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(Received for publication, October 31,
1994; and in revised form, December 29, 1994) From the
The
The G G 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 It is difficult to isolate and characterize individual members of
G Activation of PLC leads to the breakdown of phosphatidylinositol
4,5-bisphosphate (PIP In the present studies, we have expressed and purified
each subtype of the G
We have constructed a transfer vector in which both coding
regions of
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
Figure 1:
Expression in Sf9 cells of
G
We have purified the trimeric form of G proteins from cells
expressing G
Figure 2:
SDS-PAGE of fractions generated during the
purification of recombinant G
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
Fig. 3A shows the SDS-PAGE patterns of purified
G
Figure 3:
SDS-PAGE of purified recombinant G
proteins. A, purified G
Two forms of G
Figure 4:
Activation of brain PLC-
Figure 5:
Stimulation by carbamylcholine of
[
Figure 6:
Effect of carbamylcholine concentrations
on the [
GDP is known to have a lower affinity
for G
Figure 7:
Effect of GDP concentrations on
[
Figure 8:
Stimulation by carbamylcholine of
PLC-
Figure 10:
Effect of protein kinase C on the
carbamylcholine-stimulated PLC activity. A, lipid vesicles
containing m1 mAChR, G
Figure 9:
Effect of PLC-
Reconstituted vesicles containing m1 mAChR, G We have expressed three kinds of G The present reconstitution studies provide direct
evidence that G Berstein et al.(12) have presented evidence for the functional
interaction between the m1 mAChR and a mixture of G The stimulation by carbamylcholine of
IP The m1 mAChR may interact with all of G 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- In summary, we have expressed and purified three kinds
of G
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 6246-6253
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Family G Proteins G
(G
), G
(G
), and
G
Expressed in the Baculovirus-Insect Cell System (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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)
(GTPS) 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 GTPS
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.
()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) . 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
GTPS (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-. 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 GTPS 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-. 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. ) 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. 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.
Materials
[
S]GTPS
and [H]QNB were purchased from DuPont NEN,
[H]PIP was from American Radiolabeled
Chemicals, Inc., [
P]ATP was from Amersham
International plc, and [P]GTP was from the
Hungarian Academy of Science. The cDNA encoding mouse
G subunit was kindly donated by Dr. M. I. Simon from
the California Institute of Technology at Pasadena; Sf9 cells and
recombinant baculoviruses containing cDNA encoding the human m1 and m2
mAChR were donated by Dr. E. M. Ross at the University of Texas
Southwestern Medical Center at Dallas.Construction of G
The
baculovirus transfer vectors containing sequences for G protein Transfer Vectors
subunits (pVLG, pVLG,
pVLG, and pVLG) were constructed as follows.
The NcoI-SmaI fragment containing the entire
G coding region was excised from pGL1(3) ,
where the NcoI site was filled in with T4 DNA polymerase to
yield a blunt end and attached by an EcoRI linker prior to SmaI digestion. The fragment was inserted between the EcoRI and SmaI sites of the baculovirus transfer
vector pVL1392. The NaeI-EcoRI fragment, including
the G coding sequence, was excised from pGL7 (3) and ligated to the SmaI-EcoRI sites of
pVL1393. The NotI-SspI fragment (1.2 kilobase pairs)
was excised from the P3 plasmids containing the G cDNA (2) and was inserted between the NotI and SmaI sites of pVL1392. The NcoI-HincII
fragment containing the entire coding sequence of bovine
G1 that was excised from the clone
pG3 was filled in with T4 DNA polymerase, attached to
an EcoRI linker, and inserted into the EcoRI site of
pVL1392. 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 (pVL13932). 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 pVL13932.
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 pVL3R2, was
selected for further construction. The coding sequence of bovine
subunit excised from pG4 (26) was
inserted between the SmaI and XbaI sites of
pVL3R2.Isolation and Inoculation of Recombinant
Baculoviruses
Recombinant viruses were generated and isolated
according to the standard procedures(27, 28) . Spodoptera frugiperda (Sf9) cells were cultured in 75 cm flasks containing 12 ml of IPL-41 medium supplemented with 4%
fetal bovine serum at 28 °C. Cells were grown in 150 ml of the same
medium supplemented with 0.1% pluronic F-68 with constant stirring at
60 rpm. Cells were usually seeded at a density of 0.4 
10
cells/ml in 150 ml of medium and allowed to multiply for
3-4 days up to 2.5-3 10 cells/ml. Grown
cells were spun down and suspended in 5-7 ml of the same medium.
To this cell suspension, 12-14 ml of subunit recombinant
virus (6 10
plaque-forming units/ml) and 3-4
ml of recombinant virus (6
10 plaque-forming units/ml) were added. After incubation
for 1 h, the cell suspension was transferred into a spinner flask with
130 ml of the medium and cultured in an atmosphere of 50%
O/50% N for 65-72 h. Harvested cells were
stored at -80 °C until use.Preparation of G Proteins
All purification steps
were carried out at 4 °C. The cells (2-3
10) from the six spinner flasks were resuspended in 280 ml
of ice cold homogenization buffer containing 20 mM Tris-HCl
buffer (pH 8.0), 1 mM EDTA, 1 mM DTT, and a mixture
of protease inhibitors (2.5 µg/ml pepstatin, 0.002 mg/ml
phenylmethylsulfonyl fluoride, 0.02 mg/ml leupeptin, and 0.5 mM benzamidine). Cells were homogenized with 10-15 strokes in a
Potter-type homogenizer. The homogenates were centrifuged at 100,000
g for 30 min. Pellets were washed once in 280 ml of
the homogenization buffer, resuspended in 200 ml of the homogenization
buffer supplemented with 1% sodium cholate, and gently stirred for 2 h.
After centrifugation at 100,000 g for 45 min, the
supernatant fraction (210 ml) was applied to a DEAE-Sephacel column (50
ml) pre-equilibrated with the homogenization buffer containing 1%
sodium cholate (Buffer A). The column was washed with 50 ml of Buffer A
and eluted with a gradient consisting of 100 ml each of Buffer A and
Buffer A supplemented by 500 mM NaCl. The subunits,
together with subunits, were eluted between 120 and 200
mM NaCl. Aliquots of each column fraction were subjected to
SDS-PAGE and immunostaining. The G protein-rich fractions (30 ml) were
diluted 5-fold with Buffer B (20 mM Hepes-NaOH buffer (pH
7.0), 1 mM EGTA, 1 mM DTT, and protease inhibitors)
and then applied to a heptylamine column (20 ml) that had been washed
with 40 ml of buffer B supplemented with 0.2% sodium cholate. The
column was eluted with a gradient consisting of 70 ml each of Buffer B
supplemented with 0.2% sodium cholate and 200 mM NaCl and
Buffer B containing 1.75% sodium cholate and 50 mM NaCl. The G
protein trimers were eluted between 0.7 and 1.0% sodium cholate. The G
protein-rich fractions were supplemented with 5 mM potassium
phosphate and applied to a hydroxylapatite column (5 ml), which had
been pre-equilibrated with Buffer C (20 mM Hepes-NaOH buffer
(pH 7.0), 1 mM EGTA, 1 mM DTT, and 0.8% sodium
cholate) supplemented with protease inhibitors and 5 mM potassium phosphate. The column was washed with Buffer C
containing 5 mM potassium phosphate and eluted with a linear
gradient of Buffer C and the Buffer C containing 150 mM potassium phosphate. subunits were eluted at 5-25
mM potassium phosphate, and then the trimers
were eluted between 50 and 75 mM potassium phosphate.Phospholipase C Assays
Phospholipase C-
(PLC-) was purified from bovine brain by a method modified from
those of Ryu et al.(29) and Rhee et
al.(30) . PLC assay was performed according to Smrcka et al.(7) . Briefly, substrate was provided as
vesicles containing 2 µg of phosphatidylinositol 4,5-bisphosphate
(PIP), 20 µg of phosphatidylethanolamine, and 10 nCi of
[H]PIP in a Hepes buffer solution (43
mM Hepes-NaOH buffer (pH 7.0), 2 mM EGTA, 1 mM DTT, 20 mM NaCl, 30 mM KCl, 5 µM GDP, 2 mM MgCl, and 0.06% sodium cholate in
50 µl, final volume). CaCl was supplemented to yield
approximately 1 µM free Ca
. Where
necessary, 20 µM AlCl and 6 mM NaF
were added as activators of G protein. The reaction was initiated by
addition of 10-20 fmol of PLC- and proceeded for 10 min at
37 °C. The reaction was terminated by addition of 600 µl of
methanol/CHCl mixture (2:1, v/v), 200 µl of
CHCl, and 200 µl of 0.1 M HCl, followed by
separation of the aqueous and lipid phases as described(7) .
[H]IP in the aqueous phase was
quantitated by a liquid scintillation counter.Reconstitution of mAChRs and G
Proteins
Recombinant mAChRs, m1 and m2 subtypes, were purified
by a single-step affinity chromatography from Sf9 cells expressing
human m1 or m2 subtypes(31) . Reconstitution of mAChRs and G
proteins was performed as described
previously(32, 33) ., 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 GTPS 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.[
For
[H]QNB and
[S]GTPS Binding AssaysH]QNB binding, the reconstituted vesicle
containing mAChRs and G proteins (10 µl) was incubated with 1
nM QNB ([H]QNB, 50-60 cpm/fmol) in
the presence or absence of 10 µM atropine for 60 min at 30
°C in HEN buffer (20 mM Hepes-KOH (pH 8.0), 1 mM EGTA, and 160 mM NaCl in 500 µl, final volume). For
the [S]GTPS binding assay, the same
reconstituted vesicle (10-20 µl) was mixed with HEN buffer
(80-90 µl) supplemented with 1 mM DTT, 20 mM MgCl, and 50 nM GTPS
([S]GTPS, 30-40 cpm/fmol), and either
1 mM carbamylcholine or 10 µM atropine. The
mixtures were incubated for 0-15 min at 30 °C and then
diluted with chilled solution (20 mM Tris-HCl (pH 8.0), 100
mM NaCl, and 25 mM MgCl in 0.5 ml, final
volume) containing 0.1 mM GTP. Bound forms of
[H]QNB and [S]GTPS
were assayed as described previously(32, 33) .
Specific activities of purified m1 and m2 receptors were estimated to
be approximately 2 nmol/mg protein. Recoveries of m1 and m2 receptors
in the reconstitution experiments were 5-16 and 10-20%,
respectively. The specific activities of G proteins reconstituted with
m1 receptors were estimated to be 1-2 nmol/mg protein, as
assessed by [S]GTPS binding activity.GTPase Assay
The GTPase assay was performed
according to Berstein et al.(13) . Briefly,
reconstituted vesicles (10 µl) containing mAChR and G proteins were
mixed with an assay mixture (final concentrations: 20 mM Hepes-NaOH buffer (pH 7.0), 1 mM EGTA, 100 mM NaCl, 5 mM MgCl, 0.5 µM GDP, 1
µM [-P]GTP (30-50
cpm/fmol), and either 1 mM carbamylcholine or 100 µM atropine in 30 µl, final volume). The reaction was initiated
by addition of PLC- (final concentration: 0-10 nM in 10 µl, final volume) and was proceeded at 30 °C for 15
min. GTP hydrolysis was measured by charcoal
precipitation(34) .Phosphorylation by Protein Kinase
C
Phosphorylation was performed as described
previously(23) . Briefly, reconstituted vesicles (180 µl)
containing m1 mAChRs were supplemented with G (20.8 pmol),
PLC- (1.5 pmol), and protein kinase C (a mixture of , ,
and subtypes in 2.6 pmol, final volume). Protein kinase C was
purified as described previously(35) . The vesicles were
incubated with or without 100 µM ATP for 1 h at 30 °C
in Buffer D (20 mM Hepes-NaOH buffer (pH 7.0), 1 mM EGTA, 100 mM NaCl, 1 mM GTP, 1 µM GDP, 1 mM carbamylcholine, and 10 µM free
Ca). The reaction mixture was passed through a small
column of Sephadex G-50 (2 ml) to remove ATP. The PLC assay was
initiated by addition of the void volume fraction from the Sephadex
column (20 µl) to the mixture of substrate and ligands (final
concentrations: 20 mM Hepes-NaOH buffer (pH 7.0), 1 mM EGTA, 10 mM MgCl, 1 mM DTT, 1
µM GDP, 1 mM GTP, 1 µM free
Ca, and either 1 mM carbamylcholine or 10
µM atropine in 30 µl, final volume). The reaction was
performed for 0-20 min at 30 °C. To confirm incorporation of
[P]phosphates, the reconstituted vesicles were
incubated with 12.5 µM [P]ATP in
Buffer D for 1 h at 30 °C. An aliquot (20 µl) was directly
subjected to SDS-polyacrylamide gel (12%) electrophoresis followed by
autoradiography and then Cerenkov counting of the receptor band.
Phosphorylation of PLC- and G protein subunit was estimated
from the density of the autoradiography.Miscellaneous Procedures
To detect the
subunits of G proteins, Tricine-buffered SDS-polyacrylamide gel
electrophoresis was employed(36) . G protein concentrations
were determined by the protein-dye binding method(37) . The
amount of active G proteins may be less than that estimated by protein
assay, because the maximum binding of
[S]GTPS in the presence of carbamylcholine
and m1 mAChR was only 10-20% of the amount of G proteins
estimated from protein assay. Antiserum against the carboxyl terminus
of G was prepared as described
previously(3) . A Konica Immunostaining Kit or
3,3`-diaminobenzidine tetrahydrochloride was used as a substrate for
peroxidase. Free Ca concentration was estimated by a
chelation calculation program(38) .
Expression and Purification of Recombinant G
Proteins
We have expressed G in Sf9 cells
alone or simultaneously with the G protein and subunits and
have solubilized these proteins with 1% sodium cholate. Immunoblot
analysis showed that the yield of solubilized G was
approximately 10 times higher when subunits were coexpressed
as compared with when subunits were expressed alone (compare lanes1 and 2 in Fig. 1). The
coexpression of subunits also facilitated the solubilization
of G and G (not shown). Endogenous
proteins that react with anti-G antibody, probably
intrinsic G or related proteins, were also found to be
solubilized by the same procedure (lane 3 in Fig. 1).
Some degradation of recombinant G, particularly in
the preparation expressed without subunits, was detected.
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.
, 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.
. 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.
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.
, 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.
, 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.
, 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.Activation of Phospholipase C-
To determine whether each subtype of G by G
Proteins family G proteins activates PLC- or not, each of the
subunits G, G, and G was
reconstituted with PLC- in lipid vesicles. In the presence of
aluminum fluoride, G, G, or G activated PLC- 13-19-fold (Fig. 4, A-C). In the absence of aluminum fluoride, these G
proteins only slightly enhanced (1.5-3-fold) the activity of
PLC- in a dose-dependent manner. Under the assay conditions
employed, the concentrations of G, G, and
G giving a half-maximal effect were estimated to be 7.37
± 4.87, 9.47 ± 6.89, and 11.5 ± 6.3 nM,
respectively. The PLC activity in the presence of excess G proteins was
also not different among G, G, and
G. These results indicate that the
AlF
-bound forms of G,
G and G have essentially the same affinity for
PLC- and the same ability to activate PLC-. In contrast,
G only slightly activated PLC- (1.5-3-fold), and
its activation was not enhanced by the addition of aluminum fluoride (Fig. 4D).
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.
Reconstitution of mAChRs and G Proteins
We have
reconstituted m1 or m2 mAChRs with each subtype of G family
G proteins in lipid vesicles in order to test the specificity of
coupling between receptors and G proteins. The m1 mAChRs markedly
stimulated [S]GTPS binding to the G family G proteins in the presence of 1 mM carbamylcholine, but they only slightly stimulated the
[S]GTPS binding in the presence of 10
µM atropine (Fig. 5). In contrast with m1 mAChRs,
m2 mAChRs did not stimulate the [S]GTPS
bindings to these G proteins. Fig. 6shows the effect of
concentrations of carbamylcholine on the
[S]GTPS binding to G,
G, and G reconstituted with m1 mAChRs. The
concentrations of carbamylcholine giving a half-maximal effect on the
[S]GTPS binding were estimated to be 6.42
± 3.15, 5.41 ± 2.00, and 3.47 ± 1.97 µM for G, G, and G,
respectively, and these values were not significantly different from
each other. These results indicate that the agonist-bound form of m1
mAChR may activate G, G, or G with similar potency.
S]GTPS 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]GTPS 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.
S]GTPS 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.
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 GTPS binding in the presence or
absence of agonist in a dose-dependent manner, and the higher
concentration of GDP was required to inhibit the GTPS binding in
the presence of agonist. The concentrations of GDP giving 50%
inhibition of the [S]GTPS binding were
estimated to be 0.2 and 4 µM in the absence and presence
of carbamylcholine, respectively. In contrast, the concentrations of
cold GTPS giving 50% inhibition of the
[S]GTPS binding were not different in the
presence of carbamylcholine and atropine (not shown).
S]GTPS 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.
Reconstitution of m1 Receptor, G Protein, and PLC-
Fig. 8shows that the activity of PLC- reconstituted
with m1 mAChR and each member of G family G proteins was
enhanced by carbamylcholine in the presence of GTPS. A similar
stimulation was observed when 1 mM GTP was used instead of
GTPS, as shown later (see Fig. 10, B and C). The activity of PLC- in the presence of
carbamylcholine and G, G, or G was estimated to be 16, 17, or 30
fmolmin
/mg of G protein, respectively.
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 GTPS (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.
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.
Stimulation of the GTPase Activity of G
PLC- Family G Proteins by PLC- is
known to accelerate the GTPase activity of a mixture of G and G(13) . We have extended these
experiments and examined the effect of PLC- on the GTPase activity
of G, G, and G reconstituted with
m1 mAChR (Fig. 9). The GTPase activity of G,
G, and G but not of G was greatly
enhanced by PLC- in the presence of carbamylcholine. The GTPase
activity of G family G proteins was 60-90 fmol/min in
the presence of 10 nM PLC- and 1 mM carbamylcholine, whereas the GTPase activity was barely detectable
in the absence of either carbamylcholine or PLC- (1-5
fmol/min). The concentrations of PLC- giving a half-maximal effect
on the GTPase of G, G, and G were
estimated to be 1.36 ± 0.50, 2.71 ± 1.17, and 3.07
± 1.43 nM, respectively (averages and standard
deviations for three independent experiments). These results indicate
that PLC- acts not only as a G protein effector but also as a
GTPase-activating protein for all three G proteins. In sharp contrast
with G family G proteins, the GTPase activity of G reconstituted with m2 mAChR was not affected by addition of
PLC- under exactly the same conditions, although the GTPase
activity was stimulated by carbamylcholine (Fig. 9D).
It should also be noted that G exhibits a much higher
GTPase activity than G family G proteins in the absence of
PLC- and agonists.
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.
Effect of Phosphorylation by Protein Kinase C
The
experiments described above demonstrate the establishment of a
reconstitution system for the signal transduction from m1 mAChRs to
PLC-. We next examined if this signal transduction is affected by
protein kinase C, as was expected from in vivo experiments.
When the lipid vesicles containing m1 mAChR, G, and
PLC- were incubated with protein kinase C (a mixture of ,
, and subtypes) and [P]ATP, m1 mAChR
was predominantly phosphorylated. The amount of
[P]phosphate recovered in the band corresponding
to m1 mAChRs (59 kDa) was estimated to be 4 mol/mol of
[H]QNB binding site. PLC- was also
phosphorylated (0.58 mol/mol), but G,
G, G, and subunits were barely
phosphorylated (<0.01 mol/mol) (Fig. 10A). , 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.
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., 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). 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. 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 GTPS ( 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 GTPS
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.,
G, and G, but the m2 mAChR apparently did not
interact with any of them. A slight activation by the m2 mAChR of
[S]GTPS 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. 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. 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-.
), G;
G, G; G,
G; G,
G(1). G and
G are the bovine homologues of mouse
G and G, respectively.)S, guanosine
5`-O-(3-thiotriphosphate); QNB, L-quinuclidinyl
benzilate; PIP, phosphatidylinositol 4,5-bisphosphate;
IP, inositol 1,4,5-trisphosphate; PAGE, polyacrylamide gel
electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
We thank Dr. M. I. Simon of the California Institute
of Technology for the donation of cDNA of G, Dr. M. D.
Summers for permission to use the baculovirus vectors, Dr. E. M. Ross
for the m1 and m2 mAChR baculoviruses, Dr. K. Haga for purification of
m1 and m2 mAChRs, and Dr. D. W. Saffen for comments and for the editing
of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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