(Received for publication, July 13, 1995; and in revised form, September 12, 1995)
From the
Previously, this laboratory has demonstrated that epidermal
growth factor (EGF) increases adenylyl cyclase activity in cardiac
membranes and elevates cAMP accumulation in hearts and cardiac
myocytes. Since EGF does not increase cAMP accumulation in all tissues,
we investigated the possibility that the expression of a specific
isoform of adenylyl cyclase (AC) was necessary to observe EGF-elicited
stimulation of cAMP accumulation. HEK 293 cells were transfected with
different isoforms of AC, and the ability of EGF to increase AC
activity as well as elevate cAMP accumulation was determined. In cells
transfected with AC I, II, V, and VI cDNAs, neither the expression nor
the amount of the two isoforms of G (45 and 52 kDa)
were altered. Similarly, EGF-elicited phosphorylation of cellular
proteins on tyrosine residues in various transfectants was unaltered.
However, EGF increased AC activity and elevated cAMP accumulation only
in cells expressing the rat and canine ACV. EGF did not alter either AC
activity or cAMP accumulation in cells overexpressing types I, II, and
VI isozymes. As assessed by the ability of an anti-G
antibody to obliterate the effect, stimulation of AC activity in
AC V transfectants involved the participation of G
, a
finding consistent with previous data concerning EGF effects on cardiac
AC (Nair, B. G., Parikh, B., Milligan, G., and Patel, T. B.(1990) J. Biol. Chem. 265, 21317-21322). Thus we conclude that
the expression of AC V isoform confers specificity to the ability of
EGF to stimulate AC activity.
Epidermal growth factor (EGF) ()produces an array of
biological effects in a variety of tissues(1, 2) .
These effects range from the ability of EGF to increase hyperplasia and
hypertrophy(1, 2) to the actions of EGF on metabolic
processes such as glycogenolysis (3) ,
gluconeogenesis(4) , amino acid uptake(5) , and
increases in cardiac contractility and heart rate(6) .
Apparently, these pleiotropic actions of EGF are mediated by the
activation of a number of cellular second messenger systems. In this
respect, it has been well established that after binding to its
receptors, EGF increases the protein-tyrosine kinase activity of its
receptors and thereby activates other kinase cascades such as the
mitogen-activated protein kinase (MAPK) (reviewed in (7) ) and
also stimulates phosphatidylinositol metabolism by increasing the
activity of phospholipase C
following tyrosine phosphorylation of
this enzyme(8) . EGF has also been demonstrated to modulate the
cAMP second messenger system. Thus studies from our laboratory have
demonstrated that EGF produces inotropic and chronotropic actions in
the perfused rat heart by increasing cellular cAMP
accumulation(6) . An EGF-elicited increase in cAMP levels in
the heart is the result of stimulation of adenylyl cyclase activity (9) by a mechanism that involves the stimulatory GTP-binding
protein of the system, G
(10) .
Presently, eight
isoforms of adenylyl cyclase have been cloned and sequenced (reviewed
in (11) ). Based upon their regulatory properties, these eight
isoforms can be subdivided into four major groups. Hence, group 1 would
consist of the types I, III, and VIII adenylyl cyclases, which are
stimulated by calcium and calmodulin (12, 13, 14) . The types II and IV adenylyl
cyclases constitute group 2 and are stimulated by subunits
of the heterotrimeric G proteins provided that active G
is also present(15, 16) . The third group
comprises type II and type VII adenylyl cyclases, which are
phosphorylated and activated by protein kinase
C(17, 18) . Finally, the type V and type VI are the
predominant adenylyl cyclases in the
heart(19, 20, 21, 22) . Both these
isoforms, which are inhibited by low concentrations of
calcium(20, 22) , form the fourth group.
Although
the EGF receptor, G, and adenylyl cyclase are present in a
number of different cells including non-myocytes derived from hearts,
studies from our laboratory have previously shown that EGF stimulates
cAMP accumulation only in cardiac myocytes(23) . Other tissues
in which EGF stimulates adenylyl cyclase are the parotid gland (24) and luteal cells(25) . Therefore, in the present
study we have performed experiments to determine whether or not
specific isoform(s) of adenylyl cyclase confers specificity to the
ability of EGF to stimulate adenylyl cyclase. We have employed a human
embryo kidney cell line (HEK 293), which expresses approximately the
same number of EGF receptors as the heart, and the 52-kDa as well as
the 45-kDa isoforms of G
to address this hypothesis.
Essentially, data are presented to show that EGF increases adenylyl
cyclase activity via G
in HEK 293 cells stably
transfected to express rat and canine AC V isoforms; EGF did not alter
adenylyl cyclase activity in cells transfected with AC I, AC II, and AC
VI cDNAs.
Cyclic AMP accumulation in HEK 293 cells
transfected to express different adenylyl cyclase isoforms was
monitored employing two different methods. The first approach involved
monitoring the conversion of [H]ATP into
[
H]cAMP after labeling the cells with
[
H]adenine as described by Wong et
al.(27) . Essentially, cells were plated in 24-well
culture dishes (10
cells/well) and labeled overnight with 2
µCi of [
H]adenine. Labeled cells were
preincubated with 3-isobutyl-1-methylxanthine (100 µM) in
Krebs-Henseleit bicarbonate buffer (28) modified to contain 5
mM NaHCO
, 20 mM Hepes, pH 7.4, and 10
mM glucose. The cells were exposed to the test reagents for a
period of 15 min, and reactions were terminated by the addition of 10%
perchloric acid containing [
C]cAMP and
[
P]ATP to correct for recoveries of
H-labeled cAMP and ATP, respectively. The ATP and cAMP were
separated on Dowex and alumina columns as described by Salomon et
al.(26) . Cyclic AMP accumulation is presented as a
fraction of total adenine nucleotides to correct for variations in
labeling efficiency or cell numbers. As a second approach, after
treatment of cells as described above, reactions were terminated by the
addition of 2 N HCl, and the amount of cAMP accumulation in
the cells was monitored by radioimmunoassay procedure described by
Brooker et al.(29) .
Western analyses for the
expression of G were similarly performed except that
the cells were not exposed to EGF and the polyclonal anti-G
antiserum (CS1) against the carboxyl terminus decapeptide of
G
was used(10) .
The observation that EGF, by itself, increases cAMP
accumulation and stimulates adenylyl cyclase in the hearts and
cardiomyocytes(6, 9, 10, 23) ,
parotid gland(24) , and luteal cells (25) but does not
increase cAMP accumulation in non-myocytes derived from hearts (23) and in other tissues such as the liver (3) suggested that the expression of some specific signaling
element(s) was required to confer specificity to the ability of EGF to
stimulate adenylyl cyclase. Studies from our laboratory (6, 9, 10, 23, 32) have
demonstrated that the stimulation of adenylyl cyclase by EGF is
mediated by G and that the EGF receptor
protein-tyrosine kinase is also important for the effect. Presently,
only one form of the EGF receptor has been described. Although
alternative splicing of the pre-mRNA may result in four forms of
G
(33) , thus far there have been no unique
regulatory features associated with the four G
isoforms. The greatest diversity of regulation among the
signaling elements known to participate in EGF-elicited stimulation of
cAMP accumulation is that of the various adenylyl cyclase isoforms (see
the Introduction). Therefore, employing HEK 293 cells overexpressing
various types of adenylyl cyclase we have investigated whether or not
the reconstitution of the EGF/AC signaling requires the expression of a
specific isozyme. The HEK 293 cells were selected for these experiments
for the following reasons: (a) these cells express the same
number of EGF receptors as the rat heart (
11 fmol/mg protein at 50
pM EGF); (b) the HEK 293 cells express the same
isoforms (45 and 52 kDa) of G
as the cardiomyocytes (cf. Fig. 1and (34) ); and (c) EGF
does not increase cAMP accumulation in naive or control,
plasmid-transfected HEK 293 cells (discussed below).
Figure 1:
Effect of overexpression of various AC
isoforms on amounts of the G isoforms (A) and
the ability of EGF to phosphorylate cellular proteins on tyrosine
residues (B). A, cellular proteins (30 µg) were
separated by SDS-PAGE (10% acrylamide) and after transfer onto
nitrocellulose, subjected to Western analysis with the
anti-G
antiserum (CS1) as described under
``Experimental Procedures.'' Migration of the 52- and 45-kDa
isoforms of G
is indicated by the arrows. B, HEK 293 transfected with plasmid alone (V2) or cDNAs
encoding various AC isoforms were exposed to either vehicle or 100
nM EGF for 2 min as described under ``Experimental
Procedures.'' Reactions were terminated by the addition of Laemmli
sample medium, and aliquots representing 10
cells (100
µg of protein) each were separated by SDS-PAGE (7.5% acrylamide).
Western analysis of the cellular proteins with anti-phosphotyrosine
antibody (Zymed Inc.) is presented. The arrow indicates the
migration of the 170-kDa protein whose phosphorylation was most
prominently altered by EGF. Roman numerals indicate the AC
isoforms, and numbers in parentheses represent clone
number; R-ACV(11) and C-ACV(34) represent clonal
transfectants expressing rat and canine forms of AC V, respectively.
Following
transfection and selection by G418 of HEK 293 cells overexpressing AC
isoforms, experiments were performed to determine if overexpression of
adenylyl cyclases altered the amount and/or the forms of G present in these cells. As demonstrated by data in Fig. 1A, the amount of the 52- and 45-kDa isoforms of
G
was not altered in cells transfected with plasmid
alone or cells expressing the various AC isoforms. Similarly,
experiments were performed to determine if overexpression of the AC
isoforms in HEK 293 cells altered the ability of EGF to stimulate
phosphorylation of cellular proteins on tyrosine residues. The data in Fig. 1B demonstrate that the addition of EGF increased
tyrosine phosphorylation in all of the different transfectants to a
similar extent. These findings (Fig. 1) indicated that
transfection of HEK 293 cells with cDNAs encoding the various AC
isozymes did not alter the expression of either G
isoforms or functional EGF receptors, the two signaling elements
other than adenylyl cyclase required in EGF-elicited stimulation of
cAMP accumulation(10, 32) . It should be noted that in
all of the experiments described in this report, the data obtained with
naive HEK 293 cells were identical to those with cells transfected with
the plasmid alone, and therefore, the plasmid transfectants (V2) are
presented as controls.
In order to determine whether or not EGF
increased adenylyl cyclase activity in the HEK 293 cells transfected
with either the plasmid alone (V2) or the plasmid containing various AC
isoforms, experiments depicted in Fig. 2A were
performed. Essentially, lysed cells were assayed for adenylyl cyclase
activity in the presence of the GTP analog, Gpp(NH)p, with and without
either EGF or isoproterenol. It is noteworthy that in membrane
preparations or lysed cells, the addition of GTP analogs is required to
observe G protein-mediated modulation of adenylyl cyclase (see e.g. Refs. 9, 10, and 17). Moreover, the addition of GTP or its analogs
Gpp(NH)p or GTPS does not maximally stimulate adenylyl cyclase,
and the enzyme activity can be further augmented by agents that
activate receptors coupled to
G
(9, 10, 17) . In a previous
study(9) , we have shown that the optimal concentration of
Gpp(NH)p required to observe EGF-elicited stimulation of adenylyl
cyclase activity in membrane preparations is 10 µM. As
demonstrated by the data in Fig. 2A, isoproterenol (100
nM) increased adenylyl cyclase activity in all cell types
expressing either the endogenous (control, V2) or the transfected
isoforms of adenylyl cyclases. On the other hand, EGF (100 nM)
only stimulated adenylyl cyclase activity in cells overexpressing rat
and canine AC V (Fig. 2A). The ability of EGF to
stimulate adenylyl cyclase activity in cells transfected with the rat
and canine AC V cDNAs is not the result of differences in the
expression of the various isoforms of the enzyme. For instance, as
demonstrated by the ability of forskolin to stimulate enzyme activity,
AC I and canine AC V transfectants demonstrated similar activities (Fig. 2B). Similarly, aluminum fluoride stimulated
adenylyl cyclase activity in both AC II and rat AC V transfected cells
to a similar extent (Fig. 2B). Since AC VI transfected
cells did not display a very high activity with forskolin and because
activated G
along with forskolin is required to
maximally activate this form of the enzyme(35) , experiments
were conducted with forskolin in the presence of exogenously supplied
G
. As controls, vector and AC I transfectants were
employed since in the latter (AC I) the combination of G
and forskolin should not synergistically activate adenylyl
cyclase(35) . These data demonstrated that as described by
McHugh Sutkowski et al.(35) , forskolin in the
presence of exogenous G
activated type VI adenylyl
cyclase but not type I adenylyl cyclase to a larger extent than either
G
or forskolin by themselves (Fig. 2B). More importantly, these latter data also
demonstrate that the amount of adenylyl cyclase, as monitored by
activity, in AC VI transfectants is also similar to that observed in
the AC V expressing cells. Thus the effects of EGF on stimulation of
adenylyl cyclase in AC V transfectants is not the result of simply an
increase in the amount of adenylyl cyclase available.
Figure 2:
Effect of
various agonists on adenylyl cyclase activity in HEK 293 cells
transfected with plasmid alone (V2) or cDNAs encoding various AC
isoforms. A, EGF stimulates adenylyl cyclase activity in rat
and canine AC V transfectants but not in HEK 293 cells transfected with
plasmid alone or with cDNAs encoding AC I, AC II, and AC VI isozymes.
Lysed HEK 293 cells overexpressing AC I, AC II, and rat and canine AC V (R-ACV and C-ACV, respectively), AC VI, and controls
(V2) were assayed for adenylyl cyclase activity in the presence of
Gpp(NH)p (10 µM) with and without the addition of 100
nM of either EGF or isoproterenol as described under
``Experimental Procedures.'' B, effect of forskolin
(10 µM), aluminum fluoride (2 mM NaF + 30
µM AlCl), G
(100
nM), and combinations of some of the activators on adenylyl
cyclase activity in lysed control cells (V2) or cells overexpressing AC
I, AC II, rat and canine AC V (R-ACV and C-ACV,
respectively), and AC VI. Data presented are the mean ± S.E. of
three determinations in at least three experiments. Numbers in parentheses represent clone number for each AC isoform.
In order to
determine whether or not EGF increased cAMP accumulation in cells
transfected with either the vector alone or vector containing different
AC isoforms, experiments presented in Fig. 3A were
performed. In accordance with the data presented in Fig. 2A, these experiments demonstrated that EGF
increased cAMP accumulation in HEK 293 cells expressing the AC V but
not in cells overexpressing any other AC isozyme. Since AC I has been
demonstrated to be optimally stimulated by -adrenergic agonists
such as isoproterenol in the presence of
Ca
-mobilizing agonist such as the ionophore
A23187(36) , one interpretation of the data with AC I
expressing cells in Fig. 3A is that the experimental
conditions were not optimal to observe receptor agonist-mediated
alterations in cAMP accumulation in these cells. Therefore, experiments
were performed to determine whether or not the ability of EGF to
stimulate cAMP accumulation in AC I transfectants was also dependent
upon simultaneous increases in cytosolic free Ca
. The
data in Fig. 3B demonstrate that although (as reported
by Wayman et al.(36) ) isoproterenol-elicited
stimulation of cAMP accumulation in AC I transfectants was markedly
augmented in the presence of A23187, EGF did not alter cAMP
accumulation in the presence of the ionophore. Therefore, unlike
isoproterenol, the ability of EGF to stimulate cAMP accumulation in AC
I transfectants is not dependent upon the simultaneous increase in
cytosolic free Ca
. In experiments similar to those in Fig. 3B, the ionophore A23187 did not alter the ability
of any of the agonists to increase cAMP accumulation in HEK 293 cells
transfected to express type V adenylyl cyclase (not shown). However,
since in enzyme activity assays the type V isoform has been reported to
be inhibited by 100 µM Ca
(19, 20) , we investigated the effects of
Ca
on stimulation of type V adenylyl cyclase activity
by the various agonists. The data in Fig. 3C show that
adenylyl cyclase activity in canine type V transfectants was inhibited
under all conditions by 100 µM Ca
.
However, the ability of EGF and isoproterenol to stimulate the type V
enzyme was not altered when compared with the corresponding control (cf. 68% stimulation by EGF in the absence of Ca
with 97% stimulation by EGF in the presence of 100 µM Ca
) (Fig. 3C). At lower
concentrations of Ca
(10 µM), only the
ability of forskolin to stimulate type V adenylyl cyclase activity was
diminished (Fig. 3C). The lack of an effect of 10
µM Ca
on basal or isoproterenol- and
EGF-stimulated activity (Fig. 3C) is consistent with
the data of Ishikawa and co-workers(19, 20) .
Moreover, since Ca
ionophores only increase
intracellular free Ca
concentrations to
2.0
µM(37) and because inhibition of AC V requires
high micromolar concentrations of Ca
(Fig. 3C)(19, 20) , it is not
surprising that A23187 did not alter EGF- and isoproterenol-stimulated
cAMP accumulation in type V adenylyl cyclase transfected cells (not
shown). For reasons similar to those described for the experiments with
Ca
ionophore in AC I transfectants, since activation
of protein kinase C increases the response of AC II to
agonists(18) , experiments were performed with the AC II
transfectants to determine whether or not protein kinase C activation
with phorbol 12-myristate 13-acetate would allow EGF to increase cAMP
accumulation in these cells. However, EGF did not increase cAMP
accumulation in AC II transfectants whether or not protein kinase C was
activated (data not shown).
Figure 3:
EGF
increases cAMP accumulation in HEK 293 cells overexpressing AC V but
not other isoforms of adenylyl cyclase, and this effect is independent
of Ca. A, EGF stimulates cAMP accumulation
in AC V transfectants but not in control cells (V2) or cells
overexpressing AC I, AC II, and AC VI. The cells (2
10
/35-mm dish) were treated with and without EGF for 15
min, and reactions were terminated by the addition of 1 ml of HCl (2 N). Following lyophilization of the samples, the cAMP content
was determined by radioimmunoassay as described under
``Experimental Procedures.'' Data are presented as the -fold
increase over basal cAMP content and are the mean ± S.E. of six
determinations. R-ACV represents rat AC V transfectants. B, effect of ionophore A23187 on EGF- and
isoproterenol-elicited cAMP accumulation in control (V2) and AC I
transfected HEK 293 cells. Cells were labeled overnight with
H-labeled adenine and after washing incubated with medium
containing 3-isobutyl-1-methylxanthine (100 µM). Thirty
minutes later the cells were exposed to 100 nM of EGF (100
nM), isoproterenol (100 nM), or forskolin (100
µM) in the absence and presence of the ionophore A23187
(10 µM).
H-Labeled cAMP was separated from
H-labeled ATP as described under ``Experimental
Procedures.'' cAMP levels are presented as the mean ± S.E.
of four determinations and are the ratio of cAMP/(ATP + cAMP). C, effect of Ca
on the ability of EGF,
isoproterenol, and forskolin to stimulate adenylyl cyclase activity in
lysates of AC V transfectants. Lysates of HEK 293 cells transfected to
overexpress canine AC V (C-ACV(34)) were assayed for adenylyl
cyclase activity in the presence of Gpp(NH)p with and without the
addition of EGF (100 nM), isoproterenol (100 nM), or
forskolin (100 µM) as described under ``Experimental
Procedures.'' Controls (without Ca
) were
performed in the presence of EGTA (1 mM); otherwise
Ca
was added at the appropriate concentration. Data
are presented as mean ± S.D. of four
determinations.
Previous studies from this laboratory
have reported that EGF-mediated stimulation of cardiac adenylyl cyclase
activity requires the participation of G(10) .
Therefore, we investigated whether or not G
is
necessary to mediate EGF-elicited stimulation of adenylyl cyclase
activity in the AC V transfectants. The approach employed is
essentially similar to that described in our previous publication (10) and involves the use of anti-G
antiserum
directed against the carboxyl terminus decapeptide of
G
. Since the carboxyl terminus of G
is important in coupling to receptors that activate
G
(38, 39) , the anti-G
antiserum (CS1) directed against the carboxyl terminus of
G
abolishes the ability of EGF and the
-adrenergic receptor agonist, isoproterenol, to stimulate adenylyl
cyclase activity in membrane preparations(10) . The
anti-G
antiserum (CS1), however, does not alter
adenylyl cyclase activity or the coupling of G
to the
enzyme(10) . As demonstrated by the data in Fig. 4, CS1
antiserum obliterated the ability of both EGF and isoproterenol to
stimulate adenylyl cyclase activity in lysates of HEK 293 cells
overexpressing rat AC V; in control experiments with non-immune serum,
the ability of either EGF or isoproterenol to stimulate adenylyl
cyclase was not altered (Fig. 4). Results similar to those in Fig. 4were also observed with the canine AC V transfectants (not
shown). Moreover, consistent with the notion that G
participates in the actions of EGF on adenylyl cyclase, in the
absence of the GTP analog, Gpp(NH)p, neither EGF nor isoproterenol
significantly stimulated enzyme activity in AC V transfectants (Fig. 4). These findings, therefore, demonstrate that
G
is involved in the actions of EGF on AC V and are
consistent with our previous findings in rat heart membranes (10) .
Figure 4:
G is involved in the
activation of AC V by EGF. Cell lysates (500 µg of protein)
prepared from canine AC V transfectants were incubated for 60 min on
ice with 20 µg each of either anti-G
antiserum (CS1) or non-immune serum (NIS) as described
previously(10) . Adenylyl cyclase activity was then measured in
the presence and absence of Gpp(NH)p (10 µM) with and
without the addition of 100 nM EGF or isoproterenol. Data are
presented as mean ± S.E. (n =
4).
The data presented in this communication demonstrate
that the expression of AC V is required to observe EGF-elicited
stimulation of adenylyl cyclase activity and cAMP accumulation in HEK
293 cells. The naive (untransfected) HEK 293 cells express AC II, AC
III, AC VI, and AC VIII isoforms (40) and do not respond to EGF
by increasing cAMP accumulation (see e.g. data with control
vector transfectants, Fig. 2and Fig. 3). Although the
predominant adenylyl cyclase isoforms in the heart are AC V and AC
VI(19, 20, 21, 22) , overexpression
of the AC VI in HEK 293 cells does not allow EGF to stimulate adenylyl
cyclase activity. Thus in the hearts, the stimulation of adenylyl
cyclase by EGF (9, 10, 32) must represent
activity of the AC V isoform. As evident from the data in Fig. 4, the stimulatory GTP-binding protein of adenylyl cyclase,
G, is involved in the actions of EGF on AC V. However,
since G
activates all AC isoforms used in our
experiments(11) , our data would suggest that the integration
of the signal by AC V involves a regulatory component in addition to
G
, which specifically allows stimulation of this
isoform. In this respect, it should be noted that although AC I is
activated by G
, isoproterenol only stimulates cAMP
accumulation in cells expressing AC I if intracellular Ca
concentration is also elevated (Fig. 3B and (36) ). Similarly, maximal activation by G
of
AC VI is observed in the presence of forskolin (Fig. 2B and (35) ) and of AC II in the presence of
subunits(15) , respectively. Moreover, although AC I and AC VI
activities are not directly altered by phorbol esters, which activate
protein kinase C, in HT4 cells that express AC I and AC VI,
pretreatment with phorbol esters augments the stimulation of adenylyl
cyclase activity by
-adrenergic receptor agonists(41) .
Thus, it is tempting to speculate that the activation of the EGF
receptor modifies the AC V, perhaps by phosphorylation, so that the
subsequent responsiveness to G
is enhanced (i.e. increased coupling between G
and catalytic
subunit of adenylyl cyclase). In this manner, AC V may integrate
different signals and work as a ``coincidence detector'' (see e.g.(42) ). Indeed a precedent for alteration in
activities of AC isoforms by phosphorylation has been established by
the studies, which have demonstrated that phosphorylation of AC II by
protein kinase C (17) augments the activity of this isoform and
also increases the responsiveness of AC II to stimulators of its
activity (17, 18) . Similarly, in a recent
study(43) , AC V has been demonstrated to be phosphorylated and
inactivated by cAMP-dependent protein kinase. Whether or not AC V is
also phosphorylated by the EGF receptor protein-tyrosine kinase or
another kinase activated by the EGF receptor and if such a modification
increases the coupling of AC V with G
is presently unknown
and forms the subject of future investigations.