From the Department of Life Science, Pohang University of Science and Technology, Pohang, 790-784, South Korea
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ABSTRACT |
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Phospholipase C (PLC)-4 has been considered to
be a mammalian homolog of the NorpA PLC, which is
responsible for visual signal transduction in Drosophila.
We reported previously the cloning of a cDNA encoding rat
phospholipase C-
4 (PLC-
4) (Kim, M. J., Bahk, Y. Y., Min,
D. S., Lee, S. J., Ryu, S. H., and Suh, P.-G. (1993) Biochem.
Biophys. Res. Commun. 194, 706-712). We report now the isolation
and characterization of a splice variant (PLC-
4b). PLC-
4b is
identical to the 130-kDa PLC-
4 (PLC-
4a) except that the
carboxyl-terminal 162 amino acids of PLC-
4a are replaced by 10 distinct amino acids. The existence of PLC-
4b transcripts in the rat
brain was demonstrated by reverse transcription-polymerase chain
reaction analysis. Immunological analysis using polyclonal antibody
specific for PLC-
4b revealed that this splice variant exists in rat
brain cytosol. To investigate functional differences between the two
forms of PLC-
4, transient expression studies in COS-7 cells were
conducted. We found that PLC-
4a was localized mainly in the
particulate fraction of the cell, and it could be activated by
G
q, whereas PLC-
4b was localized exclusively in the
soluble fraction, and it could not be activated by G
q.
In addition, both PLC-
4a and PLC-
4b were not activated by
G-protein
-subunits purified from rat brain. These results
suggest that PLC-
4b may be regulated by a mechanism different from
that of PLC-
4a, and therefore it may play a distinct role in
PLC-mediated signal transduction.
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INTRODUCTION |
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Phosphoinositide-specific phospholipase C (PLC)1 plays a pivotal role in transmembrane signaling. In response to various extracellular stimuli such as numerous hormones, growth factors, and neurotransmitters, this enzyme catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and thereby generates two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP3) (1, 2). Diacylglycerol is a direct activator of protein kinase C, whereas IP3 induces transient release of calcium from the endoplasmic reticulum into the cytoplasm (3).
Multiple PLC isozymes have been purified from a variety of mammalian
tissues, and several PLC genes have been cloned (4, 5). As predicted
from the cDNAs, the PLC isozymes vary in size, with molecular
masses ranging from 85 to 150 kDa. Despite low overall homology among
the predicted amino acid sequences, significant sequence similarity
exists in two domains that are designated as the X- and the Y-domains.
These domains appear to constitute regions important for catalytic
activities such as the specific recognition of the substrate and the
hydrolysis of its phosphodiester bond. On the basis of the relative
locations of the X- and Y-domains in the primary structure, PLC
isozymes are classified into three types: ,
, and
. All
PLC-
types have a carboxyl-terminal 400-amino acid domain that
contains an unusually high number of charged residues. On the other
hand, the
type has a long stretch of sequence between the X- and
Y-domains, and the
type contains neither of the two additional
sequences (4-6).
As expected from their distinct structural features and their different
cellular expression patterns, the PLC isozymes are distinct in their
modes of activation in response to extracellular stimuli. The two type PLCs, PLC-
1 and -
2, but not the
and
type isozymes,
are activated through tyrosyl phosphorylation by growth factor receptor
tyrosine kinase or nonreceptor tyrosine kinases (6). On the other hand,
the PLC-
types (
1,
2,
3) have been shown in cotransfection
assays and in in vitro reconstitution experiments to be
activated by the
q-subunit of heterotrimeric G-protein
(7-10) and also by the
-subunit (11-16, 39). Additionally, it
is known that the carboxyl-terminal tail that follows the Y-domain is
involved in the activation of PLC-
type by G
q (17,
42-44).
Previously, Min et al. (18, 19) purified the 97-kDa and the
130-kDa PLC-4 enzymes from bovine cerebellum. cDNA encoding a
130-kDa PLC-
4 has been isolated (20, 33, 38). Based on these
studies, it has been suggested that PLC-
4 might be a mammalian homolog of the Drosophila NorpA PLC, which is responsible
for photosignal transduction. Furthermore, recent results obtained from
cotransfection assays and in vitro reconstitution
experiments showed that PLC-
4 could be activated by
G
q but not by
-subunits of G-proteins (21,
22).
Here we report the identification of a rat PLC-4 variant with a
different carboxyl-terminal region. We show by reverse
transcription-PCR and immunoblot analysis that this new splice variant
of PLC-
4 exists in vivo. Furthermore, we further
demonstrate that this splice variant is neither associated with the
particulate fraction of the cell, nor is it activated by
G
q.
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EXPERIMENTAL PROCEDURES |
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Isolation of cDNA--
In the course of isolating PLC-4
cDNA from a rat brain
ZapII cDNA library (20), we identified
cDNA clones (clone
4-53 and
4-52) which exhibited patterns
of restriction enzyme digestion differing from the previously described
130-kDa PLC-
4 cDNA (Fig. 1A). Further sequence analysis
of these cDNAs revealed that they were splice variants of the
PLC-
4 gene.
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DNA Sequencing--
Two clones, 4-53 and
4-52, were
plaque purified and subcloned into Bluescript vectors by in
vivo excision with R408 helper phage. The sequences were assembled
from nested deletion clones generated by the Erase-a-base system
(Promega, Madison, WI) and dideoxy chain termination sequencing
(23).
Reverse Transcription-PCR Analysis--
Total RNA was prepared
from adult Sprague-Dawley rat brain tissue using the guanidium
thiocyanate phenol-based single-step method (24). cDNA was
synthesized in a 50-µl reaction mixture containing 50 mM
Tris-HCl, pH 8.3, 5 mM MgCl2, 75 mM
KCl, 0.5 mM dNTPs, 10 mM dithiothreitol, 25 µg of oligo(dT)12-18/ml, 10 µg of total RNA, and 100 units of avian myeloblastosis virus reverse transcriptase. After a 2-h
incubation at 42 °C, the reaction was terminated by heating at
94 °C for 5 min. One µl of the reaction mixture was used for PCR
amplification. PCR was carried out in a 25-µl reaction mixture
containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.2 mg of gelatin/ml, 1 mM dithiothreitol, 200 ng of each primer, 0.2 mM dNTPs, and 1.25 units of AmpliTaq DNA polymerase
(Perkin-Elmer). The reaction proceeded for 30 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min. The two primers used
were: primer P1 (5-AAGCAAAGAGATGCGAGC-3
), encompassing amino acids
1016-1021 of the PLC-
4b transcript, and primer P2 (5
-TGTGTTTGGGACACTGCATG-3
), which is the 3
-untranslated region specific for the PLC-
4b mRNA (Fig.
2A). The amplified PCR
products were analyzed in a 2% agarose gel stained with ethidium
bromide.
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Isolation of Full-length cDNA Encoding PLC-4b--
Whole
rat brain poly(A) mRNA from CLONTECH was
reverse transcribed with PLC-
4b-specific antisense primer
(5
-CTTGTGTTTGGGACACTGCA-3
). The resulting single-stranded cDNA
was used as template for PCR amplification using the sense primer
(5
-ATCATGGCCAAACCTTACCGA-3
) located at the initiation codon of
PLC-
4a and another antisense primer (5
-CACTGCATGACAGGATTTCA-3
),
which is also specific for PLC-
4b cDNA. The amplified PCR
product was subcloned into T-vector (Novagen) and sequenced by the
Sanger dideoxy method.
Construction of Mammalian Expression Vectors--
A pBluescript
KS plasmid containing the whole open reading frame of PLC-4a was
constructed by splicing an ApaI-SalI fragment of
clone
4-19 and a SalI-XhoI fragment of clone
4-54 (the XhoI site used was originated from 3
-end
linker of
ZapII). The resulting cDNA was then subcloned into the
EcoRV site of pBluescript KS (Stratagene, La Jolla, CA) and
named pKS/
4a. The construction of a pBluescript KS vector containing
the whole cDNA for PLC-
4b (pKS/
4b) was accomplished by
replacing the SalI-ApaI fragment of pKS/
4a
with the SalI-ApaI fragment of pKS/
4-53,
which had been constructed by inserting an
SalI-XhoI (XhoI in 3
-end linker of
ZapII) fragment of clone
4-53 between the
SalI-XhoI sites of pBluescript KS. The mammalian
expression vectors for PLC-
4a and PLC-
4b were constructed by
inserting the blunt ended SmaI-ApaI fragments of
pKS/
4a and pKS/
4b into the EcoRV site of pcDNAI. The resulting plasmids were named pcDNAI/PLC-
4a and
pcDNAI/PLC-
4b, respectively. A mammalian expression vector for
mouse G
q was made utilizing the PCR technology with the
GeneAMP kit from Perkin-Elmer using mouse G
q cDNA as
template together with the sense primer 5
-CGCGGATCCATGACTCTGGAGTCCATCAT-3
and the antisense
primer 5
-GCCGGATCCTTAGACCAGATTGTACTCCT-3
(BamHI sites are underlined). PCR primers were designed to
amplify region corresponding to the open reading frame of mouse
G
q cDNA. So, amplified PCR product contain no
untranslated regions of 5
-end and 3
-end. The amplified product was
digested with BamHI and inserted into the BamHI
site of pcDNAI, and the construct was named
pcDNAI/G
q. All DNA sequences were verified by
sequencing.
Transient Transfection of COS-7 Cells-- COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transfection of the COS-7 cells was by the DEAE-dextran method (25). Cells were seeded at 1 × 106 cells/100-mm dish and transfected 24 h later by incubation with 2 ml of transfection mixture (4 µg of plasmid DNA and 500 µg of DEAE-dextran in PBS) for 1 h. Then, 7 ml of serum-free Dulbecco's modified Eagle's medium containing 100 µM chloroquine was added. After 2.5 h the medium was aspirated, and the cells were treated with 10% dimethyl sulfoxide in Dulbecco's modified Eagle's medium for 2.5 min, washed with PBS, and incubated in a CO2 incubator. The cells were harvested 48 h after transfection.
Antibodies--
Peptide 4-N (MAKPYEFNWQKE, corresponding to
residues 1-12 of PLC-
4a or PLC-
4b) and peptide 116-specific
(GKQRDASPSG, corresponding to residues 1013-1022 of PLC-
4b) were
synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry, conjugated to keyhole limpet hemocyanin with glutaraldehyde, and injected into rabbits as described before (26). Antisera were
affinity purified on protein A-agarose (Pierce Chemical Co.). The
anti-G
q rabbit polyclonal antibody used in
immunoblotting was a generous gift from Dr. Y. S. Kim (Seoul National
University, South Korea).
Identification of PLC-4b in the Cytosolic Fraction of Rat
Brain--
Twenty frozen rat brains were homogenized in a homogenizer
(Brinkmann) with 200 ml of buffer A (20 mM Tris-HCl, pH
7.6, 1 mM EDTA, 1 mM EGTA, and 0.1 mM dithiothreitol) containing 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at
23,000 × g for 1 h. The supernatant was adjusted
to pH 7.4 with 1 M Tris solution and applied to a
DE52-cellulose column (5 cm × 10 cm2) preequilibrated
with buffer A. The proteins were eluted with a 1-liter linear gradient
from 0 to 1 M NaCl in buffer A. All fractions collected
were tested by immunoblots probed with the antibody generated against
the 116-kDa PLC-
4b-specific sequence GKQRDASPSG. Fractions that
contained protein recognized by the antibody were eluted with 70-120
mM NaCl. The peak fraction showing the strongest
immunoreactivity eluted with 90 mM NaCl and was used for
analysis.
Intracellular Localization of PLC-4a and -
4b--
COS-7
cells, transfected with expression plasmids carrying the cDNA of
PLC-
4a or -
4b, were lysed in homogenization buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, and 0.2 mM phenylmethylsulfonyl fluoride) by pestle strokes. The suspension was then centrifuged at
100,000 rpm for 20 min at 4 °C in a Beckman TL-100s ultracentrifuge. The cytosolic fraction (supernatant) was separated from the particulate fraction. Samples were resolved by 6% SDS-polyacrylamide gel
electrophoresis and then electroblotted. The blots were incubated
initially with rabbit antibody (1:5,000 dilution) raised against the
NH2-terminal 12 amino acids of PLC-
4, and then the blot
was incubated with a 1:10,000 dilution of horseradish
peroxidase-conjugated anti-rabbit antibody and processed using the ECL
(enhanced chemiluminescence) system (Amersham). The expression of
recombinant PLC-
4b protein in HeLa cell by using recombinant
vaccinia virus expression system was done as described previously
(40).
Localization of PLC-4s in the Purkinje Cells in the Rat Brain
by Immunocytochemistry--
Male Sprague-Dawley rats weighing 200-250
g were anesthetized with pentobarbital (60 mg/kg, intraperitoneally)
and perfused with fixative solution that contained 2%
paraformaldehyde, 0.075 M lysine, 0.01 M sodium
m-periodate in 0.05 M sodium phosphate buffer, pH 7.4, at
room temperature. The brain was dissected, postfixed overnight in the
perfusion fixative without sodium m-periodate at 4 °C, and then cut
on a vibratome (TPI, vibratome series 1000) into frontal sections (30 µm). Sections of the cerebellum were incubated for 30 min with 2%
normal goat serum in TBS to block the nonspecific binding sites of
protein. The sections were incubated with preimmune sera and antibodies
against PLC-
4a and -
4b (diluted 1:2,000 in TBS and 2% BSA) for
16 h at 4 °C. For avidine-biotin-peroxidase immunostaining, the
sections were washed three times for 10 min each with TBS, incubated
for 2 h with biotin-labeled goat anti-rabbit IgG or anti-rabbit
IgG (Vector), diluted 1:400 in TBS and 2% BSA, washed three times for
10 min each with TBS, and then incubated for 1 h with
peroxidase-labeled streptavidin (Vector), diluted 1:400 in TBS. After
washing three times with same buffer, the preparations were reacted
with 0.05% 3,3
-diaminobenzidine (Sigma) in 50 mM TBS, pH
7.6, containing 0.006% H2O2.
Responsiveness of PLC-4a and PLC-
4b to G
q
Activation--
COS-7 cells were cotransfected with
pcDNAI/G
q and pcDNAI/
4a or pcDNAI/
4b.
After 24 h, the cells were labeled overnight with 2 µCi/ml
myo-[3H]inositol in inositol-free Dulbecco's
modified Eagle's medium. The monolayered cells were washed twice with
PBS and preincubated in serum free medium for 1 h at 37 °C.
During the last 10 min of the preincubation period, 20 mM
LiCl was added. The cells were then treated with 30 µM
AlF4
(10 mM NaF and 30 µM AlCl3) at 37 °C for 30 min. The
reaction was terminated by removal of the medium and washing the cell
with ice-cold PBS. Total inositol formation was measured as described previously (27). Cells were incubated with 3 ml of ice-cold 20 mM formic acid for 30 min on ice. The cells were scraped
off the dishes, and cell debris was removed by centrifugation. One ml
of supernatant was neutralized with 0.5 ml of 50 mM
ammonium hydroxide and loaded onto a 1-ml Bio-Rad Dowex AG 1-X8 anion
exchange column (formate form, 200-400 mesh). Free inositol was washed three times with 3 ml of distilled water (free inositol fraction, Ins),
and then the column was washed three times with 3 ml of 60 mM ammonium formate and 5 mM sodium tetraborate
removing the glycerophosphoinositol fraction. Finally, total inositol
phosphate was eluted with 6 ml of 1 M ammonium formate and
0.1 M formic acid (total inositol phosphate fraction, IPs).
0.5 ml of each of the Ins and IP fractions was mixed with 10 ml of
scintillation mixture and counted (27). The data are presented as the
quotient of IP divided by Ins plus IP. In addition, by measuring total radio activities of cell lysates extracted with formic acid, the incorporation of precursor in the transfected cell was normalized.
Activation by G-protein -Subunits--
Mammalian
expression vectors for PLC-
4a, PLC-
4b, or PLC-
2 were
transiently transfected into COS-7 cells. After 48 h, transfected COS-7 cells were rinsed twice with PBS and extracted with extraction buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM EGTA, 37 mM sodium cholate, 43 mM 2-mercaptoethanol, and 0.1 mM
phenylmethylsulfonyl fluoride) for 30 min at 4 °C. After
centrifugation, the extracted proteins (~5 mg/ml) were quick-frozen
in liquid nitrogen. For the in vitro PLC assay, detergent
extracts of transfected COS-7 cells were diluted 200-fold with buffer
containing 20 mM Tris-HCl, pH 7.5, 10 mM EGTA,
43 mM 2-mercaptoethanol, and 0.1 mM
phenylmethylsulfonyl fluoride. PIP2 lipid substrates were
made as follows. Phosphatidylethanolamine and
[3H]phosphatidylinositol 4,5-bisphosphate were mixed in a
molar ratio of 10:1. The lipids were evaporated to dryness under a
stream of N2 and then sonicated in a bath type sonicator
for 10 min in buffer containing 87.5 mM Tris-maleate, 17.5 mM LiCl, 17.5 mM EDTA, and 1.6 mM
sodium deoxycholate. The final concentration of
[3H]phosphatidylinositol 4,5-bisphosphate in the 70-µl
assay mixture was 28 µM, with 38,000-40,000 cpm/single
assay. The PLC activity was assayed at 25 °C in a mixture (70 µl)
containing 40 µl of lipid substrate, 5 µl (125 ng) of the
transfected COS-7 cells lysates, 5 µl (final 2 µM) of
the
-subunits (provided by Dongeun Park (Kwang-Joo Institute of
Science and Technology, South Korea), and 5-10 µl of 0.1 M CaCl2, adjusting the concentration of free Ca2+ to 0.1 µM. The reaction was started with
the addition of the transfected cell lysate and stopped by adding 350 µl of chloroform/methanol/concentrated HCl (500:500:3, v/v/v)
followed by vortex mixing. Samples were then supplemented with 100 µl
of 1 M HCl containing 5 mM EGTA. After
centrifugation in an Eppendorf microcentrifuge for 5 min at 4 °C,
the amount of [3H]IP3 in the supernatant was
assayed for radioactivity by liquid scintillation counting.
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RESULTS |
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PLC-4b cDNA Cloning--
We isolated two clones (clone
4-52 and
4-53) that had restriction enzyme digestion patterns
differing from the 130-kDa PLC-
4 reported previously. Subsequent
sequence analysis revealed that these clones could be a splice variant
of the 130-kDa PLC-
4 mRNA (Fig. 1A). Clone
4-53
was selected for further analysis because it contained the longer
cDNA insert. The overall cDNA structure of clone
4-53 was
almost identical to the 130-kDa PLC-
4 mRNA except that this
clone had a 176-nucleotide deletion from the region encoding the
carboxyl-terminal part of the 130-kDa PLC-
4 (Figs. 1A and
2A). This deletion caused a frameshift compared with the
reading frame of the 130-kDa PLC-
4 cDNA. As a result the
carboxyl-terminal 162 amino acids are replaced with 10 distinct amino
acids in
4-53 (Fig. 1B). As seen in Fig. 2, the variant mRNA shares most of its sequence with the 130-kDa PLC-
4
mRNA, but it also has its own 37 nucleotides that are not present
in the 130-kDa PLC-
4 mRNA and are inserted 247 nucleotides
downstream from where the 176-nucleotide deletion occurred. Because the
difference between the two mRNAs is restricted to the presence or
absence of only those two specific regions, it is possible that the
variant mRNA originated from an alternative mRNA processing
event, although it cannot be ruled out that each mRNA is the
product of a different gene.
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Detection of the Alternative Transcript by Reverse
Transcription-PCR--
To verify whether a transcript of the spliced
variant of PLC-4 exists in vivo, we performed reverse
transcription- PCR. We used total RNA from rat brain for reverse
transcription with oligo(dT)12-18. The primers used in the
PCR were designed to amplify specifically the region corresponding to
the 3
-untranslated region of the PLC-
4b transcript (Fig.
2A). As shown in Fig. 4, the
PCR product obtained from reverse-transcribed rat brain RNA, and clone
4-53 was the expected 279 bp. This suggests that a PLC-
4b
transcript does exist in vivo and that our clone was not
just an entity generated by recombination during
phage
amplification.
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Immunodetection of PLC-4b in the Rat Brain
Cytosol--
Although we successfully isolated a full-length cDNA
encoding PLC-
4b by long distance PCR, we were not successful in
isolating a
phage clone containing the whole coding region of the
splice variant, and thus we could not exclude the possibility that the PLC-
4b transcript was aberrant or a nonproductive mRNA.
Therefore, we chose a different strategy to confirm the conclusion that
PLC-
4b exists in vivo and shares the amino-terminal
region with PLC-
4a. If we could detect the 116-kDa PLC in rat brain
and if this protein would immunoreact with an antipeptide antibody
generated against the 116-kDa PLC-
4b-specific sequence, and an
antipeptide antibody generated against the amino-terminal sequence of
PLC-
4a, then this would prove that PLC-
4b is an authentic entity
in vivo. Thus, we made two antipeptide antibodies, one to
the carboxyl-terminal region of PLC-
4b (anti-116-specific antibody)
and the other to the amino-terminal region of PLC-
4a (anti-
4-N
antibody). First, we used these antibodies in an experiment where we
expressed PLC-
4b in HeLa cells after infection with vaccinia virus
containing the corresponding cDNA to test whether the molecular
mass of recombinant PLC-
4b would be the expected 116 kDa when
expressed in a eukaryotic cell. As shown in Fig.
5, the molecular mass of the recombinant PLC-
4b expressed in HeLa cells was the predicted 116 kDa, and it was
recognized by both the anti-
4-N antibody and the anti-116-specific antibody (Fig. 5, center lane in both
panels).
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Intracellular Localization of PLC-4a and PLC-
4b--
To
assess functional differences of the two forms of PLC-
4, we compared
their intracellular localization, since previous studies had suggested
that the carboxyl-terminal region of the PLC-
type is required for
their association with the particulate fraction of the cell (17,
47-49). A mammalian expression vector carrying PLC-
4a or PLC-
4b
was transiently transfected into COS-7 cells. The transfected cells
were then fractionated into a soluble fraction and a particulate
fraction, each of which was then immunoanalyzed with anti-
4-N
antibody. As seen in Fig. 6, the majority
of PLC-
4a is localized in the particulate fraction, whereas
PLC-
4b is found exclusively in the soluble fraction. In addition,
the distribution of PLC-
4b in the Purkinje cell of rat cerebellum
was examined with immunocytochemistry. PLC-
4b immunoreactivity was
distributed homogeneously in the cytoplasm of Purkinje cell (data not
shown). Moreover, we have performed immunoblot analysis of the
particulate fraction of the rat brain homogenate by using 116-kDa
PLC-
4b-specific antibody. But 116 kDa PLC-
4b was not detected in
the particulate fraction of the rat brain homogenate (data not shown).
These results are consistent with our observation that 116-kDa
PLC-
4b is found in the cytosolic fraction of the rat brain
homogenate.
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PLC-4b Is Not Activated by G
q--
Because
PLC-
4b is localized exclusively in the cytosolic fraction and
because the carboxyl-terminal region of PLC-
1 is essential for both
its association with the particulate fraction and its activation by
G
q (17), we tested PLC-
4a and PLC-
4b for their ability to be activated by G
q. COS-7 cells were
cotransfected with the mammalian expression vector for
G
q and a cDNA construct expressing either PLC-
4a
or PLC-
4b. Aluminum fluoride
(AlF4
) was added to
activate the G-protein
-subunit (28). As seen in Fig.
7A, transfection with vector
alone or with vector carrying PLC-
4a or PLC-
4b leads to a low
level accumulation of total inositol phosphates. When G
q
was transfected, there was some increase in total IP formation compared
with vector alone. When the cells were cotransfected with the mammalian
expression vectors for PLC-
4a and G
q, there was a
significant increase in total inositol formation. However, cells
cotransfected with the expression vectors for PLC-
4b and
G
q showed no increase in inositol formation compared
with that of cells transfected with the G
q expression vector alone. To exclude the possibility that the observed difference in the activating effect of G
q on PLC-
4a
versus PLC-
4b was the result of a differential expression
of intrinsic PLC-
4 s or G
q in COS-7 cells, we
performed an immunoblot analysis using anti-
4-N antibody and
anti-G
q antibody. It was confirmed that the amount of
PLC-
4a was comparable to that of PLC-
4b and that the expression
levels of intrinsic G
q and PLC-
4s in the COS-7 cells
were not affected by the cotransfection (Fig. 7B).
Furthermore, in a PI-PLC assay employing lipid vesicles the intrinsic
PI-hydrolyzing activity of PLC-
4b purified from extracts of HeLa
cells infected with vaccinia virus containing the corresponding
cDNA was 2 µmol/min/mg of protein. This specific activity was
comparable to that of the 130-kDa PLC-
4a purified from bovine
cerebellum, suggesting that the 116-kDa PLC-
4b variant was as active
as the 130-kDa PLC-
4a species (data not shown). It thus appears that
the carboxyl-terminal region of PLC-
4a is important for activation
by G
q, but it is not important for the intrinsic PLC
activity of the PLC-
4 variants.
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Both PLC-4a and PLC-
4b Are Not Activated by G-Protein
-Subunits Purified from Rat Brain--
Previously, Banno
et al. (36, 37) reported that the activation of a
carboxyl-terminal truncated form of PLC-
3 generated by calpain
cleavage can be enhanced by brain G-protein
-subunits over that
of the intact PLC-
3. We examined whether PLC-
4b, having a shorter
carboxyl-terminal tail, can be activated by G-protein
-subunits.
For this purpose, mammalian expression vectors for PLC-
2, PLC-
4a,
or PLC-
4b were transiently transfected into COS-7 cells. The
transfected cell lysates were then reconstituted with G-protein
-subunits purified from rat brain, and in vitro PLC
activity was measured. Fig. 8 shows that
in accordance with previous reports of PLC-
2 being activated by
G-protein
-subunits (21, 22), the PIP2-hydrolyzing
activity of PLC-
2 is potentiated by the G-protein
-subunits, whereas PLC-
4a and PLC-
4b are not activated by
the G-protein
-subunits. These results, therefore, suggest that
PLC-
4a and PLC-
4b are not targets for regulation by G-protein
-subunits.
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DISCUSSION |
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PLC-4 has been considered to be a mammalian homolog of the
NorpA PLC, which is responsible for visual signal
transduction in Drosophila (29, 30, 32). Recent results
obtained from in situ hybridizations of rat brain tissue
sections and from a mouse that lacks PLC-
4 suggest that PLC-
4 may
play a significant role in mammalian visual processing (31, 41). In our
study, we isolated a novel splice variant of rat PLC-
4 and named it PLC-
4b. This splice variant has a shorter carboxyl-terminal tail compared with the previously reported 130-kDa PLC-
4 (20, 33, 38).
Based on the comparison of the cDNA sequences of the two forms of
PLC-
4, we predict that the mechanism by which PLC-
4b is generated
might be alternative processing of the mRNA.
To date, identified splice variants of the PLC- type include rat
PLC-
1 (26), bovine PLC-
4 (33), Drosophila PLC-p21 (34), and Drosophila NorpA PLC (35). The splicing in all of these PLC isozymes occurs outside the X- and Y-domains. Although alternative splicing in the carboxyl-terminal regions of the rat PLC-
1 and the Drosophila PLC-p21 has been reported (26,
34), the possibility that those splice variants may be regulated
differentially based on differences in the primary structure is purely
speculative.
Previously, Wu et al. (17) have identified regions in
PLC-1 which are involved in the activation process by
G
q. By making a series of deletion mutants, they found
that the region between residues Thr-903 and Gln-1030 (P-Box) is
necessary for both association of PLC-
1 with the particulate
fraction and activation by G
q. They also found that the
region between residues Lys-1031 and Leu-1142 (G-box) is required for
interaction with the G-protein
q-subunit but is not
necessary for association of the PLC-
1 with the particulate
fraction. The P-box in PLC-
1 is the most lysine-rich region in the
entire molecule. In their study, the authors suggested that association
with the particulate fraction might occur through phospholipid or some
intermediate negatively charged membrane-bound proteins. In addition,
the importance of the COOH-terminal basic residues of PLC-
1 and
PLC-
2 for particulate association has been reported (42-44). We
found that the most lysine-rich region in PLC-
4, the P-box, is
located upstream of the carboxyl terminus in PLC-
4b (Fig.
1A). But only PLC-
4a, containing an additional 162 amino
acids in the carboxyl-terminal region, was localized in the particulate
fraction, and it could be activated by G
q. Therefore, it
seems that the P-box in PLC-
4 is insufficient for the association of
the protein with the particulate fraction, and the carboxyl-terminal
162 amino acids in PLC-
4a are necessary for the association with the
particulate fraction and activation by G
q. Because the
carboxyl-terminal 162 amino acids of PLC-
4a also contain a large
number of charged residues (basic and acidic amino acids), a major
determinant for the localization may be the appropriate spatial
arrangement of the charged residues rather than the absolute strength
of positive charge.
Banno et al. (36, 37) reported that a carboxyl-terminal
truncated form of PLC-3 generated by calpain cleavage can be activated to a greater extent by brain G-protein
-subunits than the intact PLC-
3. The authors suggested that the carboxyl-terminal region of PLC-
3 may inhibit its activation by G-protein
-subunits. Recently, Kuang et al. (45) suggested that
the Glu-435 to Val-641 region of the PLC-
2 molecule is involved in
the interaction with the G-protein
-subunits. In their report,
they further narrowed the region down to 62 amino acids (residues
Leu-580 to Val-641) after in vitro binding assays using
glutathione S-transferase-fused PLC-
2 and pure G-protein
-subunits. These residues (Leu-580 to Val-641) of PLC-
2 are
located inside the Y-domain, one of the most conserved regions among
PLC isozymes. Although it has been known that PLC-
4a cannot be
activated by G-protein
-subunits in vivo and in
vitro, previously published observations raised the question of
whether G-protein regulation of the 116-kDa PLC-
4b might occur by
-subunits. However, we found that PLC-
4b, having a short
carboxyl-terminal tail, is nevertheless unresponsive to regulation by
G-protein
-subunits. Therefore, our results suggest that PLC-
4
(PLC-
4a and PLC-
4b) does not have the sequence motif necessary to
be activated by G-protein
-subunits.
In conclusion, we have isolated a splice variant of the previously
reported rat brain PLC-4 enzyme. The two subtypes of PLC-
4 (-
4a and -
4b) are most likely generated by alternative processing of mRNA. They differ in their intracellular localization and their susceptibility to activation by the
q-subunit of
G-protein. We therefore propose that the two forms of PLC-
4 may have
distinct roles in PLC-mediated signal transduction.
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FOOTNOTES |
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* This work was supported in part by Basic Research Institute Program BSRI-954434 from the Ministry of Education and by the Biotech 2000 Program from the Ministry of Science and Technology of Korea.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) AF031370.
Present address: Dept. of Molecular Physiology and Biophysics,
Vanderbilt University School of Medicine, Nashville, TN 37232-0295.
§ To whom correspondence should be addressed. Tel.: 82-562-279-2293; Fax: 82-562-279-2199; E-mail: pgs{at}pop.postech.ac.kr.
1 The abbreviations used are: PLC, phospholipase C; IP, inositol phosphate; IP3, inositol 1,4,5-trisphosphate; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5-bisphosphate; G-protein, heterotrimeric guanine nucleotide binding protein; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; Ins, free inositol fraction; RACE, rapid amplification of cDNA ends; bp, base pair(s).
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