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INTRODUCTION |
The central nervous system is established through various
developmental steps, which include determination of cell fate to the
neuronal lineage, proliferation, and differentiation of precursor cells, migration to defined regions, and formation of neuronal interactions. Most neurons derived at the embryonic stage construct a
number of synapses during the early postnatal development of the brain,
which accompanies programmed cell death of excessively produced neurons
in order to form a proper neuronal network (1-3). A number of
molecules such as humoral factors (4, 5), receptors (6, 7), adhesion
molecules (8, 9), and transcriptional factors (10, 11) have been
reported to participate in brain development at various stages. The
molecular mechanisms for the formation of structural and functional
nervous systems are complex, and our knowledge is limited. Therefore,
we consider that many as yet unidentified genes would contribute to the
regulation of brain development at the embryonic and postnatal stages.
In the present study, we describe the isolation and characterization of
a novel gene enriched in the embryonic and postnatal stages of rat
brain development. Our cloned gene encoded an
ER1-Golgi localized protein
that was associated with GRP78, one of the heat shock proteins. We
suggest that our cloned GRP78-binding protein (GBP) plays a role in the
functional development of neurons.
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EXPERIMENTAL PROCEDURES |
Molecular Cloning of Rat GBP--
The expressions of mRNAs
in the hypothalamus, including the suprachiasmatic nucleus, of
postnatal rats (2 weeks after birth) and adult rats (10 weeks) were
compared by PCR-selected cDNA subtraction according to the
manufacturer's instructions (Clontech) (12).
Briefly, poly(A)+ RNA (0.5 µg) was extracted from the
brains at each age, converted to double-stranded cDNA (ds cDNA)
by incubation with reverse transcriptase,
5'-AAGCAGTGGTAACAACGCAGAGTACGCGGG-3' (10 µM), and
5'-AAGCAGTGGTAACAACGCAGAGTACN
1N-3' (10 µM). The ds cDNA was digested to blunt-ended
fragments by treatment with RsaI, and only ds cDNA from
2 week-rats was ligated with oligonucleotides:
5'-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3' (adapter1) or
5'-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT-3' (adapter2R). For the 1st hybridization, each adapter-ligated cDNA was respectively hybridized with an excessive amount of cDNA from adult rats. Following the 2nd hybridization by combination with each
1st hybridized solution, 2-week-specific cDNAs were amplified by
PCR using the 1st primer, 5'-CTAATACGACTCACTATAGGGC-3', 2nd primer1, 5'-TCGAGCGGCCGCCCGGGCAGGT-3', and 2nd primer2,
5'-AGCGTGGTCGCGGCCGAGGT-3'. The secondary PCR products were
cloned using the T/A cloning system and sequenced on both strands using
an ABI PRISM dye terminator cycle sequencing kit (PerkinElmer Life
Sciences) with T7 and SP6 primers. To obtain the full-length cDNA
of GBP, we produced 5'-RACE and 3'-RACE PCR products from rat forebrain
RNA and sequenced them as described above.
Cell Lines, Cell Culture, and Transfection--
Neuro2a and COS7
cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, and PC12 cells were cultured
in Dulbecco's modified Eagle's medium supplemented with 5% fetal
bovine serum and 5% calf serum. Transfection with each plasmid (2-6
µg) was performed using LipofectAMINE Plus (Invitrogen) according to
the manufacturer's instructions. Stable cell lines transfected with
each plasmid were selected and maintained by G418
(Clontech) at concentrations of 0.5-2.0 mg/ml.
Northern Blotting and RT-PCR Analysis--
Total RNA (5-10
µg) from various rat tissues was purified, resolved by agarose gel
electrophoresis under denaturing conditions, and blotted onto nylon
membranes. To investigate the expression of GBP mRNA in
the various brain regions and in the whole brain at indicated ages, we
purchased RNA-blotted membrane from Seegene Co. Ltd. An
-32P-radiolabeled rat GBP cDNA fragment containing
nucleotides 2972-3248 was hybridized in the
Clontech hybridization solution. After washing, the
membranes were exposed to a Fuji imaging plate and analyzed with
BAS2000 (Fuji Film, Japan). In order to control for the amount of RNA
present, the membranes were stripped and re-hybridized with a G3PDH
cDNA probe.
RT-PCR was also performed to estimate the mRNA levels of various
tissues and cell lines as described previously (13). Briefly, 0.5 µg
of total RNA was converted into cDNA by reverse transcriptase, and
specific DNAs were amplified by PCR using Taq polymerase. The PCR primers used in this study were as follows: GBP
sense primer, 5'-GGCTCCCAGTGCTTCAGAGA-3'; GBP
antisense primer1, 5'-TCCAGGGGCCTGGAATCCGG-3'; GBP antisense
primer2, 5'-GATCCTCTCCATGTAGTTCCGAA-3'; GBP antisense primer3, 5'-TGCAGTTCCACTACACAGGC-3'; G3PDH sense primer,
5'-TCCACCACCCTGTTGCTGTA-3'; and G3PDH antisense primer,
5'-ACCACAGTCCATGCCATCAC-3'. The level of GBP mRNA
in tissues and cell lines was detected using GBP sense primer and GBP antisense primer1 or -2, and T7 primer and
GBP antisense primer3 were used to detect the level of
Myc-tagged GBP mRNA in Neuro2a cells. The typical
reaction conditions were 0.5 min at 95 °C, 0.5 min at 60 °C, and
1 min 72 °C. The results shown represent 18-30 cycles of
amplification. After amplification, the cDNAs were separated by
electrophoresis in 2.0% agarose gels and visualized using ethidium bromide.
In Situ Hybridization--
Antisense and sense RNA probes were
prepared by in vitro transcription of an RT-PCR-amplified
fragment of GBP cDNA (nucleotides 1122-3248) subcloned into
pBSSK(+) and synthesized from these cDNA templates with T3 or T7
RNA polymerase and a digoxigenin (DIG)-labeling mixture (Roche
Molecular Biochemicals) (14). Postnatal (2-week) and adult
(10-week) rat brains were perfused and fixed with 4% paraformaldehyde
in 0.1 M phosphate buffer at 4 °C, and coronal sections
(30 µm in thickness) were cut on a cryostat. The sections were
treated with 0.1 mg/ml proteinase K (Sigma), 10 mM Tris-HCl
buffer (pH 7.4), and 10 mM EDTA for 10 min at 37 °C, 4%
paraformaldehyde in 0.1 M phosphate buffer for 5 min, and
0.25% acetic anhydride in 0.1 M triethanolamine for 10 min. The sections were then incubated in hybridization buffer
containing a DIG-UTP-labeled GBP riboprobe for 12 h at 60 °C. Following the hybridization, the sections were washed
sequentially in 2× SSC containing 50% formamide, RNase solution, and
0.4× SSC. Finally, they were processed for DIG-coloring steps using an
anti-DIG antibody, 4-nitro blue tetrazolium chloride, and
5-bromo-4-chloro-3-indolyl phosphate as described previously (14).
Fluorescence Microscopy--
COS7 cells were seeded on glass
coverslips and transfected with the indicated plasmid as described
above. For the detection of cells expressing Myc-tagged GBP, cells were
fixed with PBS containing 4% formaldehyde for 15 min. After washing,
the cells were permeabilized with PBS containing 0.2% Triton X-100 for
5 min and incubated with an anti-Myc antibody for 1 h. The cells were then incubated with a fluorescein isothiocyanate-conjugated anti-mouse IgG as the secondary antibody. The ER and Golgi apparatus were visualized by staining with BODIPY 558/568-brefeldin A for 30 min
just before fixation with PBS containing 4% formaldehyde (16).
In all cases, the cells were mounted in Vectashield (Vector
Laboratories) and observed by fluorescence microscopy (Olympus, Japan).
GST Pull-down Assay--
PC12 cells in 10-cm dishes were
preincubated with methionine-free medium for 60 min, and then 25 µCi
of [35S]methionine was added. After 6 h of
incubation, the cells were lysed with lysis buffer (Tris-HCl (pH 8.0),
150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM PMSF, and protease inhibitors) and centrifuged at 15,000 rpm for 10 min. The collected supernatants were rotated with the
C-terminal region of GBP-fused GST- or GST-immobilized beads overnight
at 4 °C (15). After extensive washing with lysis buffer, the
proteins bound to each resin were eluted with 50 mM Tris-HCl (pH 8.0) containing 10 mM GSH and separated by 8%
SDS-PAGE. After drying, the gels were exposed to a Fuji imaging plate
and analyzed with BAS2000. To extract and analyze the proteins bound to
GST-fused GBP, SDS-PAGE gels were stained with Sypro Ruby (Molecular Probes) or Coomassie Brilliant Blue, and the stained regions were digested in trypsin solution at 35 °C for 20 h for analysis by liquid chromatography/mass spectrometry/mass spectrometry
(Aproscience, Japan).
Western Blotting and Co-immunoprecipitation--
COS7 cells in
10-cm dishes were transfected with pcDNA3.1/GBP-myc.his
(Invitrogen) and resuspended in lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and protease inhibitors) containing 1%
Triton-X100 or Nonidet P-40 for 20 min on ice. After centrifugation at
15,000 rpm, the detergent-soluble fraction was collected. The
detergent-resistant fraction was resuspended and sonicated in an equal
amount of lysis buffer. An aliquot of each sample was mixed with an
equal amount of SDS sample buffer and boiled for 3 min. Western
blotting was performed as described previously using the anti-Myc
monoclonal antibody 9E10 (1:500) (Santa Cruz Biotechnology).
For co-immunoprecipitation, COS7 cells in 10-cm dishes were transfected
with pcDNA3.1/myc.his or pcDNA3.1/GBP-myc.his together with
pflag-GRP78. 24 h after the transfection, the cells were dissolved
in lysis buffer, and debris was discarded after centrifugation. Anti-Myc or anti-FLAG M2 monoclonal antibodies (Sigma) were added to
the whole cell lysates and rotated overnight at 4 °C. Then 20 µl
of protein G-Sepharose beads were added and rotated for a further
1 h. After washing the beads four times with 20 mM
Tris-HCl (pH 8.0) containing 150 mM NaCl, 1 mM
EDTA, 1 mM PMSF, 0.2% Nonidet P-40, and protease
inhibitors, the beads were resuspended in SDS sample buffer, boiled,
and analyzed by Western blotting. Each protein was detected with the
anti-Myc antibody or anti-FLAG antibody, respectively.
Measurement of Cell Viability--
Cell viability was determined
by the MTT assay as described previously (17). Briefly, each Neuro2a
cell line stably transfected with Myc-tagged GBP or empty vector
(3 × 104 cells/well) was cultured in the presence or
absence of 10% fetal bovine serum for the indicated time, and the MTT
solution was added to each well and incubated for 4 h. The cells
were lysed with 0.04 M HCl in isopropyl alcohol, and the
difference in absorbance between 595 and 655 nm was measured as an
indicator of cell viability.
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RESULTS |
Molecular Cloning of GBP--
To identify novel genes in the
hypothalamus during postnatal brain development, the mRNA
expressions in the forebrains of postnatal day (P) 14, and adult rats
at 10 weeks were compared by PCR-selected differential screening
because retinal axons grow into the hypothalamus (retinohypothalamic
tract) during the postnatal stage, and this reaches a peak at around 2 weeks (18, 19). We screened the partial sequence of a novel gene, which
was expressed at a higher level in postnatal brain compared with adult
brain. To obtain the full-length cDNA of this gene, we produced
5'-RACE and 3'-RACE PCR products from the rat forebrain RNA, and fused them together. The nucleotides and deduced amino acid sequence of this
rat cDNA are shown in Fig. 1.
According to a search with BLAST, this sequence was almost 90%
identical to a mouse one (GenBankTM accession number
BC006896). A related sequence in human (GenBankTM
accession number AK023577) was partial and about 80% identical to rat
GBP (nucleotides 1077-3066). The cDNA encoded 1021 amino acids
with a predicted molecular mass of 110 kDa. A motif search of this
protein with PROSITE predicted two transmembrane regions (amino acid
residues (aa) 14-40 and 852-868), a proline-rich region (aa
739-780), and a glutamic acid-rich region (aa 809-831). In addition,
several N-glycosylation, N-myristoylation, and
phosphorylation sites were deduced (data not shown).

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Fig. 1.
Nucleotides and predicted amino acid sequence
of rat GBP. The nucleotides and predicted amino acid sequence of
GBP cDNA are shown. Putative transmembrane (squares),
proline-rich (underline), and glutamic acid-rich
(double underline) regions are indicated.
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Enrichment of GBP mRNA in the Rat Brain--
Northern blotting
and RT-PCR analysis were performed to examine the distribution of
GBP mRNA in tissues. As shown in Fig. 2A, enriched expression of
GBP mRNA was observed in the brain compared with other
tissues. The expression in the lung was moderate, and weak signals were
detected in the heart, liver, kidney, spleen, testis, and muscle.
Within the brain, Northern blotting of total RNA isolated from
different brain regions showed that GBP was expressed at
higher levels in the cerebral cortex, thalamus, and cerebellum and at
lower levels in the pons, medulla oblongata, and spinal cord (Fig.
2B).

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Fig. 2.
Tissue distribution of GBP
mRNA by Northern blotting. Total RNAs isolated from
different rat tissues. A, lane 1, whole brain;
lane 2, heart; lane 3, lung; lane 4,
liver; lane 5, kidney; lane 6, spleen; lane
7, testis; and lane 8, muscle, and different brain
regions. B, lane 1, olfactory; lane 2,
cerebral cortex; lane 3, hippocampus; lane 4, thalamus; lane 5, hypothalamus; lane 6, midbrain;
lane 7, cerebellum; lane 8, pons and medulla
oblongata; and lane 9, spinal cord were hybridized with a
radiolabeled 277-bp fragment of GBP as described under "Experimental
Procedures." The same membrane was rehybridized with a radiolabeled
fragment of G3PDH.
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The temporal changes in the expression of GBP mRNA in
the whole brain from the embryonic stage to the adult were examined (Fig. 3). The expression of
GBP was faintly detected at E12 and gradually increased
until P0. After birth, the GBP level was almost maintained
from E20.5 to 2 weeks, and then continuously declined until 12 months.
Densitometric analysis of Northern blotting normalized by
G3PDH mRNA intensity revealed that the expression of
GBP in the 12-month-old rat brain had decreased by about
70% compared with that in postnatal brain.

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Fig. 3.
GBP mRNA levels during rat
brain development. The expression levels of GBP
mRNA in the rat brain at the indicated different embryonic
(E) (A) and postnatal (P)
(B) stages were analyzed by RT-PCR and Northern blotting,
respectively, as described under "Experimental Procedures."
C, the relative mRNA level of GBP in
B was calculated by comparison of G3PDH-normalized values
with the level of E20.5. D, day; W, week.
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To elucidate the expression pattern of GBP
mRNA in embryonic, postnatal, and adult rat brain brains, we
performed in situ hybridization histochemistry. The
expression of GBP mRNA was observed predominantly in the
gray matter, and the appearance and distribution of the GBP-expressing
cells suggested that GBP mRNA was expressed in neurons
but not in glial cells. GBP-expressing cells were detected in various
regions of the rat forebrain, including the hypothalamus, and cells
strongly expressing GBP were observed in the thalamus, cerebral cortex,
amygdala, and cerebellum (Fig. 4). In
agreement with the results of the Northern blotting, the
expression of GBP mRNA in aged rat brain was
ubiquitously decreased in comparison with that in postnatal developing
brain.

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Fig. 4.
Localization of GBP mRNA
in the rat brain by in situ hybridization.
GBP mRNA localization was detected by in situ
hybridization with a DIG-labeled antisense RNA probe derived from the
coding region of GBP. At 2 weeks, a number of GBP
mRNA-expressing cells were widely distributed in the rat
hypothalamus (a), thalamus (c), and cerebral
cortex (e), and their expressions were stronger than those
in adulthood (hypothalamus (b), thalamus (d), and
cerebral cortex (f)). g shows a higher
magnification of the rectangle in e. Control hybridization
experiments were performed using a sense RNA probe (h). The
scale bars represent 200 µm. OC, optichasm;
3V, third ventricle; LV, lateral ventricle.
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Immunostaining of COS7 Cells Transfected with GBP--
Next, we
prepared Myc- and GFP-tagged GBP constructs, and we investigated the
expression and intracellular localization in intact cells by
immunoblotting and fluorescence microscopy. As shown in Fig.
5, Myc-tagged GBP in COS7 cells was
almost solubilized by 1% Triton X-100 or Nonidet P-40, and bands
corresponding to a 170kDa protein were detected by immunoblotting. COS7
cells transfected with Myc-tagged GBP or GFP-tagged GBP showed similar
localization of GBP (Fig. 5, A and C).
Furthermore, we confirmed that GFP-tagged GBP in COS7 cells almost
overlapped with the fluorescent probe (BODIPY)-labeled brefeldin A
which specifically stains the ER and Golgi apparatus (Fig.
5C). To characterize further the localization of GBP, we
constructed three deletion mutants, consisting of the N-terminal region
(aa 1-411), the middle region (aa 375-817), and the C-terminal region
(aa 666-1021) and established stable cell lines expressing full-length
GBP or a mutant GBP. As shown in Fig. 6,
only the C-terminal region of GBP among the three mutants had a unique
cellular localization, and its pattern was similar to that of
full-length GBP.

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Fig. 5.
Cellular localization of GBP in COS7
cells. A, COS7 cells were transiently transfected
with Myc-tagged GBP. 24 h later, the cells were fixed and
incubated with an anti-Myc monoclonal antibody. The cells were then
incubated with a fluorescein isothiocyanate-conjugated anti-mouse IgG
and analyzed by fluorescence microscopy. B, 24 h
after transfection, COS7 cells were lysed with 1% Triton X-100 or
Nonidet P-40 (NP-40). After centrifugation, the supernatant
(sup), pellet (p), and total homogenates
(H) were immunoblotted as described under
"Experimental Procedures." C, 24 h after
transfection with GFP-tagged GBP or GFP vectors, the cells were
incubated with BODIPY-brefeldin A for 30 min. The cells were fixed and
analyzed by fluorescence microscopy.
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Fig. 6.
COS7 cells stably expressing GFP-tagged
full-length, deletion mutants, and empty vectors were analyzed by
fluorescence microscopy. Each GFP-GBP fusion protein corresponding
to the indicated residues (A) was stably expressed in COS7
cells and observed by fluorescence microscopy (B).
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Association Analysis between GBP and GRP78--
To investigate the
roles of our cloned gene, we used affinity methods to identify its
binding proteins. An affinity matrix was prepared by immobilization of
a GST-GBP (aa 606-913) fusion protein on glutathione-Sepharose beads,
because the roles of this region were somewhat deduced by the
observation of the COS7 cells transfected with the GFP-tagged GBP (aa
666-1021) plasmid as described above. A band corresponding to an
~45-kDa protein was mainly detected by Coomassie Brilliant Blue
staining, although this cDNA encoded 248 amino acids with a
predicted molecular mass of about 54 kDa (Fig.
7A). We considered that the
GST-fused C-terminal region of GBP in Escherichia coli would
be digested during the extraction and immobilization to the
GSH-Sepharose beads. Incubation of
[35S]methionine-labeled PC12 cell lysates with the
immobilized GST-GBP-(606-1022) fusion protein resulted in the binding
of several proteins (Fig. 7B). Among these proteins, we
identified two proteins with molecular masses of about 70 kDa from
non-labeled PC12 cell lysates (see "Experimental Procedures").
Liquid chromatography/mass spectrometry/mass spectrometry analysis of
the fragments of the lower and upper bands and comparison with the
sequences in the GenBankTM Data Bank revealed almost
complete identity to rat Hsc73 and GRP78, respectively.

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Fig. 7.
Analysis of the association between GBP and
GRP78. A, the GST-fused C-terminal region of GBP
expressed in E. coli was extracted by GSH-conjugated
Sepharose beads, resolved by SDS-PAGE, and stained with Coomassie
Brilliant Blue. B, the GST-fused C-terminal region of
GBP or GST immobilized beads was incubated with
[35S]methionine-labeled PC12 lysates as described under
"Experimental Procedures." After washing and elution, the proteins
bound to each resin were resolved by SDS-PAGE and visualized with
BAS2000. C, COS7 cells were co-transfected with
different combinations of expression constructs as indicated.
Co-immunoprecipitation was performed using the anti-Myc monoclonal
antibody as described under "Experimental Procedures."
Immunoprecipitates (IP) were analyzed using the anti-Myc or
anti-FLAG antibodies, respectively. IB, immunoblot;
Ab, antibody.
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To investigate the association with Hsc73 or GRP78 in intact cells,
COS7 cells were co-transfected with Myc-tagged GBP and FLAG-tagged
Hcs73 or GRP78. After 24 h of transfection, the cells were lysed
and anti-Myc antibody immunoprecipitates were subjected to SDS-PAGE,
transferred to polyvinylidene difluoride membrane, and immunoblotted
with an anti-FLAG antibody. Under these conditions, FLAG-tagged GRP78
was identified in the anti-Myc antibody immunoprecipitates (Fig.
7C). Therefore we named this novel gene,
GRP78-binding protein (GBP).
On the other hand, interaction with Myc-tagged GBP and FLAG-tagged
Hsc73 was very weak (data not shown). No signals were detected in
immunoprecipitates without the anti-Myc antibody or the lysates from
empty vector-transfected cells.
Overexpression of GBP Attenuates Serum Starvation-induced Cell
Death--
In order to investigate a biological role for GBP, we
established four Neuro2a cell lines stably expressing Myc-tagged GBP or
an empty vector. Two overexpressed cell lines markedly up-regulated the
level of GBP mRNA, although Neuro2a cells did
endogenously express some GBP (Fig.
8A). The band of Myc-tagged
GBP in Neuro2a cells corresponded to about 135 kDa by immunoblotting,
which was different from that in the COS7 cell lysates (Fig.
8B). Overexpression of GBP did not change the features or
growth rate of Neuro2a cells. However, overexpression of GBP attenuated
the time-dependent reduction in cell viability that was
caused by serum deprivation (Fig. 8C). After 48 h of
serum starvation, both Neuro2a cell lines overexpressing GBP were more
resistant to the cell death induced by serum starvation (Fig.
8D).

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Fig. 8.
Overexpression of GBP attenuates serum
starvation-induced cell death in Neuro2a cells. A,
Neuro2a cells stably expressing Myc-tagged GBP (GBP-1 and
-2) or empty vector (Con-1 and -2)
were established by selection with G418, and the level of Myc-tagged
GBP (myc-GBP) and total GBP
(GBP) mRNA in each clone was analyzed by RT-PCR.
B, the expression of Myc-tagged GBP in Neuro2a cells
was detected by immunoblotting. C, Neuro2a cells stably
expressing Myc-tagged GBP (triangles) or empty vector
(circles) were cultured with (filled symbols) or
without 10% serum (open symbols) for the indicated times,
and then the cell viability was measured using the MTT assay as
described under "Experimental Procedures." Each value represents
the percentage of the control value at day 0 (D0).
D, 2 days after serum starvation, the cell viability of
each clone was measured by the MTT assay. Each value represents the
mean ± S.D. of four independent cultures and is expressed as the
percentage of the cell viability of each clone cultured in the presence
of serum for 2 days. *, the difference from cells expressing the empty
vectors (Con-1 and -2) was statistically
significant by analysis of variance (p < 0.01, (Fisher's protected least significant
difference)).
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DISCUSSION |
In the present study, we isolated and characterized a novel
GRP78-binding protein, GBP, in the rat brain. GBP was predicted to have
two transmembrane regions, a proline-rich region and a glutamic
acid-rich region, but the sequence offered few clues to the possible
function. In order to identify regions likely to be functionally
important, we observed the intracellular localization of full-length
and deletion mutants of this gene in COS7 cells. We then found that
GRP78, one of the heat shock proteins, was a GBP-binding protein by the
GST pull-down assay method using the identified region of GBP. Our
results suggest that the C-terminal region of GBP would have some
function in the localization to the ER-Golgi region and interaction
with GRP78, but the precise functional domain in GBP remains to be
determined. The bands of GBP corresponding to about 135- and 170-kDa
proteins in Neuro2a and COS7 lysates, respectively, were detected by
immunoblotting although GBP encoded 1021 amino acids with a deduced
molecular mass of 110 kDa. We consider that some sites in GBP would be
individually modified by as yet unknown and different mechanisms in
Neuro2a and COS7 cells because a motif search of GBP by PROSITE
suggested that GBP contains several N-glycosylation,
N-myristoylation, and phosphorylation sites.
We demonstrated that GBP mRNA was predominantly
expressed in the neurons, but not glial cells, of the rat brain, by
Northern blotting, and in situ hybridization. GBP-expressing
cells were widely detected in various regions of the rat brain such as
the cortex, thalamus/hypothalamus, amygdala, and cerebellum.
Interestingly, in the cerebellum, granule cells but not Purkinje cells
expressed GBP mRNA (data not shown). GBP
mRNA was already detected in the E12 brain, and it gradually
increased to reach a peak within P0-2 weeks after birth. These
observations indicate that GBP would play a role in the development of
the embryonic and postnatal brain and the function of neuronal cells.
Furthermore, the age-dependent decrease in GBP expression
implies that GBP might affect neuronal susceptibility to
various extra- and intracellular stresses to cause several neuronal disorders.
GRP78 was originally identified because its expression is significantly
increased in glucose-deprived cells. Similar to other heat shock
proteins, such as Hsp40, Hsp70, and GRP98, GRP78 has been shown to be
induced by a variety of stimuli, including an increase in intracellular
calcium, oxidative stress, and ER stress (20-22). GRP78 has been
reported to exist as an ER lumen protein and to act as a molecular
chaperon that regulates protein folding and translocation into the ER
and protein secretion, and ER disruption occurred when mutated GRP78
was overexpressed (23-26). The localization of GBP in the ER-Golgi
apparatus and its association with GRP78 in our experiments is
consistent with the intracellular localization of GRP78. In the central
nervous system, the levels of GRP78 are markedly induced in response to
ischemia, axotomy, and kainate-induced seizures (27, 28).
Down-regulation of the GRP78 level by treatment with GRP78 antisense
caused sensitivity to a variety insults such as glutamate,
Fe2+, and amyloid
-peptide (29). In particular, GRP78
has been suggested to suppress oxyradical accumulation and to stabilize mitochondrial functions in PC12 cells (29), but the precise mechanisms
remain to be determined. Very recently, GRP78 has been reported to
associate with ATF6 and caspase-7/12, respectively (22, 30), and
suggested to regulate stress-related gene expression and apoptosis.
Both ATF6 and caspases are anchored in the ER by binding to GRP78 under
resting conditions, but some ER stresses cause cleavage of both
proteins and translocation to the Golgi-nucleus and cytosol,
respectively. In our preliminary experiments, the amount of FLAG-tagged
GRP78 co-immunoprecipitated with Myc-tagged GBP was hardly affected by
treatment with thapsigargin, which causes ER stress by inhibiting ER
Ca2+-ATPase, suggesting that the association of GRP78 and
GBP is constitutive. On the other hand, the expression of
GBP mRNA in neuronal cell lines was hardly modulated by
several stimuli that cause growth arrest and/or apoptosis (data not
shown). The significance of GBP in vivo under pathological
conditions remains to be determined although GBP might affect
ER-Golgi-localized proteins, such as ATF-6 and caspase-7/12, by forming
a complex with GRP78.
Furthermore, we found that stable overexpression of GBP in Neuro2a
cells suppressed serum deprivation-induced cell death, which is
consistent with the anti-apoptotic feature of GRP78. Serum deprivation
would disturb a variety of cellular signaling pathways, such as
mitogen-activated protein kinases and p53 cascades, to induce
apoptosis. Because ATF-6 and caspase-12 are reported to be
predominantly activated by ER stress reagents, we consider that GBP
would attenuate other serum deprivation-induced cascades among several
death signaling pathways in Neuro2a cells.
In conclusion, we cloned a novel GRP78-binding protein (GBP), which is
predominantly expressed in the rat brain and possesses an
anti-apoptotic property, although it is not clear how overexpression of
GBP in Neuro2a cells maintained cell viability after serum deprivation.
Apoptotic cell death is accompanied by proliferation and
differentiation of neurons and the formation of synapses at embryonic
and postnatal stages. Neuronal dysfunction during aging and neuronal
diseases is also associated with apoptotic cell death. Thus, further
studies on the functions of GBP, including GRP78, would provide new
insights to clarify not only the development and differentiation of
neuronal cells at the embryonic and postnatal stages but also
neurodegenerative disorders.