From the Department of Neurobiology, The Weizmann
Institute of Science, Rehovot 76100, Israel, the ¶ Department of
Physiological Chemistry II, University of Düsseldorf,
Düsseldorf, D-40225 Germany, and the
Metabolic Diseases
Branch, NIDDK, National Institutes of Health,
Bethesda, Maryland 20892
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ABSTRACT |
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The accepted dogma concerning the regulation of
adenylyl cyclase (AC) activity by G dimers
states that the various isoforms of AC respond differently to the
presence of free G
. It has been demonstrated that AC
I activity is inhibited and AC II activity is stimulated by
G
subunits. This result does not address the possible
differences in modulation that may exist among the different
G
heterodimers. Six isoforms of G
and
12 isoforms of G
have been cloned to date. We have
established a cell transfection system in which G
and
G
cDNAs were cotransfected with either AC isoform I
or II and the activity of these isoforms was determined. We found that
while AC I activity was inhibited by both G
1/
2 and
G
5/
2 combinations, AC II responded differentially and
was stimulated by G
1/
2 and inhibited by
G
5/
2. This finding demonstrates differential
modulatory activity by different combinations of G
on
the same AC isoform and demonstrates another level of complexity within
the AC signaling system.
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INTRODUCTION |
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The heterotrimeric G-protein has been shown to be a central
molecule that connects seven-transmembrane domain receptors to the many
downstream effector molecules whose activities they regulate. The
G-protein superfamily has been divided into many classes, originally
based on the activity that the G subunit exerted on the
effectors. For example, the G
subunit that was found to
activate adenylyl cyclase
(AC)1 was classified
G
s while the G
subunit found to inhibit AC activity was called G
i (reviewed in Refs. 1-4). In
the last few years, the role of free G
subunits
(disassociated from G
upon receptor activation) in
signal transduction has begun to be revealed (reviewed in Refs. 5-7).
In vitro membrane reconstitution assays have clearly
demonstrated that a number of AC isoforms are sensitive to
G
subunits and that AC activity can either be
stimulated or inhibited by G
subunits depending of
the AC isoform in question. For example, the activity of AC type I is
significantly inhibited while the activity of AC types II, IV, and
presumably VII are activated by G
(6-13). This
finding has led to the understanding of how it is possible that the
classically known inhibitory receptors that are coupled to
G
i/o can actually stimulate AC activity in situations
where AC isoforms that are activated by free G
subunits are present (11, 12, 14).
Six G and 12 G
isoforms have been cloned
(reviewed in Refs. 4, 5). Most of the prior experiments investigating the role of free G
on AC activity were performed in cell-free systems with either baculovirus/Sf9 recombinant
G
and G
preparations (15, 16) or with
purified brain G
preparations that consist of a
mixture of various G
heterodimers (8, 9). There is
little information about the possible variations between the effects of
various G
and G
subunits in the intact
cell. This seems to be important since the various G
and
G
subunits do not necessarily have the same regulatory
activities. Due to the recent cloning of various G
and
G
isoforms, it is now possible to study the activity of
the various G
combinations. Indeed, it has recently
been shown by us that activation of PLC-
2 by G
is G
isoform-independent
(G
1/
2 being equally effective as
G
5/
2) while MAPK/ERK and JNK/SAPK activation appeared
to be G
isoform-dependent and was much more
efficiently activated with G
1/
2 than with
G
5/
2 (17).
In this study, we have characterized the modulations of two AC isoforms
by specific G combinations. Utilizing cotransfection of G
and G
together with AC isoforms I
and II, we observed differential modulation of AC activities by
specific combinations of G
. We found that AC type I
was inhibited by both G
1/
2 and
G
5/
2. On the other hand, AC II activity was
stimulated by one isoform of G
(
1) while inhibited by another (
5).
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EXPERIMENTAL PROCEDURES |
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Cell Cultures-- COS-7 cells were obtained from ATCC (Bethesda, MD) and cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 100 units/ml penicillin and 100 µg/ml streptomycin.
Plasmids--
cDNAs of AC I and II were used in the pXMD1
vector under control of the adenovirus-2 major late promoter (18). AC I
cDNA was released from pSK-AC-I (19) using HindIII and
XbaI and ligated after "fill-in" into the
SmaI site of pXMDI. AC II cDNA was released from
pSK-AC-II (20) by EcoRI and ligated to the EcoRI
site of pXMDI. G1 and G
5 cDNAs in
pcDNAIII and G
2 in pcDM8 were described earlier (17,
21). G
2C68S in pcDM8 (a mutant that cannot undergo
prenylation) was described earlier (21, 22). cDNA for
G
-transducin (
T) was provided by Dr. J. S. Gutkind. Constitutively active G
s
(G
sQ227L) was obtained from Dr. H. Bourne (23).
Transfection of COS Cells--
COS-7 cells in 10-cm dishes were
transfected with the indicated cDNAs by the DEAE-dextran
chloroquine method (24). Vectors were added to complement the amount of
cDNA in the transfection mixture to 6-7 µg. 24 h later, the
cells were trypsinized and cultured for an additional 24 h in
24-well plates for AC activity assay or in 10-cm dishes to check
protein expression by Western blots. Transfection efficiency, as
determined by staining for -galactosidase (25) activity following
transfection with the plasmid expressing the enzyme, was 60-80%.
Adenylyl Cyclase Assay-- The assays were performed in triplicate as described (26). Cells cultured in 24-well plates were incubated for 3 h with 0.25 ml/well of fresh growth medium containing 5 µCi/ml [3H]adenine. This medium was replaced with Dulbecco's modified Eagle's medium containing 20 mM Hepes (pH 7.4), 1 mg/ml bovine serum albumin, and the phosphodiesterase inhibitors isobutylmethylxanthine (IBMX) (0.5 mM) and RO-20-1724 (0.5 mM). Unless otherwise indicated, the AC stimulants forskolin (FS) or ionomycin (which increases intracellular Ca2+ and thus increases the activity of AC I) were immediately added at 1 µM concentrations, and the cells were incubated at 37 °C for 10 min. Reaction was terminated with 1 ml of 2.5% perchloric acid containing 0.1 mM unlabeled cAMP. Aliquots of 0.9 ml were neutralized and applied to a two-step column separation procedure (27). The [3H]cAMP was eluted into scintillation vials and counted.
SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Western
Blots--
Cells were washed with cold phosphate-buffered saline
(PBS), scraped in PBS, and spun down at 5000 rpm (4 °C for 5 min),
and cell pellets were mixed with 100 µl of Laemmli sample buffer
(28), sonicated, and frozen. Prior to application on the gel,
dithiothreitol (0.1 M final) was added, and the samples
were incubated for 2 h at 37 °C. Proteins were separated on
polyacrylamide gels (8% for AC and 12% for G) and
transferred to nitrocellulose. Nitrocellulose was blocked in PBS
containing 5% fat-free milk and 0.5% Tween-20 for 1 h followed
by 1.5 h of incubation with either BBC-1 monoclonal antibody
(against AC I), BBC-4 monoclonal antibody (against AC II) (29, 30), RA
polyclonal antibody (against G
1), or SGS polyclonal
antibody (against G
5) (17). Blots were washed 3 times
with PBS containing 0.3% Tween-20 and secondary antibodies
(horseradish peroxidase (HRP)-coupled rat anti-mouse or HRP-coupled
goat anti-rabbit, Jackson Immunoresearch Laboratories, Inc.) diluted
1:20,000 in 5% fat-free milk plus 0.5% Tween-20 and incubated with
the blot for 1 h, and the blot was extensively washed (>2 h) with
PBS containing 0.3% Tween-20. Peroxidase activity was observed by the
ECL chemiluminescence technique (Amersham Corp.).
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RESULTS |
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AC I Activity Is Inhibited by G1/
2 and
G
5/
2--
We have performed cotransfection
experiments of AC I together with G
1 or
G
5 and G
2. Activation of AC I is known to
be dependent on the presence of Ca2+ ions (8). In the
experiment shown in Fig. 1, we have
assayed the activity of this isozyme in the presence of the
Ca2+ ionophore ionomycin (1 µM) together with
FS (1 µM). This combination was shown to synergistically
stimulate AC I activity (11). The endogenous AC activity present in COS
cells was not significantly affected by ionomycin and thus contains
very little Ca2+-stimulated AC isozymes. As shown in Fig.
1A, AC I activity was significantly inhibited upon
cotransfection with either G
1 or G
5
together with G
2. The individual G
1,
G
5, and G
2 subunits had no inhibitory
activity on their own. Western blots of AC I protein (see Fig.
1B) reveals that the levels of AC I were not affected by the
cotransfection of the other cDNAs. Our results thus indicate that
the inhibitory effect mediated by G
subunits,
originally found in membrane assays, can also be observed in a whole
cell system and that both G
1 and G
5 are
effective in the inhibition of AC I, provided that G
2 is
present.
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AC II Activity Is Stimulated by G1 and
G
1/
2 but Is Inhibited by
G
5/
2--
The AC II subtype is known to be
stimulated by free G
(8). We have performed
cotransfection of AC II with constitutively active G
s
(G
sQ227L) and, where indicated, with G
1 or G
5 and with G
2. The amounts of cAMP in
these cells were determined 2 days after transfection. In addition, we
have treated the cells for 10 min with a mixture of the
phosphodiesterase inhibitors, IBMX and RO-20-1724, and assayed the
amounts of cAMP following this incubation. From the results shown in
Fig. 2, it is clear that transfection
with AC II increased the amounts of cAMP present in the cells under
both conditions. As expected, transfection with G
1
caused a further increase in cAMP accumulation. This is due to AC II
activation since it was not observed when G
1 was
transfected to COS cells without the addition of AC II plasmid (data
not shown) or when it was transfected with AC I (Fig. 1). The increase
of AC II activity mediated by G
1 was not significantly
affected by the addition of G
2, which by itself had no
effect. Interestingly, in contrast to G
1, G
5 caused a siginificant inihibition of AC II activity,
and the addition of G
2 increased this inhibition. These
results show that in contrast to AC I, AC II is differentially affected by G
1 and G
5, thus demonstrating a level
of specificity within the G
family members in regards to
their ability to affect AC II activity. Western blot analysis of the AC
II protein levels under the various transfection conditions revealed
that AC II expression was not affected by the cotransfection with
G
and G
subunits.
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G Subunits Modulate AC I and AC II in a
Dose-dependent Manner--
The experiments shown in Figs.
1 and 2 were performed with relatively large amounts of
G
and G
cDNAs. They were, therefore,
repeated with G
1 and G
5 cDNAs at
various concentrations. Fig.
3A demonstrates that the
inhibition of AC I reaches maximal values when concentrations of
cDNAs of G
1 or G
5 are above 2.5 µg.
The efficiencies of inhibition by transfected G
1 and
G
5 (in the presence of G
2) were similar.
Half-maximal effect was observed with ~1 µg of G
1 and 2 µg of G
5 cDNAs. The inhibition observed for
AC I activity was dependent on the presence of G
2 (2 µg/plate) for both G
1 and G
5.
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Endogenous G in COS Has a Role in AC II
Stimulation--
Since we have demonstrated that the
G
5/
2 combination can be inhibitory to AC II activity,
it became of interest to characterize the role of endogenous
G
in COS-7 cells with respect to AC II modulation. To
investigate this question, we utilized a number of molecular tools that
were shown to interfere with G
activity. A mutant
form of G
2 that lacks the prenylation site
(G
2C68S) and which therefore cannot anchor to the
membrane has been shown to redirect G
subunits into the
cellular cytosol (21, 22). Additionally, wild-type G
subunits such as
T combines with G
and interferes with G
-mediated signaling (11, 14). Fig.
4A demonstrates the effect of
cotransfection of the above mentioned proteins on AC II activity in
COS-7 cells. A marked inhibition of AC II activity was observed in
cells cotransfected with
T. G
2C68S also produced a
significant inhibition of AC II activity although the efficacy of
inhibition compared with
T was noticeably less. The cotransfection
of
T and G
2C68S with AC II did not have any effect on
the expression of AC II (data not shown). These results suggest that
G
in COS cells has a role in the stimulation of AC II
activity. Fig. 4B demonstrates that COS cells do indeed express endogenous G
1, but appear to be devoid of
G
5. Transfection of COS cells with G
1 or
G
5 cDNAs led to a significant increase in the levels
of G
1 and G
5 protein in the cell
membranes.
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DISCUSSION |
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There are many reports that show involvement of the
G dimer complex in the regulation of various
effectors including PLC-
2, MAPK/ERK, JNK/SAPK, phosphatidylinositol
3-kinase, and several AC isozymes (5, 8, 14, 17, 31, 32). Most of these
experiments were performed either in whole cells using G
scavengers (i.e. molecules that strongly
interact with G
and block its ability to modulate
effectors) or with cell membranes. In the latter case, the
G
used for most of the experiments has been with
either baculovirus/Sf9 recombinant G
and
G
preparations or with a mixture of a large repertoire
of G
heterodimers, such as in G
preparations purified from bovine brain. Therefore, there has been
little information regarding the possible differences in activities
between the various G
and G
subunits
composing the G
dimers in intact cells. Following the
cloning of specific G
and G
isoforms, it
has become possible to dissect the individual G
and
G
combinations that affect AC activity. In a previous
study, using transfection of intact cells with G
1 or
G
5 cDNAs, we showed that G
1 and
G
5 differ in their ability to activate MAPK/ERK and
JNK/SAPK but not in their capacity to activate PLC-
2 (17). Here, we have used the same approach to investigate the role of
G
1 and G
5 in the modulation of two types
of AC isozymes: AC I, previously shown to be inhibited by
G
heterodimers, and AC II, previously reported to be
stimulated by G
(8, 15, 16).
Our results demonstrate that indeed, as reported using in
vitro reconstitution assays, G inhibits AC I
activity. We found that G
1 and G
5 do not
markedly differ in their capacity to inhibit AC I and that in both
cases, the inhibition was dependent on the cotransfection of a
G
subunit. Our observations with AC II showed that while
G
1/
2 yielded stimulation of this isoform, in
agreement with previously reported activities of G
(7, 8, 11, 13, 14). G
5/
2 caused a marked inhibition
of AC II activity, demonstrating selective effects of G
subunits on the activity of this AC isoform. Interestingly, transfected
G
1 was by itself sufficient in stimulating AC II
activity. This is probably due to its capacity for recruiting
endogenous G
present in the cell. G
5 also
had some effect on its own, although its effect on the inhibition of AC
II was significantly enhanced by cotransfection of G
2. As previously proposed (17, 22), these results suggest a difference in
the capacity of G
1 and G
5 to interact
with G
subunits.
It is known that of the six cloned G isoforms,
G
5 shares only a 53% homology compared with the other
G
isoforms (5). The unique sequence of G
5
may be the source of the different effects on AC II activity that we
have observed. It should be noted that this specific activity could
have been missed in prior studies in which G
was
tested in in vitro membrane reconstitution assays due to the
possibility that the concentration of G
5 in the pool of
G
(usually purified from bovine brain) may have been
too small. The activity of G
5 may have been masked by
other G
subunits present in the preparation, and
therefore, the levels of G
5 were not high enough to
observe any inhibitory effects on AC II activity.
In summary, G1 and G
5 represent two
distinct forms of the G
subunit, as based on their
sequence, expression pattern, and ability to affect downstream
signaling proteins (17, 33). Our results show that they also differ in
their capacity to affect the activity of AC type II. Future studies
should allow complete characterization of the various
G
combinations influencing the activity of each of
the AC isozymes and elucidate the finer regulation of
G
signaling within the AC system.
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FOOTNOTES |
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* This work was supported in part by the National Institute on Drug Abuse (DA-06265), the German-Israeli Foundation for Scientific Research and Development, and the Forschheimer Center for Molecular Genetics.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of an Israeli Ministries of Science and Arts and of Absorption Fellowship.
** Incumbent of the Ruth and Leonard Simon Chair for Cancer Research and to whom correspondence should be addressed. Tel.: 972-8-934-2402; Fax: 972-8-934-4131; E-mail: bnvogel{at}weizmann.weizmann.ac.il.
1
The abbreviations used are: AC, adenylyl
cyclase; FS, forskolin; HRP, horseradish peroxidase;
GsQ227L, constitutively active G
s
subunit; IBMX, isobutylmethylxanthine; PBS, phosphate-buffered saline;
JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein kinase;
MAPK/ERK, mitogen-activated protein kinase/extracellular signal-regulated kinase.
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REFERENCES |
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