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INTRODUCTION |
Phosphatidylinositol-specific phospholipase Cs
(PLCs)1 are enzymes that
catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two second messengers, inositol
1,4,5-trisphosphate (IP3) and diacylglycerol.
IP3 releases Ca2+ from intracellular stores,
and diacylglycerol activates protein kinase C (for review see Refs. 1,
2). PLCs are importantly involved in multiple transmembrane signal
transduction pathways that regulate numerous cellular processes (for
review see Refs. 3-5). There are three families of PLC isoforms in
mammals:
,
, and
. Each family includes more than one member
(
, 1-4;
, 1-2; and
, 1-4). The activity of these different
isoforms of PLC is controlled by different upstream regulators.
PLC-
s are activated by G protein subunits G
and
G
q/11 (6). PLC-
s are regulated by receptor or
nonreceptor protein-tyrosine kinases and PIP3 (4). PLC-
s
are regulated by a GTP-binding protein transglutaminase (7). PLC-
s
are also more sensitive to Ca2+ than the other isozymes
and, unlike PLC-
s and PLC-
s, are activated by Ca2+
alone (8).
All the ten mammalian PLC isozymes identified to date are modular
proteins. As shown in Fig. 1, the PLCs contain a pleckstrin homology
(PH) domain, four EF-hand motifs, a catalytic domain (composed of X and
Y regions separated by a linker region) and a C2 domain. PLC-
s have
an additional 400-residue C-terminal region, which is required for
activation by G
q (9, 10) and may also contribute to
membrane localization (11).
Among the PLC isozymes, only members of the PLC-
family
(PLC-
1-3) are activated by G
. Part of the
G
-binding site on PLC-
2 is located in the Y region
as shown by cross-linking (12) and copurification (13). G
can
also bind to the isolated PH domains from PLC-
2 (14).
Indirect evidence suggests that this interaction may lead to activation
of PLC-
2 (15). Despite these progresses, the mechanism
whereby G
activates PLC-
2 is still unclear. It
seems unlikely that PLC-
2 activation by G
involves
membrane translocation of PLC-
2 to the plasma membrane, because G
does not significantly alter the binding affinity of
PLC-
2 to phospholipid vesicles (16-18).
The goal of this study was to further understand the mechanism of
substrate hydrolysis of PLC-
2, its regulation by G
protein subunits, and the functional contribution of some of the
PLC-
2 domains to enzyme function. Although some PLC
domains are homologous to known domains in other proteins, and highly
homologous domains can be found among the various PLC isoforms, these
domains may have different functions in the different isoforms. For
example, PH domains are found in many proteins, but only some of them
can bind WD-repeat-containing proteins (such as G
) (for
review see Ref. 19). In addition, the PH domains of various PLC
isoforms show very different affinities to phospholipids (14, 20, 21). It is therefore necessary to test individual proteins to find out which
role(s) a particular domain plays in a particular context.
The roles of other specific PLC domains in basal and ligand-regulated
PLC catalytic activities have not yet been clearly identified. For
example, the role of the linker region between X and Y regions of the
catalytic domain is less well understood. In the crystal structure of
the PLC-
1 molecule, the X and Y regions are tightly associated to form a triose phosphate isomerase barrel-like structure (22). Although the X and Y regions are well conserved among the PLC
isozymes, the linker region possesses little similarity among the PLC
isozymes. For example, PLC-
s have a long linker region that contains
two SH2 domains, one SH3 domain, and an additional PH domain, whereas
the linker regions in PLC-
and PLC-
are less than 100 residues
long and contain no obvious structural domains within them. In the
crystal structure of PLC-
1, the linker region shows a
disordered structure (22). The linker region is not essential for PLC
catalytic activity. Coexpression of the N- and C-terminal fragments of
PLC-
1 lacking the linker region produces a catalytically
active complex with an activity substantially higher than the
holoenzyme (23). Trypsin digestion of PLC-
1 cleaves the
enzyme at the linker region and generates two associated fragments that
retain catalytic activity (24). Proteolysis at or near the linker
region of a truncated form of PLC-
2 after it had folded
into an active enzyme suggested that the linker region served as an
inhibitory element (25). In this study, the linker region was cleaved
but not removed and the exact site of tryptic or V8 protease cleavage
was not determined. Because the authors used a truncated form of
PLC-
2 that was not stimulated by G
q, it
was not possible to determine the effect of proteolysis of the enzyme
in or near its linker region on G
q-dependent
PLC activity.
It is usually straightforward to analyze the contributions of domains
at the N or C termini of a protein, because truncated forms of the
enzyme can be made, and these truncated proteins are often active. In
addition to analyzing the role of the N-terminal PH domain and the C
terminus, we were particularly interested in the linker region. Because
it is often difficult to study the function of internal domains due to
misfolding of proteins with internal deletions, we attempted to
reconstitute PLC-
2 from two separate fragments, each
containing one of the two catalytic X and Y regions. We tested whether
or not the N- and C-terminal halves of PLC-
2 could
associate to form catalytically active enzymes when expressed as two
separate polypeptides and whether reconstituted PLC-
2
could still be activated by G
and G
q. Using PLC
fragments of different lengths, we examined the functional contribution
of the PH domain, the linker region and the C-terminal region to basal
activity and G
- and G
-mediated PLC-
2
activation. Answers to these questions will expand our understanding of
the mechanism of substrate hydrolysis by PLC-
2 and its
regulation by G proteins.
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EXPERIMENTAL PROCEDURES |
cDNA Constructs--
Plasmids containing cDNA sequences
encoding various fragments of human PLC-
2 were
constructed by polymerase chain reaction. Full-length wild-type
PLC-
2 in pMT2 vector (a gift from M. Simon of the
California Institute of Technology, Pasadena, CA) was used as template.
The primer at the 5'-end included a HindIII site, a Kozak
sequence (GCCGCC), and a start codon. The primer at the 3'-end included
an EcoRI site and a stop codon. To add a FLAG or
hemagglutinin (HA) epitope tag to a construct, one of the two primers
contained the sequence encoding the epitope. The polymerase chain
reaction products were digested with HindIII and
EcoRI and cloned into an
HindIII/EcoRI-cut pcDNA3 vector. All the
sequences were confirmed by DNA sequencing.
Cell Culture and Transfection--
COS-7 cells were maintained
in complete growth medium (Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and
50 µg/ml streptomycin). Cells in 6-well plates (for
immunoprecipitation) or 12-well plates (for PLC activity assay) were
transfected using LipofectAMINE (Life Technologies). Prior to
transfection, cells were transferred to Opti-MEM I medium (Life
Technologies) for 1 h. The medium was replaced with 1 ml (6-well
plates) or 500 µl (12-well plates) of Opti-MEM I containing preformed
DNA-LipofectAMINE complexes. The final concentration of DNA in the
medium was 1.5 or 2.5 µg/ml. The exact amount of each plasmid was as
indicated in the legends to individual figures. The ratio (w/w) between
DNA and LipofectAMINE was always kept at 1:8. After 5 h, 2 ml
(6-well plates) or 500 µl (12-well plates) of complete growth medium
were added to each well. The medium was replaced with complete growth
medium the next day.
35S Metabolic Labeling and
Immunoprecipitation--
Forty-eight hours after transfection, cells
on 6-well plates were starved for 2 h in 2 ml of starvation medium
(RPMI 1640 without glutamine, methionine, and cysteine (Sigma)
supplemented with 10% dialyzed, heat-inactivated fetal bovine serum
and 2 mM L-glutamine). The cells were then
metabolically labeled in 1 ml of starvation medium containing 150 µCi
of [35S]-Express Protein Labeling Mix (NEN) for 4 h.
The cells were rinsed with PBS and lysed in 1 ml of lysis buffer (50 mM HEPES-Na (pH 7.5), 6 mM MgCl2, 1 mM EDTA, 75 mM sucrose, 3 mM
benzamidine, 1% (v/v) Triton X-100, and 1 mM
dithiothreitol) at 4 °C for 30 min. The cell lysates were precleared
with 30 µl of protein G-agarose (Roche Molecular Biochemicals) or 50 µl of protein A-Sepharose (Sigma) slurry (50% (v/v) in PBS) for 30 min. After a 10-min centrifugation, the supernatants were mixed with 2 µl of M2 anti-FLAG antibody (Sigma) or anti-HA-epitope antibody 12CA5
(Babco) at 4 °C overnight. The samples were centrifuged at
15,000 × g for 15 min. The supernatants were then
mixed with 30 µl of protein G-agarose or 50 µl of protein A-Sepharose slurry for 1.5 h. The resins were washed twice at 4 °C for 15 min each with 1 ml of lysis buffer containing 150 mM NaCl and once at room temperature for 15 min with 1 ml
of PBS. 25 µl of 3× sample buffer (187.5 mM Tris-Cl (pH
6.8), 6% SDS, 30% glycerol, 0.003% bromphenol blue) was added to
each of the final pellets, and 20 µl was loaded onto an SDS-PAGE gel.
The gel was Coomassie Blue-stained, destained, treated with
EN3HANCE (NEN), dried, and used for autoradiography with
intensifying screens at
80 °C.
Inositol Phosphate Production in COS-7 Cells--
PLC activity
was analyzed as production of inositol phosphates (26, 27). Twenty-four
hours after transfection, the medium was replaced with 1 ml of
inositol-free DMEM supplemented with 5% fetal bovine serum. Two hours
later, the medium was again replaced with the same medium containing 2 µCi of myo-[3H]inositol. After 15 min, 10 µl of 1 M LiCl was added to each well (the final LiCl concentration
was 10 mM). No difference in the uptake/incorporation of
myo-[3H]inositol was found in cells incubated with
LiCl-containing medium for 1 h and 24 h. Forty-eight hours
after transfection, the cells were washed with 1 ml of PBS and
extracted twice for 30 min each with 500 µl of 20 mM
formic acid. The extracts were combined and neutralized to pH 7.5 with
a solution containing 7.5 mM HEPES and 150 mM
KOH. The neutralized extracts were loaded onto 0.5 ml AG1-X8 (Bio-Rad)
anion exchange columns. Prior to use, the columns were washed with 2 ml
of 1 M NaOH and 2 ml of 1 M formic acid and
equilibrated with H2O to neutrality. After the extracts were loaded onto the columns, the columns were washed with 5 ml of
H2O and 5 ml of 5 mM Borax and 60 mM sodium formate. The inositol phosphates were eluted with
3 ml of 0.9 M ammonium formate and 0.1 M formic
acid. The eluates were counted in a scintillation counter.
Subcellular Fractionation--
COS-7 cells cultured in 6-well
plates were transfected and metabolically labeled with
[35S]-Express Protein Labeling Mix as described above.
The cells were washed with PBS and detached by incubation in 500 µl
of Trypsin-EDTA solution (1×) (Sigma) at 37 °C for 1 min. After
mixing with 2 ml of DMEM/8% fetal bovine serum, the cells were
collected by centrifugation at 500 × g at 4 °C for
5 min. After being washed with 3 ml of a buffer identical to the lysis
buffer used in immunoprecipitation but containing no Triton X-100, the
cells were resuspended in 600 µl of the same buffer and went through
freeze-and-thaw in ethanol/dry ice three times (3 min per period). The
broken cells were then passed trough a 23-gauge (or smaller) needle ten
times to shear DNA and centrifuged at 100,000 × g in a
Beckman SW55 rotor at 4 °C for 30 min. The supernatant was the
soluble fraction. The pellet was resuspended in 600 µl of lysis
buffer containing 1% Triton X-100 and incubated at 4 °C for 30 min.
The supernatant, after a 5-min centrifugation at 15,000 × g was the particulate fraction. Both the soluble and the
particulate fractions were later used in immunoprecipitation.
Western Blot Analysis--
Forty-eight hours after transfection,
cells in 6-well plates were washed with 2 ml of PBS and harvested in 1 ml of lysis buffer. The cells were lysed at 4 °C for 30 min. After a
10-min centrifugation at 15,000 × g, an aliquot (10 µl) of the supernatant was loaded on an SDS-PAGE mini-gel, and the
resolved proteins were wet-electroblotted to a nitrocellulose membrane
and probed with specific primary and peroxidase-conjugated secondary
antibodies using a chemiluminescence kit according to the
manufacturer's instructions (NEN).
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RESULTS |
Design of PLC-
2 Plasmids--
We constructed
several mammalian expression plasmids encoding various fragments of
PLC-
2, as shown in Fig. 1.
To further understand the mechanism of substrate hydrolysis of
PLC-
2 and its regulation by G protein subunits, we used
these constructs to determine whether the amino and carboxyl halves of
PLC-
2 could associate when expressed as two polypeptides
and, if they could, how the complexes thus formed would be regulated by
G
and G
q. These constructs were designed to allow
us to test the role of the PH domain, to compare the activity and G
protein regulation of enzyme with the linker region either attached to
the C-terminal fragment or completely removed, and to compare the
activity of reconstituted enzyme with and without the C-terminal domain
required for activation by G
q. To all these fragments
(except construct A), a FLAG or an HA epitope tag-encoding
sequence was attached at one end (see Fig. 1). Construct A
had both a FLAG tag at the N terminus and an HA tag at the C terminus
to compare the results of immunoprecipitation through the FLAG and HA
tags.

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Fig. 1.
Schematic representation of a human
PLC- 2 molecule and its constructs
with epitope tags. PH, pleckstrin homology domain;
EF, EF-hands; X and Y, X and Y regions
of the catalytic domain; L, X-Y linker region;
C2, C2 domain. The long black bar at the
C-terminal end is the C-terminal region. See text for more
details.
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Assembly of PLC-
2 Fragments Expressed in COS-7
Cells--
Antibodies directed against the epitope tags were used to
(co)-immunoprecipitate metabolically labeled PLC-
2
fragments that had been expressed in COS-7 cells. The representative
autoradiograph in Fig. 2A
shows that most of the fragments were robustly expressed. Only
A and E fragments were expressed at significantly
lower levels (about one-tenth to one-fifth) when compared with their
corresponding PH domain-containing fragments (A' fragment
and wild-type, respectively).

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Fig. 2.
Expression of
PLC- 2 fragments in COS-7
cells. COS-7 cells in 6-well plates were cotransfected with
plasmids encoding various PLC- 2 fragments. 625 ng/ml of each DNA was used. Vector DNA (pcDNA3) was added as needed
to make a total DNA concentration of 1.5 µg/ml. The cells were
metabolically labeled with [35S]methionine/cysteine. The
PLC- 2 fragments were immunoprecipitated with the
anti-FLAG tag antibody as described under "Experimental Procedures"
and resolved on two 10% SDS-PAGE gels. Positions of expressed
PLC- 2 fragments are indicated by arrows. When
more than one band of a protein is present, only the band of the
largest size was indicated. The first lane on the
left in both gels is a control (transfected with vector
only).
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PLC-
2 has an endogenous proteolytic site that cleaves
off the C-terminal region necessary for G
q activation
(10). The wild-type enzyme expressed in COS-7 cells was partially
cleaved at this site giving rise to a fragment of about the same size as A' fragment. The A' fragment was further
cleaved to a polypeptide of approximately the same size as the
B' fragment. Similarly, A and E
fragments had shorter polypeptides of the same size as the B
fragment, suggesting that there is another proteolytic site near the C
terminus of the X region. The remaining proteolytic or background bands
were not identified but represented only a small fraction of the total protein.
Lanes 8-11 of Fig. 2A show that the
B' fragment could bind C, C',
D, and D' fragments (C' and
D' fragments lacked the linker region, whereas C
and D fragments included this region). The expression level
of the B' fragment was higher when the C-terminal fragments were coexpressed, suggesting that they stabilized the B'
fragment. Immunoprecipitation through the FLAG tag on the B'
fragment was able to coimmunoprecipitate the C,
C', D, and D' fragments, indicating that the B' fragment was able to form complexes with each.
The B' fragment coimmunoprecipitated approximately equal
amounts of C and C' fragments. The numbers of
methionines in these fragments were: 15 in B', 17 in
C, 16 in C', 27 in D, and 26 in
D'. The D and D' fragments were
cleaved at or near the site described by Park et al. (10) to
generate C and C' fragments, which also bound to
the B' fragment. About 90% of D fragment and
75% of D' fragment were cleaved. The implication of this
cleavage for interpretation of activity measurements will be described
below. The B fragment, which lacked the PH domain, could
also coimmunoprecipitate a C-terminal fragment (C,
C', D, and D' fragments), but the
capacity was lower when compared with the B' fragment (Fig.
2B). Therefore, removal of the PH domain reduced but did not
block assembly. Immunoprecipitation and coimmunoprecipitation could
also be performed through the HA epitope tag on A,
C, C', D, and D' fragments,
but the efficiency was lower. For this reason, we performed the
coimmunoprecipitation in all our other experiments through the FLAG
epitope tag.
We also examined whether the PLC-
2 fragments could form
complexes after they had been synthesized. When the B'
fragment and C, C', D, or
D' fragments were expressed in COS-7 cells separately and
later mixed after cell lysis, none of the C, C',
D, nor D' fragments were coimmunoprecipitated by
the B' fragment (data not shown). Therefore, the
PLC-
2 fragments need to be coexpressed in cells to form
complexes with one another. In addition, we failed to
coimmunoprecipitate G
1
2 through a FLAG
tag on the PLC-
2 fragments (including the wild-type
PLC-
2) or to coimmunoprecipitate the
PLC-
2 fragments with G
1
2
through an HA tag on G
1 or G
2, suggesting
that the interaction was weak.
Roles of the PH Domain for the Enzymatic Activity and Subcellular
Distribution of PLC-
2 Fragments--
We next tested the
catalytic activity of the PLC-
2 fragments measured as
production of inositol phosphates. Full-length, wild-type
PLC-
2 was used as control. As shown previously in this laboratory (26, 27), inositol phosphate production increased when COS-7
cells were transfected with PLC-
2 itself (Fig.
3A). Coexpression of
G
1
2 caused a pronounced rise in PLC
activity. PLC-
2 truncated at the C terminus
(A' fragment) had basal and G
1
2-stimulated activity equal to the
full-length enzyme. Even though the wild-type enzyme was substantially
cleaved, if the activity of the wild-type enzyme was much higher than
A' fragment, the mixture should still show higher activity
than the A' fragment. These results were consistent with
previous reports (9, 10, 28). However, when the PH domain was removed
from the full-length or truncated enzyme (E fragment and
A fragment, respectively), both were inactive. Fig.
3A also shows that individual fragments containing only one
of the two catalytic regions (B, B',
C, C', D, and D') had no
PLC activity whether or not the PH domain was present. Fig.
3B illustrates that basal and
G
1
2-stimulated PLC-
2 activity could be reconstituted from two fragments each containing one
of the catalytic domains only if the PH domain was present. The
characteristics of the reconstituted activity will be discussed below.
These results indicate that the PH domain was required for
PLC-
2 to hydrolyze its substrate in COS-7 cells.

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Fig. 3.
Activity of
PLC- 2 fragments and reconstituted
PLC- 2. A, activity
of single PLC- 2 fragments. COS-7 cells in each duplicate
wells in 12-well plates were transfected with 625 ng/ml of each DNA. In
all cases, vector DNA was added to give a total DNA concentration of
2.5 µg/ml. Black bars, no
G 1 2; shaded bars, in the
presence of G 1 2. WT,
wild-type. A representative experiment analyzed in duplicate is shown.
The error bars indicate the ranges of duplicate
determinations. Each construct was tested at least three times.
B, activity of two cotransfected PLC- 2
fragments. Experimental conditions were identical to those in
A of this figure except that the concentration of each
PLC- 2 DNA was 125 ng/ml. Wild-type PLC- 2
and the A' fragment were used as positive controls.
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Because the fragments and reconstituted complexes lacking the PH domain
(i.e. E, A, and complexes formed with
the B fragment) were expressed at levels significantly lower
than those containing the PH domain (the wild-type enzyme,
A', and complexes formed with the B' fragment)
(Fig. 2), it was possible that their lower catalytic activity was
simply a result of lower expression. To test this possibility, we
compared the expression and PLC activity of B + C' at a
higher DNA dose (625 ng of DNA/ml at transfection) with those of
B' + C' transfected with one-tenth of this DNA
dose (62.5 ng/ml). We chose this pair, because, in contrast to the wild-type enzyme and the A' fragment, at the lower DNA dose
B' + C' were expressed well and had a basal
activity substantially higher than the blank. Despite a higher
expression level of the B fragment (due to higher DNA
dosage) compared with the B' fragment and a similar amount
of coimmunoprecipitated C', B + C' showed no
enzymatic activity. Similar results were also observed for other
fragments (data not shown). Although this experiment did not completely
exclude the possibility that the absence of PLC activity of
E and A was in part due to low expression, our
results indicated that removal of the PH domain abolished the function of PLC-
2 in COS-7 cells.
Removal of the PH domain also altered the subcellular distribution of
the expressed proteins (Fig. 4). When the
PH domain was present (wild-type, A', and B'
fragments), ~30% of the enzyme was found in the particulate
fraction. Constructs lacking the PH domain (E, A,
and B fragments) were found almost exclusively in the
soluble fraction. Therefore, the inaccessibility to a
membrane-associated substrate may account for the observed loss of
PLC-
2 activity in constructs lacking the PH domain.
However, using these experimental approaches in transfected cells, we
could not distinguish an intrinsic loss of catalytic activity in
truncated fragments from effects secondary to alterations in
subcellular localization.

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Fig. 4.
Subcellular distribution of
PLC- 2 fragments. Lysates of
COS-7 cells transfected with various PLC- 2 constructs
and metabolically labeled were resolved into soluble and particulate
subcellular fractions by ultracentrifugation. PLC- 2
fragments were immunoprecipitated from these fractions. S,
soluble fraction; P, particulate fraction. The error
bars indicate standard deviations of three independent
experiments. See details under "Experimental Procedures."
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Effects of G
i on G
1
2
Activation of Wild Type and Reconstituted
PLC-
2--
G
needs to dissociate from G
to
interact with its effectors. Therefore, excess G
i should
block the G
activation of PLC-
2 by scavenging free
G
to form heterotrimers (26, 27). This is an important control,
because it shows that G
is activating PLC-
2 with
characteristics expected for a heterotrimeric G protein. Fig.
5 shows that, although G
i1
itself did not exhibit any significant effect in any group, it
efficiently blocked G
1
2 activation of reconstituted PLC-
2. The PLC activity dropped to a level
equal to or slightly lower than the basal activity. Cotransfection of the COS-7 cells with plasmids encoding G
i1 and/or
G
protein subunits had no effect on the amount of
immunoprecipitated PLC-
2 fragments (data not shown).

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Fig. 5.
Regulation of
PLC- 2 fragments by
G 1 2
and G i1. COS-7 cells in
duplicate wells in 12-well plates were transfected with 125 ng/ml of
each PLC- 2 DNA, but the concentrations of
G 1, G 2, and G i1 DNA were
kept at 625 ng/ml each. In all cases, vector DNA was added to give a
total DNA concentration of 2.5 µg/ml. A representative experiment out
of four independent experiments analyzed in duplicate is shown. The
error bars indicate the ranges of duplicate
determinations.
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Effects of the Linker Region on Basal and
G
1
2-stimulated Activity of Reconstituted
PLC-
2--
The above experiments showed that the
N-terminal fragment containing the PH domain (B' fragment)
could reconstitute active enzyme when coexpressed with the C-terminal
fragments. The reconstituted complexes had higher basal PLC activities
than the wild-type PLC-
2 and, like the wild-type
PLC-
2 and A' fragment, were activated by
G
1
2 (Figs. 3B and 5). Among
the four complexes, those lacking the linker region (B'
+ C' and B' + D') had higher basal and
G
1
2-stimulated activity than those with
the linker region connected to a C-terminal fragment (B'
+ C and B' + D).
Although the reconstituted complexes had
G
1
2-stimulated activities that were equal
to or greater than that of the wild-type enzyme, this was entirely due
to increased basal activity. There was no significant change in the
increment due to G
1
2 between the
wild-type enzyme and any mutant/complex but B' + C, whose increase was higher than that of any other group. This
indicates that, although cleavage or removal of the linker region leads to increased basal activity, it does not affect the activation of
PLC-
2 by G
1
2.
Regulation of PLC-
2 Activity by
G
q--
PLC-
s are the only PLC isozymes that are
activated by G
q (9, 29-31). This activation involves
the long C-terminal region characteristic of the PLC-
isozymes. We
had shown above (Figs. 3 and 5) that the recombined
PLC-
2 complexes could be activated by
G
1
2 and tested next whether they could
also be activated by G
q (Fig.
6). We used the wild-type
PLC-
2 and A' fragments, which lacks the
C-terminal region, as positive and negative controls, respectively. Among the four complexes, no significant activation by
G
q was observed in the combinations lacking the
C-terminal region (B' + C or B'
+ C'). In contrast, B' + D and
B' + D' were activated by G
q. As
with G
activation, the increment in PLC-
2 activity
due to G
q was very similar in wild-type
PLC-
2, B' + D and B' + D'. Again, removal of the linker region (as in B' + D' and B' + C') increased the basal
activity, but the basal activity and the G
q-induced
increase in activity were additive. These results documented that the
reconstituted PLC-
2 complexes containing the C-terminal
region were still subject to regulation by G
q. We also
tested the effect of G
q on fragments and reconstituted enzymes without the PH domain (E, B + C', and
B + D'). These fragments or reconstituted proteins showed no
increase in PLC activity, providing further evidence that removal of
the PH domain abolished the function of PLC-
2 (Fig.
6B).

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Fig. 6.
Activation of
PLC- 2 fragments by
G q. A, activation
of PLC- 2 fragments containing the PH domain. COS-7 cells
in each duplicate wells in 12-well plates were transfected with 125 ng/ml of each PLC- 2 DNA. The concentration of
G q DNA was 625 ng/ml. In all cases, vector DNA was added
to give a total DNA concentration of 1.5 µg/ml. The counts of the
blank group (vector DNA only) with or without G q were
subtracted from those of the other groups under the same conditions.
The error bars indicate standard deviations of three
independent experiments. Black bars, no G q;
shaded bars, in the presence of G q.
B, absence of activation of PLC- 2 fragments
lacking the PH domain. The error bars indicate the ranges of
duplicate determinations in the same experiment, which was repeated
twice with similar results.
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|
 |
DISCUSSION |
Our study is the first to reconstitute active enzymes with PLC-
fragments. In this study, we have characterized several constructs encoding various fragments of human PLC-
2. We
reassembled PLC-
2 from enzyme fragments each containing
one of the two catalytic regions (Fig. 2) and found that the PH domain
was required for both enzymatic activity (Figs. 3) and membrane
targeting of PLC-
2 (Fig. 4). These reassembled enzymes
were still subject to regulation by G protein subunits (Figs. 5 and 6).
We identified the X-Y linker region as an inhibitory element in the
intact enzyme. However, changes at the linker region did not affect the
regulation of PLC-
2 by G protein subunits (Figs. 3, 5,
and 6).
The Roles of the PH Domain--
We found that, although the
targeting of PLC-
2 fragments lacking the PH domain to
the membrane may be impaired (Fig. 4), these fragments were still
assembled (Fig. 2). Moreover, the PH domain was essential for
PLC-
2 to hydrolyze its substrates in COS-7 cells (Fig.
3). We could not distinguish whether removal of the PH domain in
PLC-
2 prevented the enzyme from getting access to its
substrates in the plasma membrane, caused a loss in enzymatic activity,
or both. A PLC-
1 construct in which the PH domain was replaced by glutathione S-transferase has full enzymatic
activity (32), suggesting that the major role of the PH domain in
PLC-
1 is to ensure membrane localization. The PH domain
of PLC-
1 binds to the PIP2 polar headgroup
with an affinity and specificity comparable to the native enzyme (20)
and is proposed as the anchor localizing the enzyme to the plasma
membrane in the "tether-and-fix" model based on the crystal
structure of PLC-
1 (22). The PH domain of
PLC-
1 binds to PIP3 strongly and
specifically and targets the enzyme to the membrane in response to
growth factor stimulation (21). In contrast, PLC-
1,
PLC-
2 and their PH domains bind to phospholipid membrane
surfaces with lower affinities, and the binding is PIP2
concentration-independent (14). We observed that PLC-
2
fragments lacking the PH domain were not found in the particulate
fraction, whereas constructs containing the PH domain were partially
targeted to the particulate fraction (Fig. 4). Therefore, this domain
is also involved in membrane targeting of PLC-
2. Besides
binding to some inositol phosphates, PH domains identified in some
proteins bind to proteins containing WD-repeats (33). An example
is the strong interaction between
-adrenergic receptor kinase and
G
(34). The isolated PH domain of PLC-
2 binds to
G
with an affinity comparable to that of the full-length PLC-
2 (14), but the significance of this interaction for
the enzyme's regulation by G
needs to be further tested.
The Roles of the Linker Region--
The X and Y regions form the
catalytic domain of PLC. In the present study, we demonstrated by
coimmunoprecipitation experiments that, when COS-7 cells were
cotransfected with two plasmids each containing the DNA sequence
encoding one of the two catalytic regions, the two in vivo
coexpressed fragments associated tightly with each other (Fig. 2). In
contrast, fragments that were separately expressed and then combined
could not bind to each other, suggesting that the association occurs
during translation.
The reassembled enzymes possessed catalytic activity similar to or
higher than that of the wild-type PLC-
2 (Figs.
3B and 5). The most dramatic elevation of the basal
catalytic activity was found in the two combinations lacking the linker
region, B' + C' and B' + D'. These results suggest that the linker region, when present in
the intact enzyme, inhibits basal PLC-
2 activity. When
the linker region was attached to the C-terminal fragment containing
the Y domain, but (in contrast to wild-type PLC-
2) was
not linked to the X domain, thereby allowing for more flexibility, the
basal activity almost doubled (Figs. 3B and 5, compare the wild-type PLC-
2 with B' + D).
However, the linker region may still interfere with PIP2
hydrolysis, because complete removal of the linker region resulted in
even greater increase in the basal activity.
The long C-terminal region was not essential for the basal or
G
-stimulated activity of PLC-
2 (Fig.
3A). However, the highest basal activity of all was given by
B' + D' that lacked the linker region but
retained the long C-terminal domain. As was shown in Fig. 2, the
C-terminal domain did not lead to the formation of more reassembled
PLC-
2. Therefore, we conclude that the presence of the
C-terminal domain allows those complexes that do reassemble to acquire
a more active conformation.
We found that even when the linker region was cleaved or completely
removed, PLC-
2 was activated by
G
1
2 and this activation was completely
blocked by G
i1 (Fig. 5). These PLC-
2
fragments were also subject to activation by G
q.
Consistent with previous findings (9, 10), activation by
G
q was contingent upon the presence of the C-terminal
region (Fig. 6). In addition, all PLC-
2 fragments showed
similar increment in PLC-
2 activity upon activation by
G
1
2 or G
q. Therefore, it
is highly unlikely that the G protein
q and 
subunits regulate PLC-
2 by direct effects on the linker region.
Conclusions--
Our experiments show that the PH domain is
required for the basal as well as the G
q- and
G
-stimulated PLC-
2 activity in a heterogeneous
cell expression system. Like PLC-
1, functional PLC-
2 can be reconstituted from two coexpressed enzyme
fragments, each containing one of the two catalytic regions. The linker
region is an inhibitory element in PLC-
2, but cleavage
or removal of the linker region does not affect the G protein-mediated
regulation of PLC-
2. Therefore, G
and
G
q appear to activate PLC-
2 by mechanisms
other than easing the inhibition of PLC-
2 activity by
the linker region, thereby providing evidence for a regulatory pathway
for PLC-
2-involving mechanisms distinct from other PLC isoforms.