(Received for publication, October 31, 1994; and in revised form, December 29, 1994)
From the
The subunits of G
family G proteins,
G
(G
),
G
(G
), and G
were
expressed with G protein
and
subunits in insect cells using a baculovirus system. The trimeric
forms of G proteins, G
(G
),
G
(G
), and G
(G
), were solubilized by 1% sodium
cholate and purified by sequential chromatography on three kinds of
columns. G
, G
, and G
activated
phospholipase C-
purified from bovine brain in the presence of
aluminum fluoride to the same extent. Muscarinic acetylcholine receptor
m1 subtype stimulated the guanosine 5`-O-(3-thiotriphosphate)
(GTP
S) binding to G
, G
, and G
in the presence of similar concentrations of carbamylcholine.
When m1 receptor, G protein, and phospholipase C-
were
reconstituted in lipid vesicles, each subtype of G
family G
proteins mediated the activation of phospholipase C-
by
carbamylcholine in the presence of either 1 µM GTP
S
or 1 mM GTP. Phospholipase C-
stimulated the GTPase
activity of G
, G
, and G
in the
presence of m1 receptor and carbamylcholine but did not stimulate the
GTPase activity of G
. Protein kinase C phosphorylated m1
receptor and phospholipase C-
, but the phosphorylation did not
significantly affect the ability of the m1 receptor to stimulate
phospholipase C-
in the reconstitution system of purified
proteins.
The G(
)family of GTP-binding regulatory
proteins (G proteins) (
)consists of four kinds of
subunits(1) . The cDNA clones of G
and
G
were isolated from mouse brain library by
Strathmann and Simon(2) . We have identified bovine cDNA clones
encoding G
family
subunits designated
G
and G
(3) that correspond
to murine G
and G
,
respectively(2, 4) . Human G
and its
mouse version G
expressed in hematopoietic tissues
have also been identified as members of the G
family(4, 5) .
G family
subunits are thought to activate phospholipase C-
(PLC-
) in a
pertussis toxin-insensitive manner. A mixture of G
and
G
(G
/G
) purified from bovine
liver or a mixture of G
and G
from
bovine brain was reported to activate PLC-
in the presence of
GTP
S (6) or aluminum fluoride(7) . In contrast to
G
and G
, the function of
G
is as yet unknown. Whereas the G
(G
) transiently expressed in COS-7 cells was reported
to activate purified PLC-
(8) ,
G
coexpressed with metabotropic glutamate receptors
in Xenopus oocytes was found to inhibit the PLC activity in
oocytes(9) . Here, we have examined whether purified
G
stimulates or inhibits the activity of purified
PLC-
.
Muscarinic acetylcholine receptors (mAChRs) consist of
five subtypes that are coupled to their effectors via G proteins. Among
the five subtypes of mAChRs, m1, m3, and m5 subtypes activate PLC in a
pertussis toxin-insensitive manner, whereas m2 and m4 subtypes inhibit
adenylate cyclase via pertussis toxin-sensitive G
proteins(10, 11) . G family G proteins are
thought to mediate the former pathway. The mixture of G
and
G
reconstituted in lipid vesicles with m1 mAChR was
reported to bind GTP
S and subsequently activate PLC-
in an
agonist-dependent manner(12) . It has been shown that PLC-
stimulates the GTPase activity of G
/G
in the
presence of m1 mAChR and carbamylcholine(13) . It has not been
determined, however, if there are any quantitative differences between
G
and G
regarding their interactions with m1
mAChR and PLC-
. Furthermore, it is not known if
G
can also be activated by m1 mAChR or activate
PLC-
or whether its GTPase is activated by PLC-
.
It is difficult to isolate and characterize individual members of
G family G proteins because of their limited expression in
native tissues. Recently, the recombinant forms of G
,
G
, and G
have been purified from
Sf9 insect cells that coexpressed
subunits (14, 15) . We have independently found
that the coexpression of
subunits with G
family
subunits in Sf9 insect cells facilitated the solubilization of
the
-subunits and enabled us to purify the trimeric G proteins.
Activation of PLC leads to the breakdown of phosphatidylinositol
4,5-bisphosphate (PIP) and the generation of the
intracellular messengers, inositol 1,4,5-trisphosphate (IP
)
and diacylglycerol(16) . Diacylglycerol activates certain
isotypes of protein kinase C(17, 18) . Experiments in vivo have shown that the activation of protein kinase C
leads to the inhibition of mAChR-mediated PLC
activation(19, 20, 21, 22) . It
would provide the most straightforward explanation if protein kinase C
phosphorylates the components in the pathway of PLC activation and
attenuates its signal transduction. In fact, protein kinase C is
reported to phosphorylate m1 mAChR (23) and
PLC-
(24) in vitro. The effect of
phosphorylation by protein kinase C on m1 mAChR-mediated stimulation of
PLC-
can now be examined in a reconstitution system using purified
proteins.
In the present studies, we have expressed and purified
each subtype of the G family G proteins,
G
, G
, and G
, and
reconstituted them in lipid vesicles with m1 mAChR, PLC-
, or both.
Each of the G
family G proteins was found to be activated
by m1 mAChR and to activate PLC-
with essentially the same
efficiency. Protein kinase C phosphorylated m1 mAChR and PLC-
, but
the activation of PLC-
by carbamylcholine was not affected by the
phosphorylation.
We have constructed a transfer vector in which both coding
regions of and
subunits were placed downstream of the
polyhedrin promoters. A polymerase chain reaction-amplified coding
sequence of bovine
subunit (9, 25) was ligated to the SmaI-EcoRI sites of pVL1393 (pVL1393
2). The 1.9
kilobase pair NaeI-VspI fragment containing a
polyhedrin promoter, a coding sequence of the
subunit, and a poly(A) signal sequence was excised from pVL1393
2.
The fragment was filled in with T4 DNA polymerase and ligated to the EcoRV site of pVL1393. Among obtained vectors, the vector in
which two promoters were arranged back-to back, named pVL3R
2, was
selected for further construction. The coding sequence of bovine
subunit excised from pG
4 (26) was
inserted between the SmaI and XbaI sites of
pVL3R
2.
For PLC assay, m1 mAChR and G
proteins (20-80 pmol) were reconstituted in a modified HEN buffer
(20 mM Hepes-NaOH (pH 7.0), 1 mM EGTA, and 160 mM NaCl). Reconstituted vesicles (10-20 µl) were mixed with
phospholipid vesicles (10 µl) and an assay buffer (final
concentrations: 20 mM Hepes-NaOH buffer (pH 7.0), 1 mM EGTA, 10 mM MgCl, 30 mM NaCl, 1
µM GDP, and 10 µM free Ca
in 20 µl, final volume). As mAChR ligands, carbamylcholine
and atropine were also supplemented to be 1 mM or 10
µM, respectively. The assay was initiated by the addition
of a mixture of GTP
S and PLC-
(final concentrations: 100
nM and 10 fmol/tube, respectively, in 10 µl, final
volume). The reaction was performed for 0-15 min at 30 °C.
Figure 1:
Expression in Sf9 cells of
G and
subunits and
their solubilization. Sf9 cells (2-3
10
) were
infected with G
virus (lane 1),
virus (lane 3), or both (lane 2). P2 fractions were prepared from these cells 3 days
after infection and solubilized with 1% sodium cholate. An aliquot from
solubilized preparations (40 µg as protein) was subjected to
SDS-PAGE (acrylamide, 12%). Arrowheads indicate the position
of G
. A, Coomassie Blue staining; B, immunoblot staining with an antibody against a
carboxyl-terminal peptide of
G
.
We have purified the trimeric form of G proteins from cells
expressing G, G
, or
G
simultaneously with the
subunits. For
comparison, recombinant G
was also expressed in Sf9
cells and purified with
subunits using the same procedure. Fig. 2shows Coomassie staining patterns following SDS-PAGE of
the fractions in each purification step of recombinant G
.
The G
and
subunits were observed in
the particulate fractions as bands of 42 and 36 kDa, respectively. Both
G
and
subunits could be partially
solubilized with a buffer solution containing 1% sodium cholate. The
yield of solubilization was higher for
subunits compared
with
subunits.
Figure 2:
SDS-PAGE of fractions generated during the
purification of recombinant G. Aliquots (10 µg) from
each step of the purification procedure of recombinant G
were subjected to SDS-PAGE (acrylamide, 12%) followed by
Coomassie Blue staining.
The recombinant proteins were partially
purified from the cholate extract using DEAE-Sephacel and
heptylamine-Sepharose column chromatography. A hydroxylapatite column
was used to separate free subunits and
trimers, which were eluted with 5-25 and 50-75 mM potassium phosphate buffer, respectively (not shown). Table 1summarizes the results of purification of a recombinant
G
. Similar results were obtained for purification of
G
and G
. Starting with 900 ml of cell culture
suspension, final yields of trimeric G proteins ranged from 0.2 to 0.5
mg.
Fig. 3A shows the SDS-PAGE patterns of purified
G, G
, G
, and G
.
Molecular masses of
subunits were estimated to be 43, 42, 42, and
39 kDa for G
, G
, G
, and
G
, respectively. Molecular masses of
and
subunit were estimated to be 36 and 8 kDa,
respectively (Fig. 3, A and B). The intensity
of the stained band for each of the subunits G
,
G
, G
, and G
was
essentially the same as that of the stained band for each of the
copurified
subunits. These results indicate that each of these
subunits is capable of forming a stoichiometric complex with
subunit and was purified as an
trimer.
Figure 3:
SDS-PAGE of purified recombinant G
proteins. A, purified G, G
,
G
, and G
(1 µg of protein each) were
subjected to SDS-PAGE (acrylamide, 12%). B,
subunits purified from bovine brain (lane 1, 51.5 µg) or
recombinant
subunits purified from
Sf9 cells (lane 2, 34.8 µg) were electrophoresed in a
16.5% SDS-polyacrylamide gel (16% T, 3% C gel) as
described(35) . The gels were stained with Coomassie Blue. The arrowhead indicates the position of the
subunit.
Two forms of G, a major (42
kDa) and a minor (44 kDa) form, were observed. The minor form may
represent a polypeptide translated from the polyhedrin initiator
derived from the pVL1392 vector.
Figure 4:
Activation of brain PLC- by
recombinant G
, G
, and G
.
Phospholipid vesicles containing purified G proteins (0-7.7 pmol)
were incubated with PLC-
purified from bovine brain (10 fmol) for
10 min at 37 °C in the absence (
) or presence (
) of
AlF
. The assay was carried out in
duplicate. Data were fitted to a Michaelis-Menten
equation.
Figure 5:
Stimulation by carbamylcholine of
[S]GTP
S binding to G proteins reconstituted
with m1 mAChRs. The m1 (
and
) or m2 (
and
) mAChRs were reconstituted with G proteins in lipid vesicles,
and [
S]GTP
S binding activity in the
vesicles was assayed in the presence of 1 mM carbamylcholine
(
and
) or 10 µM atropine (
and
)
as described under ``Experimental Procedures.'' The amounts
of m1 and m2 mAChRs in one tube were 73.2 and 63.9 fmol, respectively.
The vesicles in one tube contained 600-650 fmol of G
proteins.
Figure 6:
Effect of carbamylcholine concentrations
on the [S]GTP
S binding. Each tube contained
14.0 fmol of m1 mAChRs and 300-400 fmol of G proteins. Each
sample was incubated for 8 min at 30
°C.
GDP is known to have a lower affinity
for G or G
reconstituted with m2 mAChR in the
presence of agonist than in the presence of
antagonist(33, 39, 40) . We have examined
whether GDP has different affinities for G
reconstituted
with m1 mAChR in the presence or absence of agonists. As shown in Fig. 7, GDP inhibited the GTP
S binding in the presence or
absence of agonist in a dose-dependent manner, and the higher
concentration of GDP was required to inhibit the GTP
S binding in
the presence of agonist. The concentrations of GDP giving 50%
inhibition of the [
S]GTP
S binding were
estimated to be 0.2 and 4 µM in the absence and presence
of carbamylcholine, respectively. In contrast, the concentrations of
cold GTP
S giving 50% inhibition of the
[
S]GTP
S binding were not different in the
presence of carbamylcholine and atropine (not shown).
Figure 7:
Effect of GDP concentrations on
[S]GTP
S binding to G
.
Experiments were performed as described in the legend to Fig. 5,
except that different concentrations of GDP were included and
incubation time was 15 min. The amounts of reconstituted m1 mAChR and
G
in each tube were 35.0 and 215 fmol,
respectively.
Figure 8:
Stimulation by carbamylcholine of
PLC- reconstituted with m1 mAChRs and G proteins. The m1 mAChRs
and G proteins were reconstituted in lipid vesicles as described under
``Experimental Procedures.'' The reaction was initiated by
addition of a mixture of PLC-
and GTP
S (final concentrations
of 0.2 and 100 nM, respectively) and proceeded for 0-10
min at 30 °C. The vesicles in one tube contained 13.4 fmol of m1
mAChRs and 300 fmol of G
, 700 fmol of G
, or
800 fmol of G
.
Figure 10:
Effect of protein kinase C on the
carbamylcholine-stimulated PLC activity. A, lipid vesicles
containing m1 mAChR, G family G proteins, and PLC-
were incubated with protein kinase C (16 nM) and
[
P]ATP (12.5 µM) for 1 h at 30
°C, and then an aliquot of the reaction mixture was subjected to
SDS-PAGE and autoradiography. The bands with apparent molecular masses
of 150, 80, and 59 kDa correspond to PLC-
, protein kinase C, and
m1 mAChR, respectively. A weakly phosphorylated band of 42 kDa
corresponds to the
subunit of G proteins. The band with an
apparent molecular mass of 29 kDa was not identified. B and C, lipid vesicles containing m1 mAChR, G
family G
proteins, and PLC-
were incubated with protein kinase C (125
nM) in the presence or absence of ATP (100 µM)
for 1 h at 30 °C, and then the vesicles were separated from free
ATP through a Sephadex G-50 column (2 ml). PLC activity of the void
volume fraction was examined in the presence of 1 mM GTP and 1
mM carbamylcholine (
and
) or 10 µM atropine (
and
), where samples treated with protein
kinase C in the presence or absence of ATP were represented by dashed lines (
and
) or solid lines (
and
), respectively. The amounts of m1 mAChR were
5.9-9.0 fmol/tube. Data in C are the means of three
independent experiments.
Figure 9:
Effect of PLC- concentrations on the
GTPase activity of G proteins. The mAChR and G proteins were
reconstituted in lipid vesicles as described under ``Experimental
Procedures.'' The reaction was started by addition of various
concentrations of PLC-
and then incubated for 15 min at 30 °C
in the presence of 1 mM carbamylcholine (
) or 10
µM atropine (
). The vesicles in each tube contained
70 fmol of m1 mAChR and 300-350 fmol of G
family G
proteins (A, B, and C) or 30 fmol of m2
mAChR and 550 fmol of G
(D). A half-maximal
stimulation of was observed in the presence of 0.78 nM PLC-
for G
, 1.87 nM PLC-
for
G
, and 1.52 nM G
in this
assay.
Reconstituted vesicles containing m1 mAChR, G, and
PLC-
were subjected to phosphorylation by protein kinase C in the
presence of 0.1 mM ATP, followed by the assay of PLC activity
in the presence of GTP. Control samples were treated in the same way
except for the omission of ATP. The PLC-
activity either in the
absence or presence of 1 mM carbamylcholine was not affected
by whether the vesicles had been treated with protein kinase C in the
presence or absence of ATP (Fig. 10B). Fig. 10C shows the dose-response curves for stimulation
by carbamylcholine of the PLC activity of samples treated with protein
kinase C in the presence or absence of ATP. The PLC activity of samples
treated in the presence of ATP tends to require higher concentrations
of carbamylcholine. The concentration of carbamylcholine giving a
half-maximal effect was estimated to be 21.0 ± 5.1 and 32.4
± 11.5 µM for samples treated with protein kinase C
in the absence or presence of ATP.
We have expressed three kinds of G family
subunits, G
, G
, and
G
, together with
and
subunits in Sf9 cells and purified each of them as an
trimer. Consistent with previous
results(14, 15) , we also found that the coexpression
of
subunits was necessary for functionally active G proteins
to be solubilized. In addition, we noticed that the yield of active G
proteins was increased by replacing air with 50% O
/50%
N
during the cell culture (41) and by infecting the
cell with
and
recombinant virus with a ratio of
3-4:1 instead of 1:1. Relatively lower amounts of
recombinant virus were used because the expression level of
subunits was found to be much higher than that of
subunits, when
a recombinant virus encoding G
,
, and
subunits in tandem was used (not shown). We could purify
trimers of G
, G
, and G
to apparent homogeneity by three-step column chromatographies,
and these purified trimers were active with respect to interaction with
m1 mAChRs and PLC-
. These results indicate that
G
, G
, or G
may
make a functionally active complex with
subunits.
The present reconstitution studies provide direct
evidence that G, as well as G
and
G
, is capable of activating PLC-
. We did not find any
significant differences among G
, G
, and
G
as far as their interaction with PLC-
. This finding
is consistent with and extends the report by Hepler et al.(14) but is not consistent with the previous report that
the potency of G
(G
) in activating
PLC-
was half as much as that of G
or
G
(8) . This discrepancy may reflect the
differences in the expression level between G
and
G
or G
. We noticed that the
expression level in COS-7 cells was lower for G
compared with G
(not shown).
Berstein et al.(12) have presented evidence for the functional
interaction between the m1 mAChR and a mixture of G and
G
and indicated that both G
and G
are responsive to the m1 mAChR. We have confirmed and extended
their results and have shown that G
, G
, or
G
may mediate the signal transduction from the m1 mAChR to
PLC-
with similar potency and efficacy. These findings, however,
are not in accord with the previous results indicating that the
formation of IP
induced by the activation of metabotropic
glutamate receptors mGluR1 (9) or thyrotropin-releasing
hormone receptor (42) in Xenopus oocytes was
accelerated by the coexpression of G
but was
inhibited by the coexpression of G
or
G
. The reason for this discrepancy is not known. A
possible explanation is that the PLC-
expressed in Xenopus oocytes has different properties from the PLC-
in bovine
brain. The amphibian PLC-
is known to be relatively distant from
any known mammalian PLC-
s, with the closest identity of 64% to
mammalian PLC-
(43) . This explanation remains
to be examined by reconstituting purified frog PLC-
with G
family G proteins.
The stimulation by carbamylcholine of
IP formation in the reconstituted vesicles containing m1
mAChR, G
, and PLC-
was observed in the presence of 1
mM GTP as well as in the presence of 1 µM GTP
S ( Fig. 8and Fig. 10). This result
indicates that the presence of three protein components is sufficient
for the signal transduction from carbamylcholine to IP
in
the presence of endogenous guanine nucleotide. Berstein et al.(12) have reported that the stimulation by carbamylcholine
of IP
formation is observed in the presence of GTP
S
but not in the presence of GTP. The reason for the discrepancy is not
known, but is not due to the lack of stimulation of GTPase activity by
PLC-
. We have confirmed and extended the result by Berstein et
al.(13) and shown that the GTPase activities of
G
, G
, and G
are stimulated by
PLC-
approximately to the same extent. Concentrations of PLC-
giving a half-maximal effect were similar among the three G proteins,
indicating that PLC-
interacts with these G proteins with similar
affinity.
The m1 mAChR may interact with all of G,
G
, and G
, but the m2 mAChR apparently did not
interact with any of them. A slight activation by the m2 mAChR of
[
S]GTP
S binding to a mixture of G
and G
in the previous report (12) may
represent a contamination by G
and G
in the
G
/G
preparation purified from brain or liver.
We have found that it is very difficult to prepare G
preparations free from G
/G
starting from
intact tissue, although it is possible to avoid the problem by using
recombinant G proteins. The PLC-
may interact with all of
G
, G
, and G
but does not interact
with G
, as was evident from the lack of stimulation by
G
of PLC activity and of stimulation by PLC of GTPase
activity of G
. These results demonstrate the strict
specificity of the interactions of G
family G proteins with
their receptor and effector, in contrast with the apparent lack of
specificity among the three G
family G proteins.
The
stimulation of protein kinase C by phorbol esters is known to lead to
the desensitization of the activation of PLC mediated by mAChRs ((20, 21, 22) ; for review see (19) ). In the present studies, we have shown that the
phosphorylation of m1 mAChR and PLC- does not affect the
stimulation of PLC-
by carbamylcholine in the reconstitution
system. We cannot, however, exclude the possibility that the
phosphorylation by protein kinase C reduces the affinity of the
interaction of G proteins with m1 mAChR or PLC-
, but the effect
has not been detected by the presence of excessive amounts of these
proteins in the reconstitution system. It is also possible that other
types of protein kinase C may be involved in the phosphorylation and
the desensitization. Alternatively, it is likely that the presence of
three components of the m1 mAChR, G protein, and PLC-
is
sufficient for the signal transduction from acetylcholine to IP
but is not sufficient for the regulation of the signal
transduction.
In summary, we have expressed and purified three kinds
of G family G proteins, G
, G
, and
G
, and reconstituted them with the m1 mAChR and PLC-
in lipid vesicles. In the reconstitution system, we have shown that 1)
these G proteins mediated the activation of PLC-
by m1 mAChR in
the presence of agonist and GTP, 2) the GTPase activity of these G
proteins is enhanced by PLC-
, and 3) the signal transduction from
the m1 mAChR to PLC is not affected by phosphorylation by protein
kinase C of the m1 mAChR and PLC-
.