(Received for publication, May 17, 1995)
From the
Regulation of basal activities of adenylyl cyclase (AC) 2 and 6,
expressed in Sf9 cells by infection with recombinant baculovirus, was
studied. An antipeptide antibody that recognizes AC2 and AC6 with equal
sensitivity was used to establish that equivalent levels were
expressed. Basal activities of AC2 and AC6 were compared at varying
concentrations of Mg or Mn
ions;
AC2 had 15- and 10-fold greater activity than AC6, respectively. At 20
mM Mg
, the K
values for ATP were 88 and 39 µM for AC2 and
AC6, respectively, whereas their V
values were
281 and 11 pmol/mg protein
min. With 100 µM forskolin
and either Mg
or Mn
, the difference
in activities between AC2 and AC6 was reduced to approximately 2-fold.
Forskolin stimulated AC6 greater than 40-fold at 0.5-2 mM Mg
, whereas AC2 was stimulated 4-6-fold.
At 20 mM Mg
, AC2 was stimulated 2-fold by
forskolin, whereas AC6 was stimulated 18-fold. With Mg
alone, activities of AC2 and AC6 were not saturable up to 20
mM and yielded curvilinear Hofstee transformations. With
forskolin, activities of both AC2 and AC6 were saturable by 10 mM Mg
and yielded linear Hofstee transformations.
These data indicate that there are substantial differences in the basal
enzymatic activities of adenylyl cyclase isoforms, due to differential
regulation by Mg
ions rather than intrinsic catalytic
capabilities. Thus the presence and relative abundance of adenylyl
cyclase subtypes could greatly affect the resting cellular cAMP levels
with consequent effects on important biological functions, such as
differentiation and proliferation.
The ambient level of intracellular cAMP is an important
regulator of biological processes, such as proliferation and
differentiation. Studies from our laboratory and others recently have
shown that a 2-fold increase, or less, in intracellular cAMP can have
profound consequences in certain cells, such as NIH-3T3, in which the
increase blocks Ras-induced transformation (1) and, through
protein kinase A, inhibits stimulation of microtubule-associated
protein kinase activity (1, 2, 3, 4, 5) . There are
several means by which ambient intracellular cAMP levels can be
regulated, including constant stimulation of adenylyl cyclase activity
and/or constant inhibition of phosphodiesterase activities. There is
little evidence, however, that either of these phenomena occur. It is
well documented that receptor or G protein stimulation of adenylyl
cyclases leads to desensitization of the stimulatory signal at the
level of the receptor (6) or at loci
downstream(7, 8) . This results in transitory
alterations in cAMP levels. If, however, the different adenylyl cyclase
isoforms had different basal activities, the ambient intracellular cAMP
concentrations could be set at a certain level simply by having the
appropriate isoform(s). The molecular diversity of effector forms in
heterotrimeric G protein-coupled systems is now established. Nine
different isoforms of mammalian G-
-stimulated adenylyl
cyclases are known(9, 10) . We chose to study the
basal properties of two of these adenylyl cyclases (AC2 and AC6) (
)in an attempt to develop the hypothesis that basal
properties of different isoforms could contribute to very different
intracellular cAMP levels. cAMP production by AC2 is stimulated by
several signals including G
-
and
-subunits(11) , and by protein kinase
C(12, 13) . In contrast, cAMP production by AC6 is
only stimulated by G
-
, and inhibited extensively by
G
-
(14, 15) , protein kinase A,
and low concentrations of Ca
( (9) and
references therein). Thus, as we had previously surmised that the
presence of AC2 could be reflective of a cell's need to have
elevated cAMP levels in response to multiple signals(14) , the
presence of AC6 could maintain low levels of intracellular cAMP. An
extension of this line of reasoning is that AC6 may also have very low
basal activities in comparison to AC2 and hence allow the cell to keep
basal cAMP levels low. To determine whether different adenylyl cyclases
have distinct basal activities, we expressed both adenylyl cyclases in
Sf9 cells using the baculovirus expression system and studied
regulation of their basal activities by Mg
and
Mn
in the absence and presence of forskolin.
AC2 cDNA was the kind gift of Dr. Randall Reed (Johns Hopkins
University). Sf9 cells and pVL-1392 were obtained by the Mount Sinai
Protein Expression Core facility from Dr. Max Summers. BaculoGold virus
was from Pharmigen. Serum-free Sf9 medium was from Life Technologies,
Inc., or Sigma. [-
P]ATP was purchased from
ICN. Forskolin was from Sigma. Sources of all other materials have been
previously described(8, 14) .
Membranes containing expressed AC2 and AC6, and control
membranes (from thyroid peroxidase-infected cells), were probed with
the AC antiserum to determine the levels of adenylyl
cyclase expressed. This antibody is an antipeptide antibody against a
14-amino acid stretch (IGARKPQYDIWGNT) that is common for the cloned
mammalian adenylyl cyclases. The antibody has been useful in the
recognition of AC2 expressed in Sf9 cells(13) . In control Sf9
cells expressing thyroid peroxidase, no major bands were seen. In
contrast, membranes from Sf9 cells expressing AC2 or AC6 showed
prominently stained bands (Fig. 1A). The size of the
bands are in accordance with the estimated masses from the cDNA clones.
AC2 is expressed as 106 KDa protein and AC6 is expressed as
132-133-KDa protein. It is noteworthy that both start sites of
AC6 (16) appear to be used, as indicated by the presence of
the doublet. When the AC
antibody was preincubated with
the immunogen peptide, no bands were visible in the AC2- or
AC6-expressing membranes (data not shown). In order to establish that
both adenylyl cyclases were being recognized by the antibody with
similar sensitivity, we tested different concentrations of antiserum on
a low (2.5 µg) level of membrane proteins from Sf9 cells. An
immunoblot with varying dilutions of antiserum is shown in Fig. 1B. The intensity of the bands was determined by
densitometry (Fig. 1C). It can be seen readily from Fig. 1, B and C, that the AC
antibody recognized AC2 and AC6 with similar sensitivity. When
different amounts of Sf9 membranes were used at a fixed (1:500)
antibody concentration, the signal intensity for both AC2 and AC6
varied with the amount of membrane protein used (Fig. 1D). The data in Fig. 1indicate that the
AC
antibody can be used to estimate the amounts of AC2
and AC6.
Figure 1:
A, immunoblot of membranes from Sf9
cells infected with thyroid peroxidase (TPO), AC2, and AC6
recombinant baculovirus. 25, 25, and 10 µg of thyroid peroxidase,
AC2, and AC6 membrane proteins, respectively, were resolved on 7%
SDS-gels, transferred to nitrocellulose paper, and probed with a 1:500
dilution of the AC antibody. Bands were visualized by a
horseradish peroxidase-coupled goat anti-rabbit IgG and the ECL system. B, immunoblot using various dilutions of antiserum. 2.5 µg
of membrane protein were used at all dilutions of antiserum. C, densitometry scans of immunoblot in Panel B. Each
band was scanned, and the area under the peak is presented in the graph
as arbitrary units. D, different amounts of AC2 and AC6
membrane proteins are immunoblotted at a fixed (1:500) concentration.
The lanes labeled 1, 2, 3, and 4 represent 10, 5, 2.5, and 1 µg of membrane protein,
respectively.
We made several batches of AC2- and AC6-expressing
membranes. Prior to studying the functional characteristics of AC2 and
AC6, we sought to ensure that equivalent amounts of AC2 and AC6 were
being expressed. We determined the amounts of expressed protein by
immunoblotting with the AC antibody (Fig. 2A), and the immunoblot and graph in Fig. 2A show that the amounts of expressed AC2 and AC6
are very similar. We then measured basal adenylyl cyclase activities of
the expressed AC2 and AC6 in the presence of varying amounts of
Mg
ions. In contrast to the equivalent levels of
expressed adenylyl cyclase protein, we found that the activities of AC6
were very low as compared to those of AC2 at all concentrations of
Mg
ions tested (Fig. 2B). In spite of
the low activity, expressed AC6 was dependent on the presence of added
Mg
ion and its activity was significantly higher than
that of control (thyroid peroxidase) infected cells (Fig. 2C).
Figure 2:
A, Immunoblot of Sf9 membranes expressing
AC2 and AC6 with AC antibody. 10 µg of each membrane
protein were used for the immunoblot. Each band was scanned, and the
area under the peak is presented in the graph as arbitrary units. B, effect of varying concentrations of Mg
on
the adenylyl cyclase activities of membranes from cells infected with
recombinant baculovirus containing either AC2 or AC6. 5 µg of
membrane protein were used in each assay. C, the data for AC6
from Panel B are plotted on an expanded ordinate scale and
compared to adenylyl cyclase activities of control membranes from
thyroid peroxidase (TPO)-infected Sf9 cells. Values are means
of triplicate determinations. Coefficient of variance was less than
10%. For other details, see ``Experimental Procedures.''
We determined whether the difference in
activities between AC2 and AC6 was observable with
Mn, an ion capable of eliciting substantial basal
activity from adenylyl cyclases in the absence of G
or
other stimulators(17) . Increasing concentrations of
Mn
ions resulted in catalytic activities for both AC2
and AC6 (Fig. 3, A and B), but an interesting
difference was observed. Increasing concentrations of Mn
ions in the range of 0.1-10 mM resulted in
proportional increases in activity of AC2 (Fig. 3A). In
the case of AC6, however, after a sharp increase in activity in the
0.1-1.5 mM range, no further stimulation was observed
with further increases in Mn
ion concentration (Fig. 3B). It is noteworthy that the ratio of
Mn
/Mg
activities of AC6 at 2 mM divalent cation is approximately 10, similar to the value seen in
cyc
S49 cell membranes(17) . We (8) and others (18) have shown that AC6 is present in
S49 cells. In contrast, the ratio of
Mn
/Mg
activities of AC2 is in the
range of 2-3.
Figure 3:
Effect of
varying concentrations of Mn ions on AC2 (A)
and AC6 (B) activities in Sf9 cell membranes. 5 µg of
membrane protein were assayed at 0.1 mM ATP. Values are means
of triplicate determinations. Coefficient of variance was less than
10%. For other details, see ``Experimental Procedures.''
As our standard assay mixture contains 0.1
mM ATP, it is possible that some of the observed differences
in activity between AC2 and AC6 are due to differences in K of the enzyme isoforms for the
substrate. Hence we measured the K
of AC2
and AC6 for ATP at a high (20 mM) Mg
ion
concentration (Fig. 4). There was a 2-fold difference in the K
for ATP between AC2 and AC6; the
difference in V
, however, was greater than
25-fold.
Figure 4:
Lineweaver-Burk plots of various
concentrations of ATP on the activity of AC2 (A) and AC6 (B) expressed in Sf9 cells. 5 µg of protein were used for
each assay. Mg ion concentration was 20 mM.
Data were processed by use of the Prophet program on a Sun workstation.
To establish whether the differences in activities between
AC2 and AC6 are due to differences in intrinsic catalytic rates or due
to differential regulation of basal activities by divalent cations, we
determined activities of the two enzymes in the presence of 100
µM forskolin, a direct stimulator of adenylyl
cyclase(19) , and varying concentrations of
Mn. Generally, Mn
plus forskolin
can be used to elicit the maximum available activity of adenylyl
cyclases. A comparison of the activities of AC2 and AC6 in the presence
of 100 µM forskolin and varying amounts of Mn
is shown in Fig. 5. Although AC6 had less activity than
AC2 at all concentrations of Mn
tested, the
difference in activity between AC2 and AC6 was only 2-fold. In
contrast, in the absence of forskolin at 20 mM
Mg
, AC2 had 15-fold greater activity than AC6 (Fig. 2, B and C), and in the absence of
forskolin at 10 mM Mn
, AC2 had 10-fold
greater activity that AC6 (Fig. 3, A and B).
Thus the observed differences in basal activities between AC2 and AC6,
in large part, are not due to intrinsic differences in catalytic
capabilities of these two isoforms but rather due to different
responses to stimulation by divalent cations. A noteworthy feature of
the experiment in Fig. 5was that, in the presence of forskolin,
the AC2 activity was saturable with respect to Mn
ions within 10 mM, in contrast to the continuous
increase observed in the absence of forskolin (Fig. 3A). Hence we determined whether AC2 and AC6
activities in the presence of forskolin were saturable with respect to
Mg
as well.
Figure 5:
Effect of
100 µM forskolin and varying concentrations of
Mn on AC2 and AC6 activities in Sf9 cell membranes. 5
µg of membrane protein were assayed at 0.1 mM ATP. Values
are means of triplicate determinations. Coefficient of variance was
less than 10%. For other details, see ``Experimental
Procedures.''
The effect of varying concentrations
of Mg in the presence of 100 µM forskolin on AC2 and AC6 activities are shown in Fig. 6A. It can be seen readily that stimulation by
increasing concentrations of Mg
is saturable by
5-10 mM Mg
. This profile contrasts
with that seen in the absence of forskolin in which the activity
increases in a continuous fashion up to the highest concentration
tested (20 mM). The fold stimulation by forskolin for AC2 and
AC6 is shown in Fig. 6B. An interesting difference
between AC2 and AC6 was observed. At all concentrations of
Mg
ion tested, forskolin was able to stimulate AC6
activity extensively, with a 45-fold stimulation at 1-2 mM Mg
(Fig. 6B). In contrast, fold
stimulation by forskolin for AC2 was much lower. Even at low
Mg
ion concentrations, only a 6-fold stimulation was
seen; increasing concentrations of Mg
ions elicited
substantial amounts of activity, such that forskolin stimulation was
very modest (
2-fold).
Figure 6:
A, Effect of 100 µM forskolin
and varying concentrations of Mg on AC2 and AC6
activities in Sf9 cell membranes. 5 µg of membrane protein were
assayed at 0.1 mM ATP. Values are means of triplicate
determinations. Coefficient of variance was less than 10%. TPO, thyroid peoxidase. B, fold stimulation of AC2
and AC6 by forskolin at indicated concentrations of
Mg
. Fold stimulation values were calculated from the
data in Fig. 2B and 6A. For other details, see
``Experimental Procedures.''
The Mg concentration
effect curves for AC2 and AC6 in the absence and presence of forskolin
were subjected to linear transformations. In the absence of forskolin,
curvilinear Eadie-Hofstee plots were obtained for both AC2 and AC6 (Fig. 7A, a, and 7B, a),
indicating the possibility of multiple sites of interactions for
Mg
. In the presence of forskolin, linear
transformations were obtained, indicating that there may be a single
allosteric site for divalent cation regulation of the catalytic
activities of AC2 and AC6 (Fig. 7A, b, and
7B, b).
Figure 7:
Hofstee transformations of the effect of
varying Mg on AC2 (A) and AC6 (B)
activities in the absence (a) and presence (b) of 100
µM forskolin. R, response in picomoles of
cAMP/mg
min. The values used are from the experiments shown in Fig. 2B and 6A. In the absence of forskolin,
the improvements in the variance ratios for AC2 and AC6 using a
two-site fit were 4.93 and 3.88, respectively. Data were processed by
use of the Prophet program on a Sun workstation.
It is now well established that the different mammalian adenylyl cyclases have unique signal recognition capabilities(9, 20) . This allows the cAMP pathway to respond to a variety of G protein-coupled receptors (21) as well as receptor tyrosine kinases that stimulate protein kinase C activity. The varied capabilities for signal input, coupled with the differential expression of the adenylyl cyclase isoforms in different cell types and tissues, allow the identity of the individual adenylyl cyclases present to impart to the hormone-regulated adenylyl cyclase system distinct features that may be in consonance with the regulatory requirements of the cell type and tissue. In addition to the varied signal recognition capabilities, the data presented here indicate that the basal activities of different adenylyl cyclases can also be sufficiently different such that the presence and relative abundance of a certain adenylyl cyclase isoform can affect the basal cAMP levels in cells and tissues. We studied AC2 and AC6. Previous studies had noted differences in basal activities between AC1 and AC3(22) . However, the reasons underlying the differences in basal activities remained unexplored until now.
The activity of AC2 is 25-fold higher
than that of AC6 at optimal Mg concentrations (Fig. 4). It is noteworthy that the differences between AC2 and
AC6 are most pronounced at low, physiologically relevant, free
Mg
ion concentrations. This difference in basal
activities between AC2 and AC6 is not due, in large part, to intrinsic
differences in the catalytic capabilities between AC2 and AC6, since in
the presence of Mn
and forskolin there is only a
2-fold difference in activity. Rather the low basal activity of AC6
arises from the inability of Mg
to elicit high levels
of catalytic activity from AC6. Thus under normal cellular conditions,
when AC6 constitutes a significant part of the complement of adenylyl
cyclases expressed, the basal cAMP production is likely to be low. This
agrees well with the previously noted propensity of AC6 to lower cAMP
levels in response to a variety of signals. As AC6 is a widely
expressed adenylyl cyclase(16, 23) , our findings may
provide an explanation for the relatively low level of basal
intracellular cAMP in many tissues. In contrast AC2, which has a high
basal activity, is most abundant in the brain, an organ known to have
relatively high basal cAMP levels(24) .
Previous studies
from our laboratory had noted the presence of an allosteric site for
divalent cations on adenylyl cyclase that was required for the
expression of G-
-stimulated activity (25) .
The data presented here extend those observations and indicate that
occupancy at the allosteric site on adenylyl cyclase may be responsible
for the expression of basal activities as well. Further, from our data
it appears that the regulation of basal activity of the different
adenylyl cyclase isoforms by Mg
ion is likely to be a
unique feature of each adenylyl cyclase.
What is the biological significance of these vastly different basal activities for the different adenylyl cyclases? The answer may be in the important role that intracellular levels of cAMP play in regulating many biological processes. Three examples are noteworthy. 1) Embryonic development: recently it has been shown that basal protein kinase A activity, and hence the ambient levels of cAMP, plays a crucial role in the formation of the compound eye, wing, and leg of Drosophila by regulating the activity of the morphogen Hedgehog (see (26) and references therein). Current evidence from analysis of the development of retina, leg, and wing (27, 28) supports the notion that the basal activity of protein kinase A is counteracted by the Hedgehog signaling pathway(26) . Similarly in vertebrate systems, long-range induction of sclerotome by Sonic hedgehog is blocked by elevation of cAMP levels(29) . Thus it appears likely that ambient levels of cellular cAMP are a crucial determinant of embryonic development in several species. It is reasonable to assume that the activity of protein kinase A in a cell is reflective of the activity of the adenylyl cyclases resident in the cell. Our data indicate that different adenylyl cyclases can have very different levels of basal activity. Thus the identity of the adenylyl cyclase present could play a determining role in regulating development by morphogens. 2) Transformation and proliferation of mammalian cells: we have shown that modest increases in cAMP levels are sufficient to block transformation of NIH-3T3 by powerful oncogenes such as H-ras(1) . Thus the presence of isoforms of adenylyl cyclase that have high basal activity could work to inhibit processes that lead to neoplastic transformation. Lowering of cAMP levels has been known to trigger proliferation of certain cells such as RAT-1 fibroblasts(30) , and in these cells it would be crucial to have adenylyl cyclases with low basal activities for proliferation to occur. 3) The addicted state: it has long been noted that the onset of the addicted state is accompanied by lowered intracellular cAMP levels in neurons(31, 32) . Such decreased cAMP levels are thought to be responsible, in part, for the onset of the addicted state(32) . It will be interesting to determine whether the lowered levels of cAMP result from decreased activity of the preexisting adenylyl cyclases or from expression of a different complement of adenylyl cyclases with lower basal activities.
The data presented in this paper, and the biological phenomena described above, indicate that the different adenylyl cyclase isoforms and their relative abundance will be able to define not only the responses of the cellular cAMP pathway to external signals but may also regulate the integral physiology of the cell by virtue of their basal activities.