From the Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
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ABSTRACT |
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To delineate the specific regions of
phospholipase C 2 (PLC
2) involved in binding and activation by G
protein
subunits, we synthesized peptides corresponding to
segments of PLC
2. Two overlapping peptides corresponding to
Asn-564-Lys-583 (N20K) and Glu-574-Lys-593 (E20K) inhibited the
activation of PLC
2 by
subunits (IC50 50 and 150 µM, respectively), whereas two control peptides
did not. N20K and E20K, but not the control peptides, inhibited
-dependent ADP-ribosylation of G
i1 by
pertussis toxin and
-dependent activation of
phosphoinositide 3-kinase. To demonstrate direct binding of the
peptides to
subunits, the peptides were chemically cross-linked
to purified
1
2. N20K and E20K
cross-linked to both
1 and
2 subunits,
whereas the control peptides did not. Cross-linking to
and
was
inhibited by incubation with excess PLC
2 or PLC
3, whereas
cross-linking to
but not
was inhibited by
r-myr-
i1. These data together demonstrate specificity of
N20K and E20K for G
binding and inhibition of effector
activation by
subunits. The results suggest that an overlapping
region of the two active peptides, Glu-574-Lys-583, mimics a region of PLC
2 that is involved in binding to
subunits. Changing a tyrosine to a glutamine in this overlapping region of the peptides inhibited binding of the peptide to
subunits. Alignment of these
peptides with the three-dimensional structure from PLC
1 identifies
a putative
helical region on the surface of the catalytic domain of
PLC
2 that could interact with
subunits.
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INTRODUCTION |
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Many transmembrane receptors coupled to heterotrimeric G proteins
can initiate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2)1 to
produce inositol trisphosphate (IP3) and diacylglycerol.
Multiple experimental paradigms provide convincing evidence that
increased PIP2 hydrolysis occurs as a result of enzymatic
activation of phosphatidylinositol-specific phospholipase C (PLC) due
to direct interactions of PLC with activated G protein or
subunits (1-3). G protein
subunits of the Gq class are thought to
be responsible for receptor-mediated activation of PLC that is not inhibited by treatment with the toxin from Bordatella
pertussis (PTX).
subunits released during activation of
Gi/o proteins are thought to activate PLC in a manner that is inhibited
by PTX, since this toxin blocks interaction of the Gi/o heterotrimer
with receptors.
PLC enzymes consist of at least nine different isoforms that have been
classified into three groups; ,
, and
(2, 4). All of these
enzymes require Ca2+ for activity. PLC
isoforms are the
primary enzymes that hydrolyze PIP2 in response to
activation by G proteins. Four isozymes of the PLC
class have been
identified and designated PLC
1,
2,
3, and
4. Each isoform
is regulated differently by G protein
or
subunits. In
in vitro enzyme assays and in co-transfection assays there
are some conflicting results, but in general PLC
1 and
4 are
regulated primarily by G
q, PLC
2 is regulated primarily by
subunits, and PLC
3 is regulated by both
and
q subunits.
Based on sequence alignments between PLC isoforms and other proteins,
some domain structure has been predicted. The first 100 amino acids are
predicted to form a pleckstrin homology domain (5). This domain in PLC
1, when expressed in isolation, binds to PIP2 and
IP3 and when removed from PLC
1, inhibits anchoring to
PIP2-containing membranes but does not inhibit catalysis
(6-8). The role of the pleckstrin homology domain in the other PLC
isoforms is unclear but is likely to be distinct from PLC
1, since
PLC
2 membrane binding is unaffected by IP3 (9). Two
highly conserved regions have been identified in all mammalian PLCs and
designated X and Y. In PLC
and
isoforms, these regions are
adjacent in the primary sequence, whereas in PLC
, the X and Y are
separated by intervening src-homology SH2 and SH3 domains. PLC
isoforms are unique in that there is an extended (40 kDa) C-terminal
domain that extends beyond the Y domain (4).
A three-dimensional crystal structure has been solved for PLC 1 that
contains the X and Y domains but is missing the N-terminal pleckstrin
homology domain (10). The structure shows that the N-terminal region
between the pleckstrin homology domain and the X domain has a
structural fold that is very similar to the EF-hand domain found in
Ca2+-binding proteins, including calmodulin. The entire X
domain and two-thirds of the N terminus of the Y domain fold to form a
catalytic core similar in structure to the
barrel found in triose
phosphate isomerase (TIM barrel). The C-terminal one-third of the Y
domain forms a domain similar to the C2 domains found in PKC and
PLA2 where they function as calcium-dependent
phospholipid binding domains. In PLC
1, the C2 domain primarily
interacts with the N-terminal EF-hand domain, and its function is
unclear.
Some progress has been made mapping the regions in the overall
sequences of the PLCs that are involved in interaction with G proteins.
Removal of the C-terminal third of PLC 1 abolishes activation by
G
q, but Ca2+-dependent activity
remains intact (11, 12). Further analysis of this region has served to
define important regions for interaction with
subunits (13).
Removal of the C-terminal third of PLC
2 does not affect its ability
to be stimulated by
subunits (14, 15). In a series of
experiments by Kuang et al. (16), segments of the PLC
2 X
and Y domains when coexpressed with PLC
2 in COS-7 cells blocked
activation by
subunits. When expressed as GST fusion proteins, a
116-amino acid polypeptide from this region bound tightly to G protein
subunits, whereas a 60-amino acid sequence of this same region
bound weakly to
subunits (16).
In this paper we further define the regions of PLC 2 that interact
with G protein
subunits. Synthetic peptides corresponding to
sections of the 116-amino acid region previously identified (16) were
tested for their ability to inhibit activation of PLC
2 by
subunits in a reconstituted, purified system. The peptides were
designed based on sequence alignment of PLC
2 and PLC
1 and
referral to the crystal structure of PLC
1. This allowed us to
identify regions within Gln-526-Val-641 that were on the surface and
potentially accessible to
subunit binding. Based on these
studies we propose a model for the structural features of PLC
2
involved in
-PLC interactions.
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EXPERIMENTAL PROCEDURES |
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Materials
Peptides were purchased from Coast Scientific or from Biosynthesis and had a purity of greater than 90% based on high performance liquid chromatography analysis and mass spectrometry. Phosphatidylethanolamine (bovine liver) and phosphatidylinositol (bovine liver) (PI) were from Avanti Polar Lipids. PIP2 was prepared from bovine brain lipids (Sigma) according to the method of Schacht (17) or obtained from Sigma. Pertussis toxin was obtained from List Biologicals. Succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC) was from Pierce.
Baculovirus for expression of the PI 3-kinase 110-kDa subunit and
101-kDa subunit were kindly provided by Len Stephens and Phil Hawkins
(Inositide Laboratory, The Babraham Institute, Babraham, Cambridge,
UK). A cDNA encoding full-length PLC
3 was provided by Dr.
Gunther Weber (Karolinska Institute, Stockholm, Sweden).
Methods
Plasmid Construction and Cloning of Recombinant
Baculoviruses--
Purified, recombinant PLC 2,
3, and PI
3-kinase proteins were prepared using a baculovirus expression system.
Construction of baculovirus for expression of PLC
2 has been
previously described (9). 6-His-tagged PLC
3 was prepared as
follows. The cDNA for PLC
3 in bluescript SK
was cut with
BamHI at base pairs 636 and 2556, and the remaining sequence
of PLC
3 was religated. This removed multiple NcoI sites
from the coding region of PLC
3. Oligonucleotides encoding an
EcoRI site (at the 5'-end) followed by the coding sequence
for an initiator methionine, an alanine, a histidine tag (6 histidines), and an NcoI site were ligated at the 5' end of
the PLC
3 sequence at the NcoI site at base pair 1 of the
cDNA coding sequence and EcoRI of bluescript. A 250-base
pair fragment was excised from this construct with EcoRI and
BstEII and ligated with the original PLC
3 cDNA cut
with BstEII and EcoRI after removal of the
insert. This fragment was then excised with EcoRI and
HindIII and inserted into the
EcoRI-HindIII site of Fastbac (Life Technologies,
Inc.). Recombinant, clonal baculoviruses were generated according to
the protocol described by Life Technologies, Inc.
Sf9 Cell Culture and Purification of Phospholipase C and
PI 3-Kinase--
Sf9 cells were grown at 27 °C in IPL-41
medium containing 10% fetal bovine serum, 0.1% pluronic acid, and 50 mg/ml gentamycin. For large scale cultures (1 liter and above), the
cells were switched into medium containing 1% fetal bovine serum and
1% lipid concentrate (Life Technologies, Inc.). Baculoviruses
directing expression of recombinant 6-His PLC 2 and 6-His PLC
3
were used to infect 1 liter of Sf9 cells at a density of
1.5 × 106 cells/ml. The proteins were purified
according to (9).
Expression and Purification of G Protein i1 and
1
2 Subunits--
For purification of
1
2, baculoviruses encoding
1,
2, and 6 × His-tagged
i1 were obtained from Alfred Gilman's laboratory. 1 liter of Sf9 cells at 1 × 106 cells/ml was
simultaneously infected with the three baculovirus constructs, and the
subunits were purified according to the published procedures
(19), which were modified as in Romoser et al. (9).
Phospholipase C and PI 3-Kinase Assays-- Phospholipase C assays were conducted as described previously (9) and in the figure legends. PI 3-kinase activity was assayed using sonicated micelles containing 600 µM phosphatidylethanolamine and 300 µM bovine liver PI as in Parish et al. (22).
ADP-ribosylation of Gi1--
ADP-ribosylation
assays were performed as described by Casey et al. (23).
Briefly, 0.4 pmol of
1
2 was mixed with 20 pmol of
i1 followed by the addition of various
concentrations of peptide. Reactions were initiated by the addition of
pertussis toxin (5 µg/ml final concentration), NAD (2.5 µM final concentration), and [32P]NAD
(750,000 cpm/assay) in a total reaction volume of 40 µl. No
phospholipids were used in the reaction. Reactions were terminated after 20 min, and proteins were precipitated in 15% trichloroacetic acid and collected on nitrocellulose filters. After extensive washing
with 6% trichloroacetic acid, the filters were dried and analyzed by
liquid scintillation counting.
Cross-linking and Immunoblot Analysis--
Peptides (30 or 150 µM) were added to 1
2 (100 nM) at room temperature in a solution containing 1 mM MgCl2, 80 mM KCl, 50 mM HEPES, pH 7.4, and 0.1% C12E10
(decaethylene glycol dodecyl ether) in a total volume of 100 µl. The
cross-linker, SMCC, was dissolved in Me2SO and diluted to 2 mM in 50 mM Pi buffer, pH 5.0, before dilution into the reaction buffer (final concentration 200 µM). The cross-linking reaction was carried out as
described in the figure legends and quenched with 10 mM
Tris, pH 8.6, and 10 mM 2-mercaptoethanol. When PLC
2,
PLC
3, or
i1 (250 or 500 nM) were added,
they were incubated with
subunits at room temperature for 10 min
before the addition of peptides and cross-linker. The cross-linked
proteins were resolved on a 12% SDS-acrylamide gel or a 17%
SDS-acrylamide gel containing 4 M urea. The proteins were
then transferred to nitrocellulose and analyzed by Western blotting
with antibodies against
1 or
2.
subunit antibody X-263 has been previously described and recognize
2,
3, and
7 (24). The
subunit antibody B600 was raised against a synthetic peptide
corresponding to the C terminus of
1,
2, and
3 (MAVATGSWDSFLKIWN) and could
recognize
4 as well. Enhanced chemiluminescence
reagents ECL (Amersham Pharmacia Biotech) were used to
visualize the proteins.
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RESULTS |
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Peptide Design--
There is significant homology between PLC 2
and PLC
1 in the 116-amino acid region of PLC
2 that was found to
bind to
subunits (16): 33% identity and 47% similarity overall
with a 70-amino acid region having 54% identity and 67% similarity (Fig. 1). Based on this high level of
homology, we predicted that PLC
2 might have a very similar fold as
PLC
l in this region. Using this assumption, we examined the
structure of PLC
1 to determine which regions of PLC
2 would be
likely to be on the surface of the protein and accessible to G protein
subunits. Three peptides were chosen for our initial studies,
corresponding to amino acids 564-583 (N20K), 584-609 (A20G), and
575-594 (E20K) of PLC
2 shown in (Fig. 1B). Also tested
as a control was a peptide composed of the same amino acids as N20K,
except the primary sequence was randomized (YVLSKNRSDLFTKAYISSEL).
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Inhibition of Stimulated PLC
2 Activity--
The
peptides were tested for their ability to inhibit the stimulation of
PLC
2 activity by
subunits (Fig.
2). In the absence of peptide,
subunits stimulated PLC
2 approximately 10-fold over basal,
Ca2+-dependent activity. The addition of N20K
inhibited
-stimulated activity with an IC50 of 50 µM with 95% inhibition of activity occurring at 200 µM. The adjacent peptide, A20G, and the scrambled peptide
had no effect on stimulation of PLC activity by
subunits at
concentrations up to 300 µM. The peptide that overlaps
the C terminus of N20K and the N terminus of A20G-E20K (E20K)
inhibited
-stimulated activity with an IC50 of 150 µM. Both peptides inhibited the basal
Ca2+-stimulated activity of PLC
2, with N20K inhibiting
activity by 58 ± 10% and E20K inhibiting activity by 43 ± 10% (n = 4 experiments). Importantly,
-stimulated activity was inhibited to a greater extent than was
basal activity. To quantitate this effect, data were normalized by
determining the fold stimulation of activity over basal activity by
subunits (activity with
divided by activity without
). The percent inhibition of the fold stimulation by
subunits in the presence of 200 µM peptides was then
calculated. N20K inhibited fold stimulation over basal by 67 ± 2%, whereas E20K inhibited fold stimulation by 42 ± 4%. We also
tested a peptide corresponding to N20K where Tyr-15 in the region that
overlaps with E20K was changed to Gln (N20K(Y15Q)). This peptide
stimulated PLC
2 activity in the absence of
subunits (data
not shown). The significance of this is unclear, but it did not allow
us to measure effects of N20K(Y15Q) peptide on
-stimulated PLC
2 activity. To prove that the inhibition of
-stimulated PLC
activity was attributable, at least in part, to binding of the peptides to
subunits, several other assays were performed to demonstrate binding of the peptides to
subunits.
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Inhibition of ADP-ribosylation--
G protein subunits block
the ability of
subunits to activate PLC by sequestering the
subunits in the heterotrimeric form. This is thought to work
because the
subunits sterically hinder interaction between the
subunits and the PLC. This predicts that peptides mimicking PLC
2 binding to
subunits could block interaction between
and
subunits. One way to measure this is to measure the
-dependent enhancement of ADP-ribosylation of
subunits by pertussis toxin. Since
binding to
subunits is
required for ADP-ribosylation of
, peptides that block
interaction between
and
subunits will inhibit
ADP-ribosylation.
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Effects of Peptides on Stimulation of PI 3-Kinase by
Subunits--
To further confirm that these peptides inhibit various
effectors by binding to
subunits, we tested the effects of these peptides on the stimulation of PI 3-kinase by
subunits. We utilized a p110
/p101 heterodimer purified from sf9 cells as
described by Stephens et al. (18). The activity of the
p110/p101 heterodimer was increased 5-fold by 150 nM
subunits. N20K or E20K inhibited the stimulation of this enzyme by 150 nM
subunits, with an IC50 of 50-100
µM (Fig. 4). A20G and
scrambled peptide had no effect. The peptides did not have a
significant effect on basal PI 3-kinase activity.
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Effects of Peptides on Stimulation of PLC 3 by
Subunits--
PLC
3 is activated by G protein
subunits at a
similar potency as PLC
2 (25). Activation of PLC
3 by
subunits and Ca2+ was measured in the presence of E20K and
N20K. Surprisingly, E20K had little effect on the activation of PLC
3 by G protein
subunits, whereas N20K inhibited activity but
not to the same extent as for PLC
2 (Fig.
5). Basal,
Ca2+-dependent activity of PLC
3 was not
measurable in these assays. We have reported previously that basal
activity of PLC
3 is much lower that for PLC
2 under these assay
conditions (25).
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Cross-linking of Peptides to Subunits--
To directly
demonstrate that these peptides bind to
subunits, we developed a
cross-linking assay that uses a heterobifunctional cross-linker to
covalently link the peptides to purified
subunits. The
cross-linker (SMCC) has a succinimide ester group that reacts with
primary amines and a maleimido group that reacts with SH moieties.
Since the peptides have no cysteine residues, the only way to
cross-link the peptides to
or
is via a primary amine on the
peptide and SH groups on either
or
subunits. Peptide cross-linking was monitored by immunoblotting for
or
subunits after electrophoresis to resolve cross-linked subunits from the unmodified subunits.
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DISCUSSION |
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Two synthetic 20-amino acid overlapping peptides that mimic a
region of PLC 2 bind specifically to G protein
subunits and
prevent interaction with different biochemical partners. An adjacent
peptide and scrambled N20K had no effect in any of the assays we have
examined. All the peptides are very similar with respect to amino acid
chemistry. Although the peptides have some effect on the basal
activities of PLC
2, this cannot explain the extent of the
inhibition of
stimulation of PLC
2 activity. It is clear
based on the other assays involving
subunits that these peptides
bind to the
subunits and that this is responsible, at least in
part, for the observed inhibition of
stimulated PLC
2.
The N20K and E20K peptides inhibit the -dependent
activation of PLC
2 with IC50s of 50 and 150 µM, respectively. These IC50s are similar to
what has been reported for a 27-amino acid peptide derived from
adenylyl cyclase type II (QEHA peptide) for inhibiting a number of
subunit-regulated effectors (26). N20K and E20K do not contain
the
binding consensus sequence identified in the QEHA peptide.
However, in the common 10 amino acids of the N20K and E20K there is a
short stretch of seven amino acids where 4 amino acids are identical to
a sequence at the C terminus of QEHA and a 5th amino acid is
conservatively substituted. When we changed one of these amino acids in
N20K, binding to
subunits was dramatically inhibited (Figs.
3B, 6A, and 7A).
The N20K and E20K peptides are more potent in inhibiting
-dependent ADP-ribosylation of
i1
(IC50 8 µM) than for inhibition of PLC
2.
This is surprising if we assume that the peptides block ADP-ribosylation of
i1 by blocking
i1
interactions with
because the affinity of
for
i1 (Kd ~ 1 nM) is much
greater than the affinity for PLC
2 (Kd ~ 100 nM). The exact mechanism for how
subunits stimulate
ADP-ribosylation of
subunits is unclear but is known to involve a
process where
subunits must cycle catalytically among a
stoichiometric excess of
subunits. Since the mechanism is not
entirely understood, it is possible that the peptides interfere with
this catalytic ADP-ribosylation in a way that is not directly related
to the known high affinity of
subunits for
subunits. One
possibility is that the peptides are not blocking
binding to
but are occupying a binding site on
required for PTX
interaction. This possibility is supported by the cross-linking data
that shows that cross-linking of the peptides to the
subunit is not
blocked by
i1-GDP.
Both N20K and E20K must cross-link directly to cysteine residues in
either the and
subunits due to the nature of the cross-linker. The site of
cross-linking is clearly cysteine 41, since this is the
only cysteine in
2. The site(s) of cross-linking of the peptide to the
subunit is unclear, but there are 14 cysteine residues in the
subunit where cross-linking could have occurred. Cross-linking of E20K and N20K to both
and
was prevented by incubation with PLC
2. PLC
3 also prevented cross-linking of N20K
and E20K but was not as effective as PLC
2, suggesting that the
binding sites for these two enzymes on
subunits do not entirely
overlap. This is consistent with the observation that the peptides were
not as effective or as potent at inhibiting PLC
3 activation by
subunits.
The cross-linking of the peptides to the subunit was not prevented
by preincubation of the
subunit with the
subunit. This suggests
that the peptide binding site on
does not entirely overlap with
the
subunit. Although it is known that
-GDP blocks activation of
PLC
by
subunits, it is probable that the binding sites for
PLC
and
subunits overlap but do not match. Thus the binding
site represented by the peptide may lie outside the overlap region.
This idea is supported by a recent study by Bluml et al.
(27) where it was demonstrated that a peptide from phosducin binds to
subunits. Visualization of the location of this peptide in the
crystal structure of
complexed with phosducin (28) shows that
this peptide would bind at a region on the
subunit that does not
overlap the
subunit binding site, whereas the N-terminal domain of
phosducin does overlap the
subunit binding site. That
-GDP
inhibits cross-linking to the
subunit may result from steric
interference with the cross-linking reaction without interfering
directly with peptide binding.
The data we have presented allows us to narrow down in the primary
sequence of PLC 2 a region of 20 amino acids that may be involved in
subunit binding. The N terminus of the E20K peptide overlaps the
C terminus of N20K peptide by 10 amino acids. The C-terminal 10 amino
acids of E20K overlaps with the N-terminal 10 amino acids of A20G,
which does not inhibit
activation in any assay. This suggests
that the potential region that is binding to
subunits can be
narrowed down to 10 amino acids, Glu-574-Lys-583. We have tested a
10-amino acid peptide corresponding to this region and found no
evidence for inhibition. A possible explanation is that this 10-amino
acid peptide may be too short to adopt the appropriate secondary
structure. This has been seen for other peptides including the peptide
from adenylyl cyclase type II (26).
Visualization of the regions homologous to our peptides in the PLC 1
structure supports the idea that the 10-amino acid overlap region is
the critical region of the peptides involved in binding to
subunits. The catalytic domain of PLC
is composed of parts of the
conserved X and Y domains that form a TIM barrel constructed from of a
series of
sheet
helix repeats. The
strands line the inside
of the barrel, and side chains from these strands project into the core
of the structure to provide the chemistry for substrate binding and
catalysis. The
helices are on the outside of this barrel structure
and in some cases are exposed to solvent. When the sequence of the N20K
is aligned with the PLC
1 sequence, the N-terminal 10 amino acids
align with a small amount of linker sequence and the T
6 strand of
the barrel (nomenclature of Essen et al. (10)). The sequence
in T
6 is very conserved between the various PLC isoforms with two
amino acids, serine 571 and phenylalanine 572 (
2 sequence), being
conserved in all known PLC isoforms. For this reason we predict that
this sequence in PLC
2 will occupy a similar position to that
observed in the PLC
sequence. Since this region is on the inside of
the TIM barrel in PLC
and is directly involved in substrate
binding, it would be unlikely to be accessible to interaction with
subunits. The region homologous to Glu-574-Lys-583 of PLC
2
(overlap region between N20K and E20K) forms an
helix on the
surface of PLC
1, and because of the significant sequence homology
in this region, would likely form the same type of structure on PLC
2. The surface exposure of just this 10-amino acid region of N20K
further suggests that this overlap region corresponds to a portion of
PLC
2 that is important for interaction with and regulation by G
protein
subunits. The location of this binding site directly
adjacent to structures that contribute amino acids to substrate binding and catalysis suggests a mechanism by which
subunits could activate PLC
. If the binding of
subunits to the helix on the
surface caused movement of the adjacent
strands, this could position amino acids so they are more favorable for catalysis there by
increasing enzymatic activity.
The region we have defined only overlaps the portion of PLC 2
(Leu-580-Val-641) originally defined by Kuang et al. (16) as a
binding domain by 4 amino acids (580-583). Using
two-hybrid analysis, Yan and Gautam (29) used the N-terminal 100-amino acid region of the
subunit to demonstrate interactions with this
62-amino acid domain. One peptide (A584-G604) overlaps extensively with
this domain yet had no effect in any of our assays for
subunit
interactions. This suggests that either only a very short 4-amino acid
region is necessary for some
binding or that other regions
within a 62-amino acid region are also involved in PLC
2-
interactions.
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ACKNOWLEDGEMENTS |
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We would like to thank Monica Schertler for
technical assistance. We would also like to thank Dr. Roger Williams
for providing the coordinates for PLC 1.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant 1-R01-GM53536-01 (to A. V. S.) and GM53162 (to D. W.).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.
To whom correspondence should be addressed: Dept. of Pharmacology
and Physiology, University of Rochester School of Medicine and
Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.; 716-275-0892; Fax: 716-244-9283; E-mail: Smrcka{at}pharmacol.rochester.edu.
1 The abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; PI, phosphatidylinositol; PLC phospholipase C; G protein, GTP-binding protein; PTX, pertussis toxin; IP3, inositol 1,4,5-trisphosphate; SMCC, succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1- carboxylate.
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REFERENCES |
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