(Received for publication, January 31, 1995; and in revised form, June 13, 1995)
From the
Type III adenylyl cyclase is stimulated by -adrenergic
agonists and glucagon in vitro and in vivo, but not
by Ca
and calmodulin. However, the enzyme is
stimulated by Ca
and calmodulin in vitro when it is concomitantly activated by the guanyl nucleotide
stimulatory protein G
(Choi, E. J., Xia, Z., and Storm, D.
R. (1992a) Biochemistry 31, 6492-6498). Here, we
examined regulation of type III adenylyl cyclase by
G
-coupled receptors and intracellular Ca
in vivo. Surprisingly, intracellular Ca
inhibited hormone-stimulated type III adenylyl cyclase activity.
Submicromolar concentrations of intracellular free
Ca
, which stimulated type I adenylyl cyclase,
inhibited glucagon- or isoproterenol-stimulated type III adenylyl
cyclase. Inhibition of type III adenylyl cyclase by intracellular
Ca
was not mediated by G
, cAMP-dependent
protein kinase, or protein kinase C. However, an inhibitor of CaM
kinases antagonized Ca
inhibition of the enzyme, and
coexpression of constitutively activated CaM kinase II completely
inhibited isoproterenol-stimulated type III adenylyl cyclase activity.
We propose that Ca
inhibition of type III adenylyl
cyclase may serve as a regulatory mechanism to attenuate
hormone-stimulated cAMP levels in some tissues.
Adenylyl cyclases are regulated by extracellular and
intracellular signals including neurotransmitters, hormones, and
intracellular Ca (reviewed in Tang and Gilman(1992)
and Choi et al. (1993a). Each of the eight adenylyl cyclases
that have been cloned (Krupinski et al., 1989; Feinstein et al., 1991; Bakalyar and Reed, 1990; Gao and Gilman, 1991;
Ishikawa et al., 1992; Katsushika et al., 1992;
Yoshimura and Cooper, 1992; Krupinski et al., 1992; Cali et al., 1994; Watson et al., 1994) has distinct
regulatory properties. For example, I-AC (
)(Tang et
al., 1991; Choi et al., 1992a), III-AC (Choi et
al., 1992a), and VIII-AC (Cali et al., 1994) are
stimulated by Ca
and calmodulin (CaM) in vitro but II-AC, IV-AC, V-AC, VI-AC, and VII-AC are not.
In contrast
to I-AC and VIII-AC which are directly stimulated by Ca and CaM in vitro, III-AC is not stimulated by
Ca
and CaM unless it is also activated by GppNHp or
forskolin (Choi et al., 1992a). Furthermore, the
concentrations of free Ca
for half-maximal
stimulation of I-AC and III-AC are 150 nM and 5.0 µM Ca
, respectively. These data suggested that
III-AC might be synergistically stimulated by intracellular
Ca
- and G
-coupled receptors in vivo. To test this hypothesis, we examined the sensitivity of III-AC to
G
-coupled receptor activation and intracellular
Ca
in HEK-293 cells. Contrary to our expectations,
intracellular Ca
inhibited glucagon- and
isoproterenol-stimulated III-AC activities in vivo.
Figure 1:
Synergistic stimulation of III-AC by
Ca and activated G
in membrane
preparations. A, stimulation of III-AC by Ca
and CaM was assayed as a function of GppNHp concentration in
membranes. B, stimulation of III-AC by Ca
and CaM was assayed as a function of glucagon concentration in
membranes. Membranes were prepared from HEK-293 cells stably expressing
III-AC and the glucagon receptor (III-AC-G cells) and adenylyl cyclase
was assayed as described under ``Experimental Procedures.''
When present, free Ca
and CaM were 200 nM and 2.4 µM, respectively. Data were corrected for
endogenous adenylyl cyclase activity by subtracting activity obtained
from HEK-293 cells expressing only the glucagon receptor (293-G).The
data are mean ± S.D. of triplicate
assays.
To determine if Ca and
receptor-activated G
will also synergistically stimulate
III-AC in membranes, the sensitivity of the enzyme to CaM and
Ca
was analyzed in the presence of glucagon (Fig. 1B). In membrane preparations, III-AC was
stimulated 4.1 ± 0.1-fold by glucagon with an EC
of
7 nM. Glucagon-stimulated III-AC activity was enhanced 45
± 6.1% by CaM and Ca
; however, the EC
for glucagon was not significantly affected by CaM. These data
suggested that Ca
stimulation of III-AC is
conditional upon G
activation, and that Ca
and hormones might synergistically activate the enzyme in
vivo.
Figure 2:
Ca inhibits
glucagon-stimulated III-AC activity in vivo. A,
HEK-293 cells expressing no glucagon receptor (293), the glucagon
receptor (293-G), I-AC and the glucagon receptor (I-AC-G), or III-AC
and the glucagon receptor (III-AC-G) were treated with increasing
concentrations of glucagon and assayed for cAMP accumulation as
described under ``Experimental Procedures.'' cAMP
accumulation assays were performed in triplicate, and the data are the
mean ± S.D. of triplicate assays. B, HEK-293 cells
expressing III-AC and the glucagon receptor (III-AC-G) were treated
with increasing concentrations of glucagon in the presence or absence
of 10 µM A23187 and 1.8 mM CaCl
and
assayed for cAMP accumulation as described under ``Experimental
Procedures.'' The data are the mean ± S.D. of triplicate
assays.
From in vitro data using isolated membranes, we expected that glucagon and
intracellular Ca would synergistically stimulate
III-AC in intact cells. In fact, increases in intracellular
Ca
, generated by A23187 and extracellular
Ca
, inhibited glucagon-stimulated III-AC activity 60% (Fig. 2B). A23187 and extracellular Ca
had no effect on the basal activity of III-AC or endogenous
adenylyl cyclase activity (data not shown). Ca
inhibited glucagon-stimulated III-AC activity in several
different III-AC stable cell lines and was not due to clonal variation.
With different cell lines, Ca
inhibition of
glucagon-stimulated III-AC activity varied from 40-60%.
The
Ca dependences for inhibition of III-AC and
stimulation of I-AC in vivo were compared using A23187 and
varying amounts of extracellular Ca
(Fig. 3).
In this experiment, III-AC-G or I-AC-G cells were treated with 100
nM glucagon, but only III-AC was stimulated by glucagon.
Glucagon-stimulated III-AC activity was inhibited by Ca
concentrations which stimulated I-AC, and the curves were almost
mirror images of each other. The concentration of free intracellular
Ca
for half-maximal inhibition of glucagon-stimulated
III-AC activity was estimated at 150 to 200 nM using Fura-2
imagining.
Figure 3:
Ca concentration
dependence for inhibition of glucagon-stimulated III-AC activity in
vivo. HEK-293 cells stably expressing I-AC (I-AC-G) or III-AC
(III-AC-G) were treated with glucagon (100 nM), 10 µM A23187, and increasing concentrations of CaCl
as
described under ``Experimental Procedures.'' Relative cAMP
accumulations were determined as described under ``Experimental
Procedures.'' The III-AC and I-AC data are presented as percentage
of the ratio (cAMP/[ATP + ADP + AMP])
100
with no added CaCl
and are the mean ± S.D. of
triplicate assays.
Figure 4:
Ca inhibits
isoproterenol- stimulated III-AC activity in vivo. HEK-293
cells expressing III-AC were exposed to increasing concentrations of
the
-adrenergic agonist isoproterenol, in the absence or presence
of 10 µM A23187 and 1.8 mM CaCl
as
described under ``Experimental
Procedures.''
In the experiments
described above, intracellular Ca was elevated using
A23187 and it was of interest to determine if Ca
generated by physiologically relevant signals would also inhibit
hormone stimulated III-AC. HEK-293 cells contain muscarinic receptors
coupled to the mobilization of intracellular Ca
.
Treatment of HEK-293 cells with 10 µM carbachol elevates
intracellular Ca
to approximately 300 nM free Ca
and stimulates I-AC (Choi et
al., 1992b). Carbachol alone did not significantly affect III-AC
activity but did inhibit isoproterenol-stimulated activity 43 ±
5% (Fig. 5). Inhibition of isoproterenol-stimulated III-AC
activity by carbachol was insensitive to pertussis toxin and therefore
not due to endogenous muscarinic receptors coupled to III-AC through
G
(data not shown). These data indicate that
physiologically relevant concentrations of intracellular Ca
inhibit hormone-stimulated III-AC activity in vivo.
Figure 5:
Inhibition of isoproterenol-stimulated
type III adenylyl cyclase by carbachol. HEK-293 cells expressing III-AC
(III-AC-G) were exposed to increasing concentrations of the
-adrenergic agonist isoproterenol in the presence or absence of 10
µM carbachol. Under these conditions, carbachol increased
intracellular free Ca
from approximately 50 nM to 300 nM. cAMP accumulations were monitored as described
under ``Experimental Procedures,'' and the data are the mean
± S.D. of triplicate assays.
Figure 6:
Ca inhibits
forskolin-stimulated III-AC activity in vivo. HEK-293 cells
stably expressing III-AC (III-AC-G) were treated with increasing
concentrations of forskolin in the presence or absence of 10 µM A23187 and 1.8 mM CaCl
. cAMP accumulations
were monitored as described under ``Experimental
Procedures.'' The data are the mean ± S.D. of triplicate
assays.
Figure 7:
Effect of KN-62 on Ca
inhibition of III-AC in vivo. HEK-293 cells expressing the rat
glucagon receptor and III-AC were pretreated for 1 h with increasing
doses of KN-62, an inhibitor of CaM kinases. The cells were then
treated with 100 nM glucagon in the presence and absence of 10
µM A23187 and 1.8 mM CaCl
to
quantitate Ca
inhibition of glucagon-stimulated
III-AC activity. cAMP accumulations were monitored as described under
``Experimental Procedures,'' and the data are presented as
percentage inhibition of cAMP accumulation caused by A23187 and
Ca
. KN-62 blocked Ca
inhibition of
glucagon-stimulated III-AC activity. The data are the mean ±
S.D. of triplicate assays.
To determine if CaM kinase II inhibits
the activity of III-AC activity in vivo, we made stable
transfectants in HEK-293 cells expressing CaM kinase II under the
control of a metallothionein promoter. The CaM kinase II used in this
experiment (KII-290) contains a point mutation that truncates the
protein, removes its autoinhibitory domain, and makes it constitutively
active (Matthews et al., 1994). These cells were then
transiently transfected with a construct encoding III-AC, and the
sensitivity of the adenylyl cyclase to CaM kinase II was evaluated by
inducing the expression of the kinase with Zn.
Zn
treatment of cells not expressing KII-290 had no
effect on basal, isoproterenol, or forskolin-stimulated III-AC
activities. However, induction of CaM kinase II activity in KII-290
cells expressing III-AC completely inhibited isoproterenol (Fig. 8A) and forskolin-stimulated (Fig. 8B) III-AC activities. These data suggest that
Ca
inhibition of III-AC in vivo may be
mediated by CaM kinase II. Thus far, we have been unable to inhibit
hormone stimulation of III-AC in membrane preparations using purified
CaM kinase II suggesting this kinase may not directly phosphorylate
III-AC. However, further experimentation is required to elucidate the
mechanism for CaM kinase II regulation of adenylyl cyclase activity.
Figure 8:
Inhibition of isoproterenol and
forskolin-stimulated type III adenylyl cyclase by CaM kinase II-290.
KII-290 cells stably transfected with the inducible, constitutively
active CaM kinase II-290 which were transiently transfected with
III-AC, were exposed to either isoproterenol (A) or forskolin (B) ± induction of CaM kinase II-290 by
Zn. cAMP accumulations were determined as described
under ``Experimental Procedures.'' The data are corrected for
endogenous adenylyl cyclase activity as described under
``Experimental Procedures.'' The data are the mean ±
S.E. of triplicate assays.
The adenylyl cyclases exhibit diverse regulatory properties
that provide a number of interesting mechanisms for regulation of
intracellular cAMP by extracellular and intracellular signals. Several
of the adenylyl cyclases are synergistically stimulated by signals
arising from different pathways and therefore can generate enhanced
cAMP signals in response to signal convergence. For example, the
complex from G proteins stimulates
G
-activated II-AC and IV-AC (Tang and Gilman, 1992)
providing a mechanism for signal integration. I-AC is synergistically
activated by Ca
and neurotransmitters in vivo (Wayman et al., 1994), a regulatory property that may be
important for some forms of synaptic plasticity and spatial memory in
mice (Wu et al., 1995). Although synergistic stimulation of
adenylyl cyclases by two or more signals may be important for some
physiological process including cAMP-mediated transcription (Impey et al., 1994), mechanisms for inhibition of adenylyl cyclase
activity and optimization of cAMP levels may be equally important. The
data in this study identify a new mechanism for regulation of adenylyl
cyclase activity; physiologically significant levels of intracellular
Ca
attenuate hormone stimulation of III-AC.
III-AC
is stimulated by Ca and CaM when it is activated by
G
in vitro, but hormone-stimulated III-AC is
inhibited by Ca
in vivo. Glucagon,
isoproterenol, and forskolin-stimulated III-AC activities were all
partially inhibited by physiologically relevant concentrations of
intracellular Ca
(100 to 300 nM free
Ca
). The mechanism for Ca
inhibition of III-AC activity was not dependent upon the activity
of cAMP-dependent protein kinase, protein kinase C, or G
.
However, KN-62, an inhibitor of CaM kinases, blocked Ca
inhibition suggesting the interesting possibility that
Ca
activation of CaM kinases may directly or
indirectly inhibit III-AC activity in vivo. Furthermore,
expression of constitutively active CaM kinase II completely blocked
hormone stimulation of III-AC activity in vivo.
To date,
five Ca-regulated adenylyl cyclases have been
identified: I-AC, III-AC, V-AC, VI-AC, and VIII-AC. I-AC and VIII-AC
are stimulated by intracellular Ca
in vivo (Choi et al., 1992b; Cali et al., 1994) and
mutagenesis of the CaM binding domain of I-AC has established that
Ca
stimulation is mediated by CaM (Wu et
al., 1993). Neither I-AC nor VIII-AC is stimulated by
G
-coupled receptors in vivo (Wayman et
al., 1994; Cali et al., 1994). Although I-AC is
synergistically regulated by intracellular Ca
and
hormones in vivo, VIII-AC is not (Cali et al., 1994).
In contrast, V-AC and VI-AC are directly inhibited by Ca
in membranes (Yoshimura and Cooper, 1992; Katsushika et
al., 1992), and VI-AC is inhibited by submicromolar Ca
in vivo (Cooper et al., 1994). Regulation of
III-AC by Ca
and hormones is distinct from all of the
other adenylyl cyclases characterized thus far; it is stimulated by
hormones in vivo, and increases in intracellular
Ca
inhibit this response.
Although in vitro studies using isolated membrane preparations or purified
recombinant adenylyl cyclases and G proteins have provided valuable
insight concerning mechanisms for regulation of adenylyl cyclases, it
is becoming increasingly evident that conclusions drawn from in
vitro data do not necessarily apply in vivo. For example,
purified I-AC or I-AC in membranes is stimulated by addition of
relatively high levels of activated recombinant G-
,
demonstrating that this enzyme has a G
-
interaction
domain (Tang et al., 1991). However, I-AC is not stimulated by
activation of G
-coupled receptors in HEK-293 cells (Wayman et al., 1994) or in cultured neurons. (
)VIII-AC is
synergistically stimulated by CaM and recombinant G
in
vitro, but it is not synergistically stimulated by Ca
and G
activation in vivo (Cali et
al., 1994). Characterization of mechanisms for regulation of
III-AC described in this study also demonstrates the importance of
defining the regulatory properties of each adenylyl cyclase in
vivo.
The physiological significance of Ca inhibition of hormone-stimulated III-AC activity remains to be
established. Adenylyl cyclase activity in most tissues is inhibited by
millimolar levels of Ca
which has been attributed to
formation of complexes between ATP and Ca
, or binding
of Ca
to a Mg
regulatory site on
adenylyl cyclases (Steer and Levitzki, 1975). Several tissues including
heart muscle (Potter et al., 1980) have been reported to
contain adenylyl cyclase activity that is inhibited by submicromolar
Ca
. It is interesting that III-AC (Xia et
al., 1992) and VI-AC (Yoshimura and Cooper, 1992; Katsushika et al., 1992) are both expressed in heart. The presence of
III-AC activity in heart may provide a mechanism whereby the positive
ionotropic and chronotropic effects of
-adrenergic agonists are
attenuated by increased intracellular Ca
. The
development of transgenic mice strains deficient in III-AC should
provide valuable information concerning the physiological functions of
the enzyme and the significance of this regulatory mechanism for
specific physiological processes including heart muscle contractility
and olfactory signal transduction.
In summary, this study describes a novel mechanism for regulation of adenylyl cyclase activity and is the first report showing that CaM kinases can regulate adenylyl cyclase activity in vivo. This regulatory mechanism may be important for a variety of physiological processes including heart muscle contractility and attenuation of neurotransmitter-stimulated cAMP levels in neurons.