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.

EXPERIMENTAL PROCEDURES

Transfection of HEK 293 Cells and Selection of Stable Lines

Measurements of Adenylyl Cyclase Activity and cAMP Formation in Cells

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) .

Assay for Functional EGF Receptors

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) .

Materials

RESULTS AND DISCUSSION

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.

FOOTNOTES

*

This work was supported in part by National Institutes of Health Grant HL48308 and by a grant from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported by National Institutes of Health Training Grant HL 07641.

¶

To whom correspondence should be addressed: Dept. of Pharmacology, University of Tennessee, The Center for Health Sciences, 874 Union Ave., Memphis, TN 38163. Tel.: 901-448-6006; Fax: 901-448-7300.

()

The abbreviations used are: EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis; AC, adenylyl cyclase; G-protein, GTP-binding regulatory protein; G, stimulatory GTP-binding regulatory protein of adenylyl cyclase; G, subunit of G; Gpp(NH)p, guanyl-5`-yl imidodiphosphate; GTPS, guanosine 5`-3-O-(thio)triphosphate.

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