(Received for publication, March 21, 1995; and in revised form, June 7, 1995)
From the
G is a G protein
subunit with biochemical
properties that distinguish it from other members of the G protein
subunit family. One such property is its ability to be
stoichiometrically phosphorylated by protein kinase C (PKC), both in vitro and in intact cells. The site of this phosphorylation
has been mapped to a region near the N terminus of G
,
but no functional significance of the modification has been
established. To investigate this question, we have developed a
baculovirus/Sf9 cell expression system to produce G
.
The protein purified from Sf9 cells is functional as assessed by its
ability both to bind guanine nucleotide in a
Mg
-sensitive fashion and to serve as a substrate for
phosphorylation by PKC. Furthermore, addition of the G protein
complex purified from bovine brain inhibits phosphorylation
of G
in a dose-dependent manner. Conversely,
phosphorylation of G
inhibits its ability to interact
with
subunits. These results establish a functional
consequence for PKC-catalyzed phosphorylation of G
and
suggest a mechanism for regulation of signaling through G
by preventing reassociation of its subunits.
G is a member of the family of trimeric guanine
nucleotide-binding regulatory proteins (G proteins) (
)which
generally function by coupling receptor(s) for extracellular ligands to
intracellular effector(s)(1, 2, 3) . The
mechanism through which G proteins link receptors to effectors involves
a complex cycle. The activation phase of this cycle consists of
receptor-catalyzed guanine nucleotide exchange (GTP for GDP) on the
subunit, resulting in dissociation of
-GTP from the
complex; GTP hydrolysis and subsequent subunit reassociation
are associated with the deactivation phase.
As with other
heterotrimeric G proteins, G is classified by the identity
of its
subunit(4, 5) . Although its specific
signaling function has not been established, several studies have
supported the idea that G
can couple membrane receptors to
intracellular effectors. Activated m2-muscarinic receptor can stimulate
nucleotide exchange on G
when co-reconstituted in
lipid vesicles(6) . Cotransfection of G
with
either the A
-adenosine,
-adrenergic, or
D
-dopamine receptor into hEk 293 cells results in pertussis
toxin-insensitive inhibition of adenylyl cyclase in response to
activation of each of these receptors(7) . In the latter
experiments, insensitivity to pertussis toxin confirmed the involvement
of G
, as this G protein is not a substrate for the
toxin(8) . Additionally, activated G
can
directly inhibit G
-stimulated adenylyl cyclase in
vitro(9) . However, since adenylyl cyclase inhibition in
tissues that express G
is generally pertussis
toxin-sensitive, and since the above receptors ordinarily induce
pertussis toxin-sensitive effects, the role of G
in
adenylyl cyclase inhibition in vivo is uncertain(10) .
Although the physiologic function of G remains obscure,
what is clear is that G
is quite distinct from other G
proteins with regard to the biochemical properties of its
subunit (8) , and these properties may provide some clue as to the
function of G
. G
exhibits a very slow
intrinsic rate of nucleotide exchange which is almost completely
suppressed by physiological levels of Mg
. The protein
also hydrolyzes bound GTP quite slowly relative to other
subunits
and thus may retain activity for many minutes following activation.
Additionally, the expression of G
is highly
constrained. Northern and immunoblot analyses have shown that
G
is found predominantly in neuronal tissues and in
platelets(4, 5, 8, 11, 12) .
Another potentially important property of G is that
it is an effective substrate for phosphorylation catalyzed by protein
kinase C (PKC). This phosphorylation has been demonstrated both in
vitro and in intact platelets treated with PKC-activating agents
such as thrombin, thromboxane A
analogs, and phorbol
esters(13, 14) . Phosphorylation is rapid and nearly
stoichiometric, suggesting a mechanism for cross-talk between PKC- and
G
-mediated processes. The functional consequence of this
phosphorylation, though, has remained elusive. The primary site for
phosphorylation of G
has been mapped through both
biochemical and mutational analyses of G
to
Ser
(15) . Since Ser
lies within the
region implicated as a contact site between the
subunit and the
complex of G proteins(2) , we considered it possible
that phosphorylation of G
might influence its
interaction with
. To test this hypothesis and to produce
protein for future studies aimed at identifying signaling processes
controlled by G
, we have expressed G
by
recombinant baculovirus infection of Spodoptera frugiperda (Sf9) cells. We chose the baculovirus-Sf9 expression system
because this cell type is known to be able to myristoylate G protein
subunits(16) , and this N-terminal modification likely
plays an important role in subunit interactions for G
, as
it does for other G proteins(17, 18) . Using purified
G
from this source, we have examined the effect of
phosphorylation of G
on its interaction with
complexes. We report that the
complex inhibits
the PKC-catalyzed phosphorylation of G
, and,
furthermore, that phosphorylation of G
directly
interferes with its ability to bind
. We discuss the
potential relevance of these findings to signaling through
G
.
The procedure for further
purification was adapted from that used by Kozasa and Gilman (9) and is based on the ability to selectively adsorb
-H
, along with the associated
and G
in the G protein heterotrimer, onto
immobilized Ni
resin. The cholate extract was diluted
5-fold with 20 mM Hepes (pH 8.0), 100 mM NaCl, 1
mM MgCl
, 10 µM GDP, 10 mM
-mercaptoethanol, and 0.5% polyoxyethylene 10-lauryl ether
(C
E
) (Buffer A). After incubation on ice for
20 min, the diluted extract was loaded onto 3 ml of the immobilized
Ni
resin Ni-NTA (Qiagen) pre-equilibrated with Buffer
A. The column was washed consecutively with 20 volumes of Buffer A
containing 300 mM NaCl and 5 mM imidazole (Buffer B)
and then 5 volumes of Buffer A containing 20 mM Hepes (pH
7.0), 5 mM imidazole, and 0.1% C
E
(Buffer C). The column was warmed to room temperature for
10
min, and G
eluted from the column with 3 volumes of 20
mM Hepes (pH 7.0), 100 mM NaCl, 10 µM GDP, 10 mM
-mercaptoethanol, 30 µM AlCl
, 50 mM MgCl
, 10 mM
NaF, 5 mM imidazole, and 1% sodium cholate (Buffer D). The
inclusion of Al
and F
results in
activation of the heterotrimer and thus release of
subunits
(predominantly the expressed G
) from the
-H
.
The elution from
the Ni-NTA column was diluted 3-fold with 20 mM Hepes (pH
7.0), 1 mM EDTA, 3 mM DTT, 10 µM GDP, 5
mM MgCl, 0.7% CHAPS (Sigma) and injected onto a
Mono S HR5/5 FPLC column (Pharmacia Biotech Inc.). The column was
washed with 5 ml of the same buffer containing 50 mM NaCl and
eluted with a 20-ml gradient of 50 to 1000 mM NaCl in the same
buffer. The gradient conditions were as follows: 50 to 335 mM NaCl over 5 ml, 335 to 525 mM over the next 10 ml, and
525 to 1000 mM over 5 ml. G
eluted at
400 mM NaCl as assessed by GTP
S binding and
immunoblot analysis. The peak fractions were pooled, supplemented with
bovine serum albumin to a final concentration
2 mg/ml, and
concentrated in a Centricon-30 concentrator (Amicon). The protein was
divided into aliquots, flash-frozen in liquid nitrogen, and stored at
-80 °C.
Figure 3:
-Mediated inhibition of
phosphorylation of G
: time course and reversal.
Purified G
(0.5 pmol) was preincubated with either 2.5
pmol of
(+) or the appropriate volume of
buffer(-) at 0 °C for 2 h as described under
``Experimental Procedures.'' The components necessary for
phosphorylation were then added, and each reaction was incubated at 30
°C for the times indicated. Reactions were stopped by placing the
tubes on ice and adding Laemmli sample buffer. The samples were
processed by SDS-PAGE and autoradiography. A, time course of
-mediated inhibition of phosphorylation. The autoradiogram
from one representative experiment is shown. The film was exposed for 7
h. Excision of the G
band in lane 9 and
analysis by liquid scintillation spectrometry indicated a
phosphorylation stoichiometry of 50% based on the quantity of
G
processed. B, quantitation of
phosphorylation. Radioactivity in the bands representing phosphorylated
G
was quantitated densitometrically from a scanned
image of the autoradiogram in A. The quantity for the band in lane 9 was arbitrarily assigned a value of 100, and the
remaining densities are expressed as a percentage of this value.
Phosphorylation of G
either in the absence (
) or
presence (
) of
is plotted versus time. C, reversal of
-mediated inhibition of
phosphorylation. The solid bars represent the phosphorylation
of G
. Reactions were performed for 25 min as described
above, except that the preincubation contained either buffer
(represented by bar 1), a 5-fold excess of
over
G
(2), or the same quantity of
plus an additional 5-fold excess of a mixture of G
and
G
relative to
(3). The control
experiment (4) contained the mixture of G
and
G
, but neither G
nor
. The
amount of phosphorylation of G
in condition 1 was arbitrarily assigned a value of 100, and conditions 2-4 were expressed as a percentage of this value.
Data represent the mean of three separate determinations. The cross-hatched bars represent the phosphorylation of histone
in the absence (5) or presence (6) of an approximate
5-fold excess of
relative to histone. Reaction conditions
and processing of samples were identical with those above except that
incubations were 10 min. The amount of histone phosphorylation in the
absence of
was arbitrarily assigned a value of 100, and
phosphorylation in the presence of
was expressed as a
percentage of this value.
For experiments examining the interaction of
phosphorylated G with immobilized
,
recombinant G
(1-2 pmol) in volume of 12.5
µl was subjected to phosphorylation by incubation for 30 min at 30
°C in 50 mM Hepes (pH 7.5), 0.4 mM EDTA, 0.4
mM DTT, 0.04% Lubrol, 10 mM MgCl
, 8
mM CaCl
, 50 µM GDP, 12 µg/ml
diolein, 120 µg/ml phosphatidylserine,
100 units/ml PKC, and
0.25 mM [
-
P]ATP (
1000
cpm/pmol). The reaction mixture was then diluted with 100 µl of ice
cold Buffer A and mixed with 50 µl of
-H
resin
(pre-equilibrated in Buffer A). After a 1-h incubation at 4 °C, the
resin was washed consecutively with 2 volumes of Buffer A, 4 volumes of
Buffer B containing 600 mM NaCl, and 4 volumes of Buffer B
containing 100 mM NaCl and 0.25% sodium cholate instead of
0.5% C
E
. The resin was then washed at room
temperature with 4 volumes of Buffer D followed by 4 volumes of 20
mM Hepes (pH 7.0), 100 mM NaCl, 10 µM GDP, 10 mM
-mercaptoethanol, 1 mM MgCl
, 150 mM imidazole, and 1% sodium cholate
(Buffer E). The imidazole elutes the
-H
from the resin by
competing for Ni
binding. The control reaction was
performed in an identical fashion, except that ATP was excluded. For
phosphorylated G
, equivalent volumes of each fraction
were processed by SDS-PAGE and autoradiography. For nonphosphorylated
G
, the fractions were subjected to precipitation with
trichloroacetic acid (24) and processed by SDS-PAGE and
immunoblot analysis using the G
-specific antisera,
P961.
Figure 1:
Purification of
G. G
was purified from Sf9 cells
simultaneously infected with recombinant baculoviruses encoding
G
,
, and
-H
as described under ``Experimental Procedures.'' A, aliquots from the Ni-NTA column were processed by 12%
SDS-PAGE and stained with Coomassie Blue: lane 1, cholate
extract; lane 2, flow-through; lane 3, high salt/low
imidazole wash; lane 4, low salt/low imidazole; lane
5, 1% cholate, Al
, and F
elution. Lane 6 shows purified G
obtained from Mono S chromatography of the material in lane
5. The arrows on the left represent the
migration positions of molecular weight markers processed on the same
gel. B, samples identical with those in A were
processed by SDS-PAGE followed by immunoblot analysis with the
G
-specific antisera, P961.
The
major protein obtained from the elution step migrates at 40 kDa by
SDS-PAGE. The presence of G in this band was confirmed
by immunoblot analysis (Fig. 1B), but it also contained
a significant amount of a G
-immunoreactive protein (data
not shown). However, further chromatography of the Ni-NTA elution on an
anion exchange Mono S column resulted in an essentially homogenous
protein of 40 kDa being obtained (Fig. 1A, lane
6) in which only G
immunoreactivity could be
detected (data not shown). Quantities of G
sufficient
for analysis were purified in this manner (see Table 1).
The
guanine nucleotide binding properties of recombinant G were assessed by the trypsin protection assay. This procedure
takes advantage of the fact that activation of G protein
subunits
by the GTP analog, GTP
S, partially protects them from cleavage by
trypsin, such that only a small portion of their N terminus is removed.
By contrast, in their basal (i.e. GDP-liganded) state,
subunits are rapidly degraded by trypsin. Thus, this assay provides a
very sensitive means of assessing the nucleotide-bound state of a G
protein. The assay is also very specific, as the protein is detected by
immunoblot analysis. Purified G
was incubated with
either GTP
S or GDP in the presence of either 1 µM free Mg
or 10 mM free
Mg
. Following incubation with nucleotide, the samples
were incubated either with or without trypsin. The data in Fig. 2show the results of these experiments. The binding of
GTP
S protected G
from trypsin digestion (lane
2). However, in the presence of 10 mM free Mg
this protection is not evident (compare lanes 2 and 4), which is expected since 10 mM free Mg
suppresses G
's intrinsic rate of
nucleotide exchange to almost zero(8) . These data confirm that
the protein which was purified is active.
Figure 2:
Demonstration of magnesium suppression
of nucleotide exchange by trypsin protection. Purified G (1 pmol) was incubated at 30 °C for 90 min in the presence of
either GDP (lanes 1, 3, 4, and 6)
or GTP
S (lanes 2 and 5) as described under
``Experimental Procedures.'' In lanes 1-3, the
free Mg
was 1 µM, while in lanes
4-6 it was 10 mM. After 90 min, MgCl
to
10 mM was added to the samples in lanes 1-3,
trypsin was added to the samples in lanes 2, 3, 5, and 6, and the incubation was continued for
another 10 min at 30 °C. Half of each sample was processed by
SDS-PAGE and transferred to nitrocellulose. This nitrocellulose
membrane was processed by immunoblot analysis with antisera 2921, a
G
-specific antisera.
The -dependent
inhibition of G
phosphorylation was examined in
further detail by determining the concentration dependence of this
suppression (Fig. 4). This analysis revealed that inhibition of
PKC-catalyzed phosphorylation by
is dose-dependent, and that
50% inhibition was observed upon addition of a roughly equimolar amount
of
. Higher concentrations of
resulted in almost
complete suppression of phosphorylation, providing very strong evidence
that association of
with G
prevents the
phosphorylation of G
by PKC.
Figure 4:
Dose dependence of -mediated
inhibition of phosphorylation of G
. Purified
G
(0.5 pmol) was preincubated with either
or buffer as described in the legend to Fig. 3, except that
quantities of
were varied as indicated. The approximate
molar ratios of
:G
are indicated above the
respective lanes. Reactions were conducted for 25 min at 30 °C and
processed as described in the legend to Fig. 3. A,
inhibition of G
phosphorylation with increasing doses
of
. The autoradiogram from one representative experiment is
shown. The stoichiometry of phosphorylation of G
in lane 1 was determined to be 50%. The film was exposed for 8 h. B, quantitation of phosphorylation. Radioactivity in the bands
representing phosphorylated G
in A were
quantitated densitometrically as described in the legend to Fig. 3. The density of the band in lane 1 was
arbitrarily assigned the value of 100, and the remaining bands are
expressed as a percentage of this value. The quantity of phosphorylated
G
is plotted versus the molar ratio of
:G
.
G was incubated
with PKC both in the presence and absence of
[
P]ATP; the latter condition served as the
control reaction. Following the phosphorylation reaction, both
phosphorylated and nonphosphorylated proteins were incubated with the
-H
resin, and the elution
patterns of each were analyzed (Fig. 5). As expected, since this
was the route to its initial purification, nonphosphorylated
G
bound quite tightly to the
-H
resin; it did not flow
through the resin nor was it washed off with either high salt or 0.25%
cholate (Fig. 5A, lanes 2-4). Washing
the column with buffer containing 1% cholate and Al
and F
results in activation of the heterotrimer
and, thus, the release of nonphosphorylated G
from the
immobilized
(Fig. 5A, lane 5). A
small portion of G
eluted when the resin was washed
with 150 mM imidazole, a treatment which strips the
-H
and any other
associated protein from the resin by competing for Ni
binding (Fig. 5A, lane 6). In contrast,
the majority of the phosphorylated G
incubated with
-H
resin either flows
through or is washed off in the high salt and 0.25% cholate washes (Fig. 5B, lanes 2-4), indicating that
its binding to the immobilized
is much weaker than for the
nonphosphorylated protein. While a small, but significant, amount of
the phosphorylated protein remains associated with the resin after the
first two elution conditions and is eluted by 1% cholate and
Al
and F
(Fig. 5B, lane 5), this fraction largely represents nonspecific
interaction of phosphorylated protein with the resin, as phosphorylated
G
elutes in a near-identical pattern from resin which
has been denatured by treatment with 8 M urea (data not
shown). Quantitation of the amounts of both nonphosphorylated and
phosphorylated G
eluted from the
resin
under each of the conditions is shown in Fig. 5C.
Figure 5:
Effect of phosphorylation of G on its interaction with immobilized
. Identical
aliquots of purified G
(1 pmol) were incubated with
PKC, Ca
, and phospholipids either in the absence (A) or presence (B) of
[
-
P]ATP for 30 min at 30 °C as
described under ``Experimental Procedures.'' Samples were
then incubated with
-H
resin for 1 h at 4 °C, and the resin was sequentially
subjected to the indicated treatments as described under
``Experimental Procedures.'' A, G
treated in the absence of ATP. The resultant fractions were
collected, concentrated by trichloroacetic acid precipitation, and
processed by SDS-PAGE and immunoblot analysis with P961. The blot from
one representative experiment is shown. B, G
treated in the presence of ATP (i.e. phosphorylated
G
). Equivalent portions of each fraction were
processed by SDS-PAGE and autoradiography. The autoradiogram from one
representative experiment is shown. The film was exposed for 12 h. C, quantitation of the elution patterns for phosphorylated and
nonphosphorylated G
. G
in the
fractions eluted from each resin were quantitated as described in the
legend to Fig. 3. Quantities are expressed as a percentage of
the total G
eluted in each condition, i.e. phosphorylated or nonphosphorylated.
To
further investigate the interference of G-
association by phosphorylation, we used sedimentation through sucrose
density gradients. As for other G proteins(32) , association of
G
with
is expected to shift its
sedimentation constant relative to the monomeric G
subunit. If phosphorylation interferes with the
G
-
association, then this modification
should prevent a
-dependent shift in the sedimentation
position of G
in a density gradient. The results,
shown in Fig. 6, demonstrate that this is in fact the case. Both
phosphorylated and nonphosphorylated forms of G
were
incubated with
and then subjected to centrifugation through
a 5-20% sucrose gradient. For nonphosphorylated
G
, the addition of
markedly shifts its
sedimentation. However, addition of
to phosphorylated
G
does not significantly shift its sedimentation;
rather, it sediments at nearly the same position as free,
nonphosphorylated G
. These data provide convincing
evidence that phosphorylation of G
prevents its
interaction with
.
Figure 6:
Sedimentation profile of G and phosphorylated G
in the presence of
. Purified G
(6 pmol) was incubated with
PKC, Ca
, and phospholipids either in the presence
(
) or absence (
) of ATP for 30 min at 30 °C as described
under ``Experimental Procedures.'' Samples were then
incubated with 60 pmol of
and subjected to centrifugation
through a 5-20% sucrose gradient. Successive fractions were taken
from the top of each gradient and 15% of each fraction was processed by
SDS-PAGE and immunoblot analysis with P961. The bands representing
G
were quantitated as described in the legend to Fig. 3. Quantities are expressed as a percentage of the total
G
fractionated in each condition. The arrow represents the peak migration position for G
treated in the absence of both ATP and
and processed
through an identical gradient.
One of the more intriguing properties of G is its ability to be rapidly phosphorylated by activated PKC both in vitro and in stimulated platelets. While previous studies
had defined the primary site for this
modification(14, 15) , no functional consequence of
phosphorylation had been established. Since the location of the primary
phosphorylation site lay within the N-terminal domain, a region known
to be important for interaction of
subunits with
, we
hypothesized that phosphorylation may play a role in regulating
G
-
interaction. In order to test this
hypothesis, we required a source of purified G
. Escherichia coli expression, however, was not feasible because
G
from this source does not efficiently interact with
(8) . This is presumably due to the inability of
bacteria to myristoylate the protein, since N-terminal myristoylation
is known to be important for subunit interactions of G
proteins(18) . Thus, we turned to expression in a eukaryotic
system with documented ability to myristoylate G proteins, the
baculovirus/Sf9 expression system(16) . Successful production
and purification of recombinant G
was greatly aided by
the use of the hexahistidine-tagged
, as coexpression
of G
with the
subunit and
-H
provided an efficient, rapid means of
purification by affinity chromatography.
PKC-catalyzed
phosphorylation of G was indeed influenced by the G
protein-
complex. The ability of the
complex to
suppress phosphorylation suggests that formation of the
G
-
trimer prevents phosphorylation by
blocking access of the enzyme to the phosphorylation site.
Additionally, once phosphorylated, G
has a greatly
decreased ability to associate with
. The most likely basis
for the reduced association of G
with
following phosphorylation is that the presence of the phosphate group
on the N-terminal domain of G
could prevent oligomer
formation through either steric interference or charge repulsion in the
contact interface between G
and
.
Since
phosphorylation influences the interaction between G and
, this modification may play a role in modulating
signaling through G
. A model for this potential modulation
is as follows. Activation of G
by an appropriately liganded
receptor would result in GTP binding and subsequent subunit
dissociation. The GTP-bound G
could interact with an
as yet unidentified effector, after which the GTPase activity of
G
could convert it to the GDP-bound form, which would
then dissociate from the effector molecule. In this form
(G
-GDP), the protein can be readily phosphorylated by
activated PKC(14) . Once phosphorylated, G
could not reassociate with
and therefore could not
recycle to interact again with receptor, resulting in further signaling
through G
being prevented. In summary, the prediction of
this model is that simultaneous activation of G
- and
PKC-controlled processes would result in rapid attenuation of signaling
through the G
pathway.
This regulatory model may have
some general applicability to signaling through other G proteins, as
several reports indicate that another G protein, G, can
also serve as a substrate for PKC. The G
subunit is
phosphorylated in hepatocytes in response to PKC
activation(33, 34) . Houslay and colleagues (35) identified the specific G protein subtype phosphorylated
in this system as G
, and several groups
observed a correlation between phosphorylation of
G
and inactivation of signaling through
this G protein(36, 37, 38) . Further, they
observed that treatment with a phosphatase restored signaling through
G
(37, 39) . Our model
offers a possible biochemical explanation for their observations, in
that phosphorylation of G
could also
interfere with its subunit reassociation. Indeed, an amino acid that
serves as a secondary site for PKC phosphorylation in
G
, that of Ser
(15) , is also
found in G
and several other G proteins,
and this amino acid also lies within the N-terminal domain implicated
in interaction with
. Thus, prevention of subunit
reassociation through PKC-catalyzed phosphorylation could be a
mechanism for the observed inactivation of signaling through
G
.
In the model presented here,
phosphorylation would directly attenuate signaling through
G. In addition, it is certainly possible that
signaling through G
might directly or indirectly lead
to activation of PKC, and that phosphorylation of G
by
this kinase is a means of feedback regulation. Such attenuation of G
protein-mediated signaling by phosphorylation is well-documented in the
case of desensitization at the level of the receptors involved in many
of these processes(40) . These data provide new leads for
developing experimental approaches to identifying components and events
in G
-mediated signaling.