(Received for publication, August 14, 1995; and in revised form, September 22, 1995)
From the
We have determined the primary structure of a novel
subunit (
, previously designated
)
of G protein purified from bovine spleen. The mature
protein composed of 68 amino acids had acetylated serine at the N
terminus and geranylgeranylated/carboxylmethylated cysteine at the C
terminus. This was consistent with the C-terminal prenylation signal in
the amino acid sequence, which was predicted from
cDNA isolated from a bovine spleen cDNA library. Western blots with the
specific antibody against
showed that
is present in all tissues examined. Among various
subunits
(
,
,
,
, and
),
has a
unique property to be phosphorylated by protein kinase C. The
phosphorylated amino acid residue was Ser
(or
Ser
). The phosphorylated
associated
with G
more tightly than the unphosphorylated form.
Exposure of Swiss 3T3 and aortic smooth muscle cells to phorbol
12-myristate 13-acetate and NaF induced phosphorylation of
. Stimulation of aortic smooth muscle cells with
natural vasoactive agents such as angiotensin II and vasopressin also
induced phosphorylation of
. The extent of
phosphorylation of
in vitro was
suppressed by a complex formation with G
, which was
relieved by the addition of guanosine
5`-O-(3-thiotriphosphate) or aluminum fluoride. These results
strongly suggest that
is phosphorylated by protein
kinase C during activation of receptor(s) and G protein(s) in living
cells.
Heterotrimeric G proteins ()play a major role in the
transduction of signals from cell surface receptors to cellular
effectors(1) . G proteins are composed of
,
, and
subunits, and the
and
subunits form a tight complex
under non-denaturing conditions. Upon activation, the GTP-bound
subunit interacts directly with various effectors. The
complex also regulates several effectors, which include K
channels, adenylyl cyclase, phospholipase A
,
phospholipase C-
, receptor kinases, and phosphatidylinositol
3-kinase(1, 2) .
Analysis of purified proteins and
cloned cDNAs has revealed the existence of multiple forms of and
subunits in addition to many isoforms of the
subunit(1, 2) . The amino acid sequences of the five
isoforms of
subunit (
-
)
are 53-90% identical to one another(1, 2) ,
while nine kinds of mammalian
subunit,
,
,
,
,
,
, two
s(3, 4, 5, 6, 7, 8, 9, 10, 11) ,
and
(previously designated
, (12) ), have more diverged sequences. Functional differences
among various forms of
complexes have been attributed to the
rather than to the
subunit(12, 13, 14, 15) .
Especially, the biological properties of
, a
complex of transducin, is noticeably different from those of
the other
complexes(12, 13, 14, 15) . Although
the properties of the latter
complexes resemble one another,
their tissue distribution varies. The
and one of the
s are specifically expressed in retinal rods and
cones, respectively(3, 4, 11) . Another
is expressed only in olfactory and vomeronasal
neuroepithelia(10) , while the
is localized
only in the brain(7, 9, 16, 17) . By
contrast,
,
, and
are distributed in a variety of
tissues(7, 9, 16, 17) .
It is
well known that many hormones, neurotransmitters, and growth factors
activate certain isozymes of phospholipase C that hydrolyze
phosphatidylinositol 4,5-bisphosphate to produce inositol
1,4,5-trisphosphate and diacylglycerol. They regulate the release of
Ca ions from intracellular stores and the activity of
protein kinase C (PKC), respectively(18, 19) .
Ca
ions and PKC play crucial roles in the signal
transduction mechanisms(19) . In many cell lines, the
agonist-induced phospholipase C reactions are inhibited by phorbol
esters such as PMA, a potent activator of PKC(19) . Such
inhibition by PKC activators appears to provide a feedback control on
agonist-stimulated phospholipase C. The activation of PKC can also
affect the function of the receptor-adenylyl cyclase reactions in
different cell types(19) . Although the mechanisms of this
inhibition by PKC has not been clarified, several groups have
investigated the effect of PKC on G
proteins(20, 21, 22, 23, 24) .
Treatment of human platelets with PMA or thrombin induced the selective
phosphorylation of G
and attenuated the ability of
agonists both to suppress formation of cAMP and to stimulate hydrolysis
of phosphoinositides(21) . Similarly, PMA and agonists such as
glucagon, vasopressin, and angiotensin II caused to phosphorylate
G
and to attenuate to inhibit adenylyl cyclase in
hepatocytes(22) . However, there is no direct evidence to show
that phosphorylation of
subunits of G
or G
indeed decreases their physiological activities. In addition to
subunit, phosphorylation of unknown isoforms of
and
subunits of G protein was observed in
vitro(23, 24) , but it has not been shown that
PKC phosphorylates these subunits in intact cells nor that the
phosphorylation changes their biological activities.
In this study,
we found that a novel subunit of G protein,
,
was a good substrate for PKC among various forms of
examined in vitro. The phosphorylation of
by PKC was
confirmed in intact cells, supporting its physiological relevance.
PKC was purified from rat or rabbit brains by the method
of Kitano et al.(28) with a modification. PKC
was separated from PKC fraction of the rabbit brain by a hydroxyapatite
column(29) . Other isozymes of PKC (PKC
,
,
, and
) were highly purified from recombinant baculovirus-infected Sf21
cells(30) . 1 unit of PKC is defined as the amount of enzyme
that catalyzes the transfer of 1 nmol of phosphate from ATP to histone
type III-S per min at 30 °C.
A peptide N-acetyl-SSKTASTNNC corresponding to residues
Ser-Asn
of
appended
with cysteine for coupling purpose was synthesized. Antisera against
were raised in rabbits by the injection of the
synthetic peptide conjugated to keyhole limpet hemocyanin. The antibody
was purified from antisera by the use of a column of Sepharose to which
peptide had been covalently coupled. Antibodies
against the other
subunits have been previously generated with
the individual peptides(17) . Antibodies against
,
, and
were
specific for the respective
subunit, while the antibody against
, raised with the C-terminal peptide of
Gly
-Cys
, reacted not only with
but also with
,
,
and
(17) .
Approximately 40 µg of
complex were incubated at 30 °C for 30 min
with 40 µg of µ-calpain (from porcine erythrocytes, >120
units/mg protein; Nacalai Tesque) in 50 mM Hepes-NaOH (pH
7.4), 1 mM dithiothreitol, 1 mM CaCl
, and
0.06% sodium cholate. The reaction was terminated by the addition of
Lubrol PX, EGTA, and E64 (an inhibitor of calpain) at final
concentrations of 0.05%, 7 mM and 45 µg/ml, respectively.
The reaction mixture was then dialyzed against 10 mM Tris-HCl
(pH 7.5), 0.1 mM dithiothreitol, and 0.05% Lubrol PX and
loaded onto a reversed-phase Cosmosil 5C8 column (4.6
150 mm;
Nacalai Tesque). Proteolytic fragments were eluted with a linear
gradient of 10-90% acetonitrile in 0.1% trifluoroacetic acid.
Phosphorylated and unphosphorylated (0.25
µg of protein) were incubated at 30 °C for 30 min with various
amounts of µ-calpain in the same medium mentioned above. The
reaction was stopped by the addition of an equal volume of 2
sample buffer for Tricine/SDS-PAGE(31) , and the reaction
mixture was subjected to electrophoresis.
For electrophoretic analyses, various
complexes (0.5 µg each) in a solution containing sodium cholate
(final concentration of 0.06-0.12%) were incubated with PKC (0.03
units) in a reaction mixture that contained 20 mM Tris-HCl (pH
7.5), 5 mM magnesium acetate, 10 µM [
-
P]ATP (500-1,000 cpm/pmol),
0.5 mM CaCl
, 40 µg/ml phosphatidylserine, and
0.8 µg/ml diolein (total volume 75 µl) at 30 °C for 1 h,
unless otherwise specified. After incubation, 3 µl of a solution of
10% SDS was added to each reaction mixture, which was then lyophilized.
The residue was incubated in sample buffer for Tricine/SDS-PAGE at 40
°C for 30 min and subjected to Tricine/SDS-PAGE. The gel was
stained with silver or Coomassie Blue, dried, and subjected to
autoradiography at -80 °C. The amount of phosphate
incorporated into
was estimated by Cerenkov counting
of the appropriate band cut out from the gel.
For functional
analyses, (25 µg) was treated with PKC
(0.15 units) in the presence and in the absence of 100 µM unlabeled ATP (total volume, 150 µl) at 30 °C for 20 min.
After incubation, each reaction mixture was dialyzed against 20 mM Hepes-NaOH (pH 8.0), 0.1 mM dithiothreitol, 0.3% sodium
cholate at 4 °C for 4 h. The resultant samples are referred to as
phosphorylated and unphosphorylated
,
respectively.
Figure 1:
Amino acid sequence of bovine
protein determined by Edman degradation and mass
spectrometry. A, the amino acid sequence of bovine
and summary of the sequence analysis. The
protein was digested with µ-calpain, S.
aureus V8 protease (V8), and Arg-C, and fragments were
analyzed with Edman degradation (fragments 1-3) or mass
spectrometry (fragment 4). Two cysteine residues at positions
42 and 68 were predicted from a molecular mass of the intact
(see text). B, CAD spectrum of an Arg-C
fragment of
. A doubly charged ion of the fragment (m/z = 746.1) was analyzed by capillary LC/MS/MS. Ions
of type y" and b are labeled. The amino acid sequence reconstructed
from these product ions is shown in the figure by the single letter
code.
The primary structure
of thus predicted was perfectly matched with that
deduced from the nucleotide sequence of
cDNA
isolated from bovine spleen cDNA library (data not shown). The cloned
cDNA had a 216-base pair open reading frame encoding a 72-amino acid
protein. A comparison of the deduced amino acid sequence of
and other known sequences of
subunits is shown
in Fig. 2. The sequence identity between the translated products
of
and the other
subunits ranged from 36%
(
) to 76% (
). The deduced amino acid
sequence Cys-Thr-Ile-Leu at the C terminus of
coincided with the consensus sequence for geranylgeranylation at
the cysteine residue(16, 37, 38) . The
C-terminal leucine selects geranylgeranyl like
,
,
,
, and
instead of farnesyl to be linked to
and
ending with
serine(11, 36) . Taken together, we concluded that
is composed of 68 amino acid residues having
acetylated serine at N terminus and C-terminal cysteine modified with
geranylgeranyl and methyl groups.
Figure 2:
Comparison of amino acid sequences of
various isoforms of subunit of G protein. The amino acid sequence
of
is aligned with the sequences of
(3, 4) ,
(5, 6) ,
(7) ,
(8) ,
(9) ,
(localized in
olfactory neurons; (10) ), and
(localized in cone, (11) ). The sequences of three
additional
subunits,
,
, and
have been recently identified(51) . Amino
acid residues conserved among more than six sequences are boxed.
During the sequence analysis, we
noticed that µ-calpain caused a limited proteolysis in (see Fig. 5), which resulted in truncation of the
N-terminal tripeptide including the site of PKC phosphorylation (see
below).
Figure 5:
Proteolysis of phosphorylated and
unphosphorylated by µ-calpain.
Unphosphorylated (A) and
P-phosphorylated (B and C)
(0.25 µg) were
incubated at 30 °C for 30 min with various amounts (lanes
1-4: 0, 0.5, 1, and 2 µg, respectively) of calpain in
the presence of 1 mM CaCl
. Each reaction was
terminated by the addition of 7 mM EGTA, and the samples were
subjected to Tricine/SDSPAGE. The gels were stained with silver (A and B) or subjected to autoradiography (C).
Figure 3:
Reactivity of the antibody against
and tissue distribution of
in the
rat. A, five forms of the purified
complex (0.5
µg each) and free
(0.1 µg) were subjected to
Tricine/SDS-PAGE, and the gel was then stained with Coomassie Blue
(
) or silver (other
complexes or
) or it was immunoblotted with antibody against
. Immunoreactive proteins were visualized with
3,3`-diaminobenzidine. B, cholate extracts (30 µg of
protein) of various rat tissues and the purified bovine
(10 ng) were subjected to Tricine/SDS-PAGE and
immunoblotted with the antibody against
.
Immunoreactive proteins were visualized by a chemiluminescence
reaction. Seminal v., seminal
vesicle.
Figure 4:
Phosphorylation of by
PKC. A, phosphorylation of various forms of the
complex of G proteins by PKC. Various
complexes containing
different
subunits (0.5 µg each) were incubated at 30 °C
for 1 h with PKC (0.03 units) in a reaction mixture that contained 20
mM Tris-HCl (pH 7.5), 5 mM magnesium acetate, 10
µM [
-
P]ATP, 0.5 mM CaCl
, 40 µg/ml phosphatidylserine, and 0.8
µg/ml diolein. The reaction was terminated by the addition of SDS,
and samples were subjected to Tricine/SDS-PAGE. Proteins were
visualized by staining with Coomassie Blue (
) or
silver (other
complexes). The gel was dried and subjected to
autoradiography at -80 °C. Numbers on the right indicate molecular masses in kDa. B, phosphorylation of
by various isozymes of PKC. Purified
(0.5 µg) was incubated at 30 °C for 1 h
with various isozymes of PKC (0.01 units) in a reaction mixture that
contained 20 mM Tris-HCl (pH 7.5), 5 mM magnesium
acetate, 10 µM [
-
P]ATP, 0.5
mM CaCl
, 40 µg/ml phosphatidylserine, and 50
ng/ml PMA. Samples were subjected to Tricine/SDS-PAGE and then to
autoradiography. C, time course of phosphorylation of
by PKC. Purified
(3.5
µg) was incubated at 30 °C with PKC (0.21 units) in the
reaction mixture described in A, in a total volume of 525
µl. At the indicated times, aliquots (75 µl) were withdrawn for
analysis by Tricine/SDS-PAGE.
As shown in Fig. 4A, the PKC-catalyzed phosphorylation gave two
protein bands of in the Tricine/SDS-PAGE, and the
more slowly migrating band coincided with the phosphorylated one
identified by the autoradiogram (Fig. 4A). This was
clearly shown by the time course study of the phosphorylation of
by PKC (Fig. 4C). The time-dependent
increase in density of the upper band paralleled the phosphate
incorporation, indicating the phosphorylation slightly slowed down the
electrophoretic mobility in the gel. Maximal incorporation of phosphate
was about 0.8 mol/mol of
, suggesting that a single
phosphorylation site in
.
To identify the
phosphorylated amino acid in ,
P-labeled
was isolated by HPLC, hydrolyzed with 5.7 M HCl, and subjected to two-dimensional thin layer electrophoresis,
which identified a phosphoserine (data not shown). The phosphoserine
seemed to be in the N-terminal region, since the antibody against the
N-terminal peptide of
did not react with
phosphorylated
(see Fig. 6B).
Consistently, the phosphorylation site was estimated to be Ser
or Ser
, because
P-labeled
lost radioactivity upon treatment even with a
smaller amount of µ-calpain (Fig. 5, B and C), which specifically removed Ser
-Lys
from
(Fig. 1A). The N-truncated large fragment of
(Fig. 5B) gave no positive signal in the autoradiogram (Fig. 5C). In N-terminal two serines, Ser
is more likely to be the phosphorylation site, because Ser
fulfills the consensus sequence of PKC (39) .
Figure 6:
Phosphorylation of in
cultured cells that contain
as a major
subunit. A, identification of
isoforms of G protein in
Swiss 3T3 and aortic smooth muscle cells. As standard proteins (lane 1 in each panel), various isoforms of the
subunit (or
complex) were subjected to Tricine/SDS-PAGE
(from left to right panels): 10 ng of
, 10 ng of
, 2 ng of
, 10 ng of
, and a mixture of
10 ng each of
(upper band) and
(lower band). In all of the panels, the cholate extracts (15 µg of protein) of Swiss
3T3 (lane 2) and aortic smooth muscle cells (lane 3)
were electrophoresed and immunoblotted with antibodies against
,
,
,
, and
. The bands stained with the
antibody against
(lanes 2 and 3 in
the right end panel) were assigned as
because (i) the antibody against
cross-reacted
with
(17) and (ii) the mobility of the
positive band in each lane was identical to that stained with the
antibody against
. B, effect of PMA on the
phosphorylation of
in Swiss 3T3 cells. Swiss 3T3
cells were incubated with 0.1 µM 4
-PMA (lane
3) or 0.1 µM PMA (lane 4) for 1 h. Cholate
extracts of cells (15 µg of protein) and standard proteins (10 ng
each; lane 1, unphosphorylated
; lane 2, phosphorylated
) were subjected
to Tricine/SDS-PAGE and immunoblotted with antibodies against
and
. C, effect of PMA,
NaF, and various hormones on the phosphorylation of
in Swiss 3T3 and aortic smooth muscle cells. Swiss 3T3 (3T3) and aortic smooth muscle cells (SMC) were
labeled with [
P]orthophosphate for 1 h and then
incubated with 0.1 µM 4
-PMA (lane 1), 0.1
µM PMA (lane 2), 40 mM NaF (lane
3), 1 µM angiotensin II (lane 4), or 1
µM arginine vasopressin (lane 5) for 1 h. Cholate
extracts isolated from the stimulated cells were immunoprecipitated
with antibodies against
as described under
``Experimental Procedures,'' and an aliquot of the
immunoprecipitate was subjected to Tricine/SDS-PAGE with subsequent
autoradiography. The position of the phosphorylated form of bovine
is indicated by an arrow. Numbers on the right indicate molecular masses in
kDa.
Here, we
should note the difference in susceptibility of to
µ-calpain between phosphorylated and unphosphorylated states. In
comparison with unphosphorylated
(Fig. 5A), higher concentration of µ-calpain
was required to cleave the N-terminal part of
in
phosphorylated
(Fig. 5B). This
observation suggests that the PKC-catalyzed phosphorylation possibly at
the N-terminal region of
induced a conformational
change of
.
In these two cultured cell systems, we tested whether
is phosphorylated by PKC in vivo. First,
Swiss 3T3 cells were treated with a direct activator of PKC (PMA), and
cholate extracts of these cells were analyzed by Western blots with
both antibodies against
and
(Fig. 6B). Although the antibody against
did not react with phosphorylated
, the results showed that PMA treatment decreased the
amount of unphosphorylated
in the cells (lane 4 in left panel). By contrast, the antibody against
could react both unphosphorylated and phosphorylated
, and the extract from PMA-treated cells showed an
additional upper band corresponding to phosphorylated
, indicating that PMA induced phosphorylation of
in the intact cells. Pretreatment of cells with
staurosporine, an inhibitor of PKC(40) , completely blocked
PMA-stimulated phosphorylation of
(data not shown).
In addition, down-regulation of PKC by long-term pretreatment of Swiss
3T3 cells with PMA (41) attenuated PMA-induced phosphorylation
of
(data not shown). These results suggested that
phosphorylation was catalyzed by PKC.
To examine whether
is indeed phosphorylated in the cells, Swiss 3T3 and
aortic smooth muscle cells were prelabeled with
[
P]orthophosphate and then tested with PMA, NaF,
angiotensin II, or vasopressin, which are known to stimulate PKC either
directly or indirectly. Cholate extracts of these stimulated cells were
immunoprecipitated with antibody against
, and the
precipitated proteins were subjected to Tricine/SDS-PAGE and
autoradiography. Exposure of both lines of cells to PMA significantly
stimulated the phosphorylation of
, while the
inactive isomer, 4
-PMA, had little effect (Fig. 6C). In the aortic smooth muscle cells, the
phosphorylation of
was also stimulated by the
addition of natural vasoactive agents such as angiotensin II and
arginine vasopressin, which are known to activate phospholipase
C(42) . In addition, direct activation of G protein(s) in both
lines of cells by NaF induced moderate phosphorylation of
. All these data strongly suggested that
is phosphorylated by PKC during activation of receptors and G
proteins in vivo.
Figure 7:
Effect
of phosphorylation of on the interaction of
with G
. A, time
courses of ADP-ribosylation reaction of G
in the
presence of phosphorylated and unphosphorylated
. The ADP-ribosylation of G
was
performed at 30 °C in a reaction mixture that contained 200
nM G
, 1 µM
[
H]NAD, 1 µg/ml preactivated pertussis toxin,
and 5 nM (
,
) or 10 nM (
,
)
of phosphorylated (
,
) or unphosphorylated (
,
)
. At an appropriate time of incubation, aliquots
were withdrawn from the mixture to measure the amount of
[
H]ADP-ribose incorporated into
G
. B and C, elution profile of
phosphorylated and unphosphorylated
from
G
-agarose column. Phosphorylated (
) and
unphosphorylated
(
, 3 µg each) were
loaded onto the G
-agarose column (0.1 ml), which was
pre-equilibrated with TEN containing 0.05% Lubrol PX, and washed with
the same buffer (fractions 1-2). Then the column was
successively washed with the TEN buffer containing 0.4% Lubrol (B) or 1% Lubrol PX (C) (fractions
3-5), and with the TEN buffer containing 0.05% Lubrol (fractions 6-10). The protein bound to the column was
eluted with 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.3 M NaCl, and 5 µM GDP and AMF (fractions
11-14). The amount of
in each
fraction (0.4 ml) was quantitated by an immunoassay. The total recovery
of the affinity chromatography was between 60 and
100%.
These results are consistent with the idea we
have described (Fig. 5) that the phosphorylation of induces a conformational change of
. In
fact, the
-
interaction domain in
seemed to involve the phosphorylation site of
,
since the phosphorylation of
by PKC was
markedly inhibited by G
(Fig. 8). This
inhibition was relieved by the addition of GTP
S or AMF, a reagent
converting G
into GTP-bound form to be released from
. These results imply that, upon activation of G protein, the
dissociated
becomes a substrate for PKC and
that the phosphorylation in turn raises the affinity of
for
subunit.
Figure 8:
Effect
of G on the phosphorylation of
by PKC. Prior to phosphorylation,
(0.5
µg) was incubated with (lanes 2-4) or without (lane 1) an equimolar amount of G
at 0 °C
for 10 min, and then it was incubated at 30 °C for 10 min in the
absence (lanes 1 and 2) or presence of 6.5 µM GTP
S and 10 mM magnesium acetate (lane 3)
and AMF (lane 4, magnesium acetate was used instead of
magnesium chloride). After incubation, the mixtures were subjected to
phosphorylation by PKC at 30 °C for 20 min and then to
Tricine/SDS-PAGE with subsequent autoradiography. The position of the
phosphorylated
is indicated by an arrow.
A novel form of subunit,
, was
originally purified from bovine spleen, partially sequenced, and
designated
previously(12) . In the present
study, we have determined the complete structure of intact
protein, which exactly matched with that predicted from the cDNA
sequence. The CXXL motif found at the C terminus of
also agreed with geranylgeranylation of the
C-terminal cysteine in mature
(16) . After
the prenylation, three amino acids, Thr-Ile-Leu, are known to be
removed, and newly exposed cysteine is carboxylmethylated(2) .
The
subunits other than
and
(36, 43) have been speculated to be N-acylated,
because their N termini is blocked and those of
and
were able to be truncated by an acylamino
acid-releasing enzyme(44) . Here, we have identified N-acetylated serine at the N terminus of
.
Co- or post-translationally, the N-terminal methionine could be cleaved
to expose serine to be N-acetylated.
In contrast to
specific localization of various subunits including
,
, and two
s(3, 4, 7, 9, 10, 11, 17) ,
showed ubiquitous distribution in rat tissues as
well as
(9, 17) . Taking our previous
study (17) on the tissue distribution of various
subunits
into consideration,
seems to be a major
subunit in many tissues other than neural tissues, in contrast with
and
enriched in the brain. These
results suggested that
may be involved in the signal
transduction common to the various cellular function.
We should
emphasize the unique property of being
phosphorylated at Ser
(or Ser
) by PKC in
vitro. Even in cultured cells having
as a major
subunit,
was phosphorylated by treatment of
cells with PMA, a direct activator of PKC. Among various isozymes of
PKC tested,
was efficiently phosphorylated by
diacylglycerol- (or PMA-)dependent PKC such as cPKC
, cPKC
,
and nPKC
but not by diacylglycerol-independent aPKC
in
vitro. This is compatible with PMA-induced phosphorylation of
in vivo. Since cPKC
, nPKC
,
nPKC
, and aPKC
are expressed in both Swiss 3T3 and aortic
smooth muscle cells(45, 46, 47) , it is
speculated that cPKC
and nPKC
would participate in the
phosphorylation of
in these cells. In addition,
similar selectivity for isozymes of PKC was observed with a
physiological substrate, myristoylated alanine-rich protein kinase C
substrate, which has the same phosphorylation site domain
(S*XK) as the predicted site of
(39) . It should be also stressed that
physiological vasoactive ligands such as angiotensin II and vasopressin
for aortic smooth muscle cells induced the phosphorylation of
endogenous
. These extracellular signals are known to
activate the cellular phospholipase C (42) possibly via
G
-type G proteins. These results strongly suggest that
is phosphorylated by PKC, which is activated in the
cells by physiological agonists.
It is important to identify the
subtype of the subunit associating with
in vivo. If the partner is a G
-type
subunit activating phospholipase C-
or alternatively
by itself regulating the phospholipase C-
,
the phosphorylation of
stimulated by the product
(diacylglycerol) of the signaling pathway may have a feedback role.
Alternatively,
may transduce signal other than
the phospholipase C pathway together with
subunits such as
G
and G
, and the dual pathways may
interact at the site of the phosphorylation of
.
In fact, one of the physiological partners of
should be G
and G
capable of
interacting with
in a phosphorylation-sensitive
manner. Although we have not examined this, some
subunits of G
protein may also be phosphorylated in these cells stimulated as shown
in other cells (21, 22) and may affect the coupling of
-
subunits. In all cases, identification of
/
-mediated signaling pathway will help to
assess the possibilities described above concerning the physiological
role of the reinforced
-
interaction due to the
phosphorylation.
Like the other complexes playing
signal-transducing roles by itself,
was able to
inhibit the Ca
/calmodulin-stimulated adenylyl cyclase
in rat retinal membranes and stimulate the activity of phospholipase C
in the cytosol of HL60 cells (data not shown). Thus,
itself had potency to transmit some signals, although the
phosphorylation of
gave almost no effect on
these regulations. These observations suggest that a possible
conformational change of
due to the
phosphorylation is localized at a contact site with G
without affecting the
-effector interaction.
Comparison of amino acid sequences of various isoforms of
revealed diverged residues concentrated at the N-terminal region, which
seems to participate in individual functions of
complexes.
The experimental evidence that the N-terminal 15 residues of
subunits specifies the interaction with G
s (33) is in line
with the regulatory role of the extreme N-terminal phosphorylation of
in the
-
interaction.
Another
interesting aspect of the present study is the
Ca-dependent cleavage at a specific site
(Lys
-Thr
) of
by
calpain. Calpain, a widely distributed enzyme, absolutely requires
Ca
ion for proteolyzing specific endogenous
substrates, such as enzymes, membrane proteins, cytoskeletal proteins,
and calmodulin binding proteins(48, 49) . Stimulation
of phospholipase C by a specific receptor/G protein system
simultaneously induces activation of PKC and mobilization of
intracellular Ca
ion, which probably lead to
activation of calpain(50) . The
is a common
target of these two enzymes. As shown in Fig. 5, however, the
proteolysis of
by calpain was noticeably inhibited
by the PKC-catalyzed phosphorylation. We speculate that one of the
physiological roles or an exclusive one of the phosphorylation of
might be the protection of
from
calpain attack when
was dissociated from G
upon G protein activation. Whether the calpain-induced degradation of
occurs in vivo and whether this degradation
is protected by PKC-induced phosphorylation are intriguing questions
that should be clarified in future experiments.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U37561[GenBank].