(Received for publication, March 28, 1997)
From the Department of Pharmacology, Mount Sinai School of Medicine, New York, New York 10029
Regulation of adenylyl cyclases 1, 2, and 6 by
Gs was studied. All three mammalian adenylyl
cyclases were expressed in insect (Sf9 or Hi-5) cells by baculovirus
infection. Membranes containing the different adenylyl cyclases were
stimulated by varying concentrations of mutant (Q227L) activated
G
s expressed in reticulocyte lysates. G
s
stimulation of AC1 involved a single site and had an apparent Kact of 0.9 nM. G
s
stimulation of AC2 was best explained by a non-interactive two site
model with a "high affinity" site at 0.9 nM and a
"low affinity" site at 15 nM. Occupancy of the high affinity site appears to be sufficient for G
stimulation of AC2.
G
s stimulation of AC6 was also best explained by a
two-site model with a high affinity site at 0.6-0.8 nM and
a low affinity site at 8-22 nM; however, in contrast to
AC2, only a model that assumed interactions between the two sites best
fit the AC6 data. With 100 µM forskolin,
G
s stimulation of all three adenylyl cyclases showed
very similar profiles. G
s stimulation in the presence of
forskolin involved a single site with apparent
Kact of 0.1-0.4 nM. These
observations indicate a conserved mechanism by which forskolin
regulates G
s coupling to the different adenylyl
cyclases. However, there are fundamental differences in the
mechanism of G
s stimulation of the different adenylyl
cyclases with AC2 and AC6 having multiple interconvertible sites. These
mechanistic differences may provide an explanation for the varied
responses by different cells and tissues to hormones that elevate cAMP
levels.
Nine adenylyl cyclases have been cloned and characterized (1-3).
Though these adenylyl cyclase isoforms display distinct signal
receiving capabilities from Ca2+ (4, 5), G (6), and
protein kinase C (7), they all share the common capability to be
stimulated by G
s. Due to its expected nature, regulation
by G
s has received somewhat less attention than other
modes of regulation. Little is known about the regions of adenylyl
cyclases involved in G
s interactions. Early studies with
the purified olfactory adenylyl cyclases showed that G
s
could be covalently linked to adenylyl cyclase (8). As a prelude to the
mapping of adenylyl cyclase regions involved in interactions with
G
s, we have studied regulation of recombinant AC6, AC2,
and AC1 by mutant (Q227L) activated G
s. Much to our surprise, we find G
s regulation of the three different
adenylyl cyclase appears to be mechanistically different. Stimulation
of AC1 is the simplest and appears to involve a single site of
interaction. Stimulation of AC2 and AC6 appears to be best explained by
multiple sites of interaction. This complex regulation is observable
only in the absence of forskolin. In the presence of forskolin,
stimulation by G
s of all three adenylyl cyclases tested
appears to involve a single high affinity site.
In vitro translation kits and Flexi
rabbit reticulocyte lysates and reagents for RNA synthesis were from
Promega (Madison, WI), and [-32P]ATP was from ICN.
Most biochemicals were from Sigma, and cell culture supplies were from
Life Technologies, Inc. All other reagents were the highest grade
available.
The wild-type Gs(short
form without the Ser) cloned from a human liver library (8) and the
Q227L-G
s
(
s*)1 in pAGA were the gift
of Dr. Juan Codina (UT Health Sciences Center, Houston). As described
previously by Sanford et al. (9) pAGA is derived from
pGEM-3Zf(
) and contains the 5
-untranslated sequences of the alfalfa
mosaic virus RNA (for sequence, see Ref. 10) upstream of the
s* sequence. Transcription and translation was according
to the method of Sanford et al. (9). The vector containing
pAGA-
s* was linearized with XhoI, and RNA was
transcribed using T7 RNA polymerase. The newly transcribed mRNA was
purified by phenol/chloroform extraction, precipitated by ethanol, and stored in RNase-free water until use. The
s* mRNA
was translated in rabbit reticulocyte lysates with all 20 amino acids
(20 µM) and approximately 3 × 105 cpm
of [35S]Met (~1,200 Ci/mmol). The translation product
was washed by 10-fold dilution and concentrated in a Centricon-10
concentrator. After three washes, the proteins in 25 mM
Na-Hepes, 1 mM EDTA, and 20 mM-
-mercaptoethanol were frozen in a dry-ice acetone
bath and stored at
80 °C until used. There are seven Met in
G
s. Assuming that all seven methionines were labeled,
specific activity of the translated G
s was calculated by
measurement of percent incorporation of 35S-labeled
L-Met into trichloroacetic acid precipitable material.
AC2 and AC6 were expressed in Sf9 or Hi-5 cells as described previously (11, 12). Bovine AC1 was tagged with the FLAG epitope at the N terminus using a strategy similar to that used for AC2 (11). The recombinant F-AC1 containing virus was then constructed by use of the shuttle vector pVL1392. Sf9 cells were grown in serum-free insect medium while Hi-5 cells were grown in Grace's medium supplemented with L-glutamine, yeastolate, and lactalbumin with 10% fetal bovine serum and 10 µg/ml gentamycin. Cells in log phase growth were infected with a multiplicity of 5-10 and harvested 48 h postinfection.
Preparation of Adenylyl Cyclase-containing MembranesInfected cells were collected by centrifugation at
4 °C. All further procedures were at 4 °C. Cells were washed in
lysis buffer containing 20 mM Na-Hepes, pH 8.0, 5 mM EDTA, 1 mM EGTA, 150 mM NaCl, 2 mM dithiothreitol, and a mixture of protease inhibitors including 1 mM phenylmethylsulfonyl fluoride (freshly
prepared), 1 mM o-phenanthroline, 3.2 µg/ml
each leupeptin and soybean trypsin inhibitor, and 2 µg/ml aprotinin
and then resuspended in 10 volumes of lysis buffer. Cells were lyzed by
nitrogen cavitation in a Parr bomb at 600 p.s.i. for 30 min.
Lysates were centrifuged at 1000 × g for 10 min
(without break) to pellet the cell debris. The 1000 × g supernatant was spun at 100,000 × g for
45 min. The 100,000 × g pellet was resuspended in 20 mM Na-Hepes, 1 mM EDTA, pH 8.0, 200 mM sucrose, 2 mM dithiothreitol, and the
protease mixture to obtain a final concentration of 3-5 mg/ml protein. Aliquots were frozen in a dry-ice acetone bath and stored at
80 °C.
Membranes containing AC2 and AC6 were blotted with the ACcomm antiserum at 1:2000 dilution. The bands were visualized by horseradish peroxidase-coupled second antibody using the ECL system.
Adenylyl Cyclase AssaysSf9 or Hi-5 cells containing
adenylyl cyclase was assayed for activity in the presence of 0.1 mM[-32P]ATP (~1000 cpm/pmol), 10 mM MgCl2 and other additives as described previously (12). Typically, 3-5 mg of crude membrane protein was
assayed in a volume of 30-50 µl for 15 min at 32 °C. The
concentration of G
s used is described in the individual
experiments.
The Gs concentration effect
curves were analyzed using the program PROPHET (Version 4.3) on a Sun
Sparc work-station. Prophet is sponsored by the NIH and distributed by
BBN Systems Technology. Initial determinations of the models to be used
for fitting were made by visual inspections of the Hofstee plots. AC1
data were fitted to a single-site model using the equation
V = Vmax*A/(Kact + A), where V is the activity of adenylyl cyclase
at A, the concentration of the activator G
s,
Vmax is the maximum reaction rate, and
Kact is the concentration of the activator at
half Vmax. Initially, the AC2 data were also
fitted to a one-site model. However, a significant improvement in the
fit was obtained when the data were fit to a noninteractive two-site
model using the equation V = Vmax1*A/(Kact1 + A) + Vmax2*A/(Kact2 + A), where V is the activity of AC2 at
concentration A of G
s and the parameters for the two sites are labeled with corresponding subscripts. The AC6 data
were first fit to a one-site model and then to a noninteractive two-site model. Neither model fit the data adequately. Hence, the AC6
data were then fitted to an interactive two-site model. At low
concentrations of G
s, the data are fitted with function, V = Vmax1*A/(Kact1 + A), since only one (the high affinity) site would be
effective at low concentrations of G
s. We then assumed that the low affinity site would become operative only above a certain
threshold concentration of G
s. The data points above this threshold concentration were then fitted to a modified two-site model described by the equation V = Vmax1*A/(Kact1 + A) for 0
A
Ath and
Vmax1*A/(Kact1 + A) + Vmax2*(A-Ath)/(Kact2 + A-Ath) for A
Ath, where Ath represents
the threshold concentration for G
s and
Vmax2 = Vmax(obs)
Vmax1. The threshold concentration was estimated
by initial visual inspection of the G
s
concentration-effect curve followed by an iterative fitting process to
obtain a threshold value that generated a curve that best fit the data
points. Three parameters were used to evaluate the model that best fit
the data. The first was the p values of fitted constants,
the second was the residual mean squares (RMS) value of the fit, and
the third was the F-test statistics to show the significance of the
improved fit. Plots of the data points and the fitted curves were
generated by Prophet. The printed plots were exported to the Canvas
program in a Mac 8100 by use of an optical scanner. The plots were
labeled and assembled within Canvas. The final plots as shown were
printed as Canvas files.
The wild type and mutant activated Gs expressed in
reticulocytes was tested for its ability to stimulate S49
cyc
membranes. Wild-type G
s did not
stimulate adenylyl cyclase activity in the presence of GTP. However,
extensive stimulation was seen with the nonhydrolyzable GTP analog
Gpp(NH)p (data not shown). However, with
s*, extensive
stimulation was observed with or without added GTP (Fig.
1). In contrast, control lysate did not stimulate the
cyc
adenylyl cyclase. These results indicated that we had
synthesized an activated G
s, which we used in further
studies. When we studied different adenylyl cyclase isoforms expressed
in Sf9 cells, we were cognizant of the activity of the endogenous
adenylyl cyclases of Sf9 cell membranes. In the case of AC1 and AC2,
the endogenous activities represented 3-5% of the total activity
under all conditions and hence were not subtracted. In the case of AC6,
however, the endogenous activity was between 20-30%. Hence for all
AC6 experiments, the difference between control (Sf9 cells infected
with baculovirus containing thyroid peroxidase) and AC6-containing
membranes for all concentration points was taken as AC6 activity.
The effect of varying concentrations of Gs* in the
presence and absence of forskolin on AC1 expressed in Sf9 cells is
shown in Fig. 2. In the absence of forskolin,
G
s* stimulated AC1 with an apparent
Kact of 0.9 nM. Stimulation by
G
s* showed a simple one-site Michaelis-Menten mechanism,
and a standard Hofstee transformation yielded a straight line (Fig.
2A, inset). Addition of 100 µM
forskolin decreased the apparent Kact of
G
s* 6-fold to 0.16 nM; however, the nature
of the curve was unaltered (Fig. 2B).
We studied the effect of varying concentrations of Gs*
in the presence and absence of forskolin on AC2. In the absence of forskolin, stimulation by G
s* did not saturate even at
35 nM (Fig. 3A). Upon Hofstee
transformation, a curvilinear profile was observed (Fig. 3A,
inset). The data best fit a Michaelis-Menten noninteracting
two-site model. The apparent Kact for the two
sites were 0.9 and 15 nM. To determine if this
heterogeneity of interactions arose from multiple forms of AC2 due to
differential post-translational modification, we analyzed the
AC2-containing membranes by immunoblotting with the ACcomm
antiserum. A single labeled band of approximately 120 kDa was observed.
This profile indicates that the expressed AC2 is not a mixture of
differentially modified proteins. We next determined the profile of the
G
s response in the presence of forskolin. With 100 µM forskolin, G
s* stimulation of AC2
yielded a simple profile (Fig. 3B); the Hofstee
transformation was linear, with a single apparent
Kact of 0.39 nM measured (Fig.
3B, inset). In Fig. 3B, the activity
due to forskolin alone (313 pmol/min/mg) had been subtracted prior to
data analysis and fitting. We next tried to define the conditions of
G
s interaction with AC2 that was optimal for G
stimulation. Without G
s, no significant stimulation of
AC2 by G
was observable (data not shown). At 2.0 nM,
G
s stimulation by G
was seven-fold. In contrast,
at 20 nM G
s, G
stimulated only
three-fold over G
s (Fig. 3C). These results suggest that occupancy of the high affinity site may be sufficient for
G
stimulation.
We next studied Gs stimulation of AC6. G
s
stimulation of AC6 was biphasic (Fig. 4A).
Hofstee transformation yielded a curvilinear profile (Fig.
4A, inset II). The G
s
concentration-response data for AC6 best fit to a two-site model.
However, the two-site fitting for AC6 was different from that used for
AC2. At low concentration of G
s, the data showed a
single-site interaction profile (Fig. 4A, inset
I) The second site was observable only at concentrations of
G
s above a certain threshold value. For the experiment
shown in Fig. 4A, the threshold value was 3 nM.
AC6 activities above 3 nM G
s are the
summation of interactions at both sites. Hence a modified two-site
model was used where, for activities above 4 nM
s*, the activity due to the second site is the
difference between the observed activity and the maximal activity due
to occupancy of the first site. This method of fitting is suggestive of
interaction between the sites and may imply that the high affinity site
needs to be fully occupied to permit occupancy of the low affinity
site. Given the very pronounced biphasic profile, we analyzed the
AC6-containing membranes from multiple forms of AC6. However, as shown
in the immunoblot in Fig. 4A, a single band was seen with
the ACcomm antibody. The observed band was at 135 kDa as
expected for AC6. We then compared the effect of varying concentrations
of G
s on AC6 activity in the presence and absence of
forskolin in the same experiment. In the absence of forskolin (Fig.
4B), the profile was very similar to that in Fig.
4A. In the presence of forskolin, stimulation of AC6 by
G
s was simple (Fig. 4C). Hofstee
transformation yielded a straight line (Fig. 4C,
inset), indicating that the data could be fit to a one-site model. G
s activated AC6 with an apparent
Kact of 0.2 nM in the presence of
forskolin. Furthermore, even in the presence of saturating G
s, forskolin potentiated AC6 activity by about 30%. To
ascertain if the unusual profile of G
s stimulation seen
with AC6 was possibly due to aggregation in the insect cell membranes,
we solubilized the AC6-containing membranes with dodecyl maltoside and
then assayed the solubilized AC6 in the presence of varying
concentrations of G
s. The solubilized enzyme had lower
activity, and the G
s response was right shifted. However
the biphasic nature of the curve was not affected by solubilization
(Fig. 4D). These observations indicate that the biphasic
nature of the AC6 response is not due to nonspecific aggregation.
Effect of varying concentrations of
s* on AC6 activity. 5 µg of Hi-5 cell membranes
containing AC6 was assayed in the presence of indicated concentrations
of
s*. The Prophet program was used to analyze the data
and obtain the fitted curves. Insets (except I in
panel A) show Hofstee transformations of the data. The
apparent Kact and Vmax obtained from the fitting are shown in the boxes. A, profile of stimulation by
varying concentrations of G
s. Inset I is a
magnified plot of the data at lower concentrations of
G
s. Immunoblot of the AC6-containing membranes with the
ACcomm antiserum is shown to the right of the
curve in panel A. 5 µg of membrane protein was
used for immunoblotting. The p values for the constants
using the interactive two-site model were Vmax1 < 0.001, Kact1 < 0.001, Vmax2 < 0.001, and Kact2 < 0.001. The RMS value of the one-site fit was 208, the
non-interactive two-site fit was 17.8, and the interactive two-site
model was 5. The F statistic for the one-site model was 38, for the non-interactive two-site model was 275, and for the interactive
two-site model was 1377. B, G
s stimulation
was measured in the absence of forskolin. The p values for
the constants using the interactive two-site model were less than < 0.001. C, G
s stimulation was measured in
the presence of 100 µM forskolin. These data and those in
Fig. 4B were part of one experiment. The p value for
constants was less than 0.01. D, stimulation of solubilized
AC6 by different concentrations of AC6. AC6 was solubilized in 0.8%
dodecyl maltoside as described previously (11). The 60,000 × g supernatant was used for the assay. Detergent
concentration in the assay 0.03%. 5 µg of solubilized membrane
protein was used for the assay. The experiments in panels
A-C are representative of five other experiments The experiment in
panel D was repeated twice. For other details, see
"Experimental Procedures."
Stimulation through Gs is the major mechanism by
which mammalian adenylyl cyclases are activated and cellular cAMP
levels elevated. Given the ubiquitous nature of this effect, one might expect the existence of a conserved mechanism for G
s
activation of the different mammalian cyclases. The data shown here
indicate otherwise. G
s stimulates AC1 in a monotonic
fashion and is the simplest of the three isoforms studied here.
Forskolin increases the affinity of G
s for AC1 without
altering the profile of the activity curve. It is noteworthy that the
apparent kact for G
s is similar
to the Ki for G
(13), thus for all practical purposes Gs-coupled receptors would be unable to activate
AC1, as has been recently demonstrated by Storm and co-workers (14). Stimulation of AC2 and AC6 by G
s is more complex. In the
absence of forskolin, G
s stimulation of AC2 and AC6 is
best explained by a two-site model. However, forskolin not only
increases the affinity of G
s but also renders
stimulation monotonic, indicating that both the low and high affinity
sites can be converted to a higher single affinity site in the presence
of forskolin. The single G
s site in the presence of
forskolin indicates that the two sites displayed by AC2 and AC6 are not
due to two populations of enzymes when expressed in Sf9 cells.
For AC2, occupancy of the "high affinity" Gs site
appears to be sufficient for G
stimulation, since maximal-fold
stimulation by G
is observed at 2 nM
G
s. This may indicate that some of the determinants for
G
s binding are in the cytoplasmic tail. Interaction of
G
s with these determinants could open up one of the
regions involved in G
binding. Such a model would provide a
molecular explanation for why G
only stimulates AC2 activated by
G
s.
Though Gs stimulation of both AC2 and AC6 best fit a two
site model, our kinetic analysis indicates that there may be crucial mechanistic differences in G
s stimulation of the two
adenylyl cyclases. The AC2 data can be fit to Michaelis-Menten type of equations, suggesting two noninteracting sites. In contrast, the model
used to fit the AC6 data required an iterative two-site fitting
procedure where the estimated Vmax of the high
affinity site had to be subtracted from the observed activity at higher G
s concentrations to fit the second site so as to obtain
an overall best fit. Such a fitting protocol may indicate interactions
between the two sites such that the high affinity site needs to be
occupied to obtain occupancy of the low affinity site.
The kinetic descriptions of Gs interactions with the
various adenylyl cyclases provided here should not be construed as an indication that each molecule of AC2 or AC6 contains two
G
s binding sites. This is one possible explanation. In
such a case, AC1 would either have only one G
s site or
two sites with very similar affinities. Studies by Neer and co-workers
on the hydrodynamic properties of the G
s-adenylyl
cyclase from bovine caudate which yield molecular weights for
G
s-adenylyl cyclase complex in the presence of forskolin of 197-225 kDa (15) may support the stoichiometery of 2 G
s/adenylyl cyclase. This possibility needs to be
explored further in detail for the different adenylyl cyclases. There
also exist data that do not support 2 G
s sites/adenylyl
cyclase model. Cross-linking of G
s to the olfactory
adenylyl cyclases suggest a 1:1 stoichiometery (8). If each adenylyl
cyclase molecule contains one G
s site, our data may
suggest that adenylyl cyclases have a propensity to dimerize in the
membrane environment. If different adenylyl cyclases have differing
capabilities to form homodimers, then our data would suggest that AC1
had a very low capability to form homodimers while AC2 and AC6 are more
likely to dimerize. Future studies analyzing cross-linking
G
s to adenylyl cyclase as well as accurate molecular
size determinations of the G
s-adenylyl cyclase complex
within the membrane are required to determine the stoichiometery of
G
s-adenylyl cyclase interactions. Irrespective of the
details that emerge from these studies, it has become abundantly clear
that each adenylyl cyclase can respond differently to G
s and that such differential responses could be crucial factors in
determining why different tissues and organs show specialized capability to regulate cAMP elevation.
We thank Dr. John Hildebrandt for
help in analyzing some of the early data and for useful suggestions in
designing experiments and Dr. Juan Codina for providing the
Gs clones and protocols for in vitro
translation.