and the mixture was
immunoprecipitated (IP) with anti-
immobilized chemically
to protein G plus/protein A-agarose. Protein G plus/protein A-agarose
without a linked antibody was used as a control. Western blotting was
carried out using various antibodies. Target proteins for Western
blotting are shown on the right. Immunoprecipitated and
control materials were processed simultaneously under similar
conditions. b, PSD proteins (20 µg) were solubilized for 5 min by boiling in the solubilization buffer containing 2% SDS and 1 mM dithiothreitol, five volumes of 2% Triton X-100 were
added, and the mixture was incubated with peptide
(C-terminal
10-amino acid peptide with N-terminally added lysine; see Fig.
1b) immobilized to Affi-Gel 10. Pull-down material was
separated in polyacrylamide gel and stained with silver or
immunoblotted with various antibodies specific to the proteins shown on
the right.
-IN and NF70
refer to
-internexin and neurofilament of 70 kDa,
respectively.
subunit in the PSD fraction, was pulled down (Fig. 10b, left panel). We then confirmed the pull-down
material as a CaMKII
subunit by Western blotting.
-Internexin
and
-actin were not detected, which was different from the findings
of the co-immunoprecipitation study (Fig. 10a). The CaMKII
subunit was not bound to peptide
. The binding of the CaMKII
subunit was judged to be specific because the band was not observed
when using only Affi-Gel or when free peptide
(500 µg/tube) was
added during the pull-down.
subunit binding to peptide
. CaMKII in the PSD fraction was
autophosphorylated in the presence of Ca2+ and calmodulin
and incubated with peptide
immobilized to Affi-Gel 10, as shown in
Fig. 10b. Autophosphorylation of the CaMKII was confirmed by
Western blotting using anti-phosphorylated CaMKII peptide antibody
(Fig. 11, PSD panels). The
autophosphorylated CaMKII
subunit was not pulled down by peptide
, under similar conditions where the nonphosphorylated CaMKII
subunit was pulled down (Fig. 11, Pulldown panels).
View larger version (28K):
[in a new window]
Fig. 11.
Effect of CaMKII autophosphorylation on the
CaMKII subunit binding to peptide
. PSD proteins (20 µg) were incubated at
25 °C for 10 min with Ca2+ and calmodulin either in the
presence or absence of 500 µM of ATP, and the reaction
was terminated with solubilization buffer for SDS-PAGE
containing SDS and mercaptoethanol. The PSD proteins were processed for
the pull-down assay as described in the legend for Fig. 10b.
Western blotting was done using either anti-phosphorylated CaMKII
peptide antibody (
-P-CaMKII) or anti-CaMKII
antibody (
-CaMKII). The lanes
marked as PSD show the Western blotting of PSD proteins (20 µg) that were incubated in the same way but without pull-down
treatment. The arrowhead indicates the P-CaMKII
subunit,
and
and
indicate the
and
subunits
of CaMKII, respectively. WB refers to Western
blotting.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-immunoreactive bands with slightly different molecular sizes were detected as shown in
Fig. 10a. Heterogeneity of the synGAP proteins may add differential roles to these proteins in various brain regions.
isoform as a CaMKII
subunit. The C-terminal portion of synGAP
proteins is important, especially for their targeting to specific sites
in the neuronal cells, because previously reported synGAP possesses the
(S/T)XV motif in the C-terminal end, and the protein
interacts with the first or second PDZ domain of PSD-95 via this motif.
Thus, the (S/T)XV motif-containing synGAPs can be localized
to PSD through this interaction. The difference in the binding partners
between the (S/T)XV motif-containing synGAP and the
synGAP-
form may explain the difference in their subcellular distribution. Unexpectedly, the (S/T)XV motif-lacking
isoform was more restricted to the PSD fraction than the
(S/T)XV motif-containing synGAP, which suggested that there
should be a specific binding molecule in the PSD fraction. In fact, the
unique synGAP-
C-terminal sequence was found to bind to the CaMKII
subunit, which was a well known PSD protein (14). Therefore,
synGAP-
isoforms may be concentrated in the PSD fraction through
binding to the CaMKII
subunit. The interaction between the CaMKII
subunit and the synGAP
form could be regulated by synaptic
activity, because peptide
did not interact with the
autophosphorylated
subunit of CaMKII (Fig. 11).
isoform are localized in the postsynaptic sites including spines. Close interaction of synGAP-
with the CaMKII
subunit may
have specific functional significance in addition to that as a
substrate for CaMKII. The functional significance of this interaction
awaits further study.
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FOOTNOTES |
---|
* This work was supported in part by a grant-in-aid for scientific research from the Japanese Ministry of Education, Science and Culture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB016962.
To whom correspondence should be addressed: Dept. of
Neuroplasticity, Research Center on Aging and Adaptation, Shinshu
University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan.
Tel.: 81-263-37-2683; Fax: 81-263-37-2725; E-mail:
suzukit@sch.md.shinshu-u.ac.jp.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M010744200
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ABBREVIATIONS |
---|
The abbreviations used are:
PSD, postsynaptic
density;
anti-1, anti-synGAP-
1 antibody;
anti-
, anti-synGAP-
antibody;
CaMKII, Ca2+/calmodulin-dependent protein kinase II;
GAP, GTPase-activating protein;
GST, glutathione
S-transferase;
MAPK, mitogen-activated protein kinase;
RT, reverse transcriptase;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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