Regulation of guanylate cyclase by a guanine nucleotide binding protein, G alpha 2, in Dictyostelium discoideum.

Binding of cyclic AMP (cAMP) to the cell surface receptor induces a transient activation of guanylate cyclase in Dictyostelium discoideum. A frigid mutant (HC85) which lacks G alpha 2, a guanine nucleotide binding protein, does not respond to cAMP. We found that 2,3-dimercapto-1-propanol (BAL) induced a continuous activation both in the frigid and in its parents. Therefore, the BAL-induced continuous activation of guanylate cyclase is independent of G alpha 2. We also found that cAMP enhanced the BAL-induced continuous activation in the frigid mutant. This result suggests that an unidentified signal transduction mechanism from the cAMP-receptor besides the one involving G alpha 2 plays a role in the enhancement of activation. Lastly, we found that the BAL-induced continuous activation was terminated by cAMP in the parental strain, but not in the frigid mutant. Therefore, the cAMP-induced suppression on the BAL-induced continuous activation is mediated through G alpha 2.     

Reducing reagent-induced activation of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum.

Binding of an intrinsic agonist (cAMP) to specific receptors on the cell surface induces transmembrane signals for activation and desensitization (adaptation and down regulation) of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum. It is generally believed that dithiothreitol (DTT) induces the activation through interaction between the receptor and gradually accumulated cAMP, since DTT is known to inhibit cAMP-phosphodiesterase which degrades cAMP. In the present paper, we investigated the mechanism of activation of adenylate cyclase by the thiol-reducing agents, DTT and 2,3-dimercapto-1-propanol (BAL). We found that BAL activated adenylate cyclase transiently even under conditions where the intrinsic agonist supersaturated the cAMP-receptors and competitively inhibited phosphodiesterase. This result is inconsistent with the generally accepted notion. We conclude that BAL has an independent effect from those of the intrinsic agonist (cAMP) and phosphodiesterase in activation of adenylate cyclase. Since BAL could induce activation just after the activation induced by a supersaturating concentration of the intrinsic agonist had ceased, the independent effect of BAL is not a simple enhancement of the cAMP-induced activation. Our result also suggests that the cAMP-induced adaptation (but not down regulation) suppresses the BAL-induced activation while BAL itself does not induce adaptation to cAMP or BAL. We propose that the thiol-reducing reagent induces or modifies the transmembrane activation signal for adenylate cyclase.     

Reducing reagent-induced activation of guanylate cyclase in the cellular slime mold, Dictyostelium discoideum.

Binding of folic acid (an intrinsic agonist) to the cell surface receptors evokes transmembrane signals for activation and adaptation of guanylate cyclase in Dictyostelium discoideum. The activation signal activates this enzyme and then the adaptation signal terminates the activation. As a result, these two signals cooperatively induce a transient activation of guanylate cyclase. We investigated transmembrane signal transduction for guanylate cyclase using 2,3-dimercapto-1-propanol (BAL, a thiol-reducing reagent) since BAL induces or modifies the transmembrane signal(s). We found that BAL induced prolonged or continuous activation of guanylate cyclase. Thus, the mode of the activation is drastically different (transient versus continuous) between folic acid and BAL. We also found that the BAL-induced continuous activation was not observed when the cells were stimulated with BAL + folic acid, while folic acid + BAL transiently induced more cGMP accumulation than folic acid alone. We lastly showed that K252a, a protein kinase inhibitor, enhanced both the folic acid-induced and the BAL-induced activation of guanylate cyclase. Our results suggest that BAL induces or mimics the activation signal for guanylate cyclase. The lack of termination in the BAL-induced activation suggests that BAL does not induce the adaptation signal or that the adaptation does not inhibit the BAL-induced activation. The former possibility is more likely since folic acid suppresses the BAL-induced continuous activation. The effect of K252a suggests that protein phosphorylation plays a role in suppression of guanylate cyclase.     

Adenosine 3', 5'-monophosphorothioate (Rp-isomer) induces down-regulation of surface cyclic AMP receptors without receptor activation in Dictyostelium discoideum

cAMP induces the activation and subsequent desensitization of adenylate cyclase in Dictyostelium discoideum. cAMP also induces down-regulation of surface cAMP receptors. Desensitization of adenylate cyclase is composed of a rapidly reversible component (adaptation) and a slowly reversible component related to down-regulation of surface cAMP receptors (Van Haastert, P.J.M. (1987) J. Biol. Chem. 262, 7700-7704). The agonistic and antagonistic activities of the cAMP derivative adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) for these responses were investigated. (Rp)-cAMPS competes with cAMP for binding to different receptor forms with an apparent Ki = 5 microM. (Rp)-cAMPS does not activate adenylate cyclase and antagonizes the cAMP-induced activation with an apparent Ki = 5 microM. (Rp)-cAMPS induces down-regulation of surface cAMP receptors with EC50 = 5 microM. (Rp)-cAMPS induces desensitization of adenylate cyclase, which is not rapidly reversible. These results indicate that desensitization of adenylate cyclase by (Rp)-cAMPS is due to down-regulation of surface cAMP receptors and not to adaptation. We conclude that down-regulation of surface cAMP receptors does not require their activation or modification involved in adaptation.     

Inhibition by EDTA and enhancement by divalent cations or polyamines of the dithiothreitol-induced activation of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum.

Binding of cAMP to cell surface receptors evokes the transient activation of of adenylate cyclase in Dictyostelium discoideum. Dithiothreitol is also known as an activator of this enzyme. We found that the dithiothreitol-induced activation was specifically enhanced by extracellular polyamines or divalent cations. Furthermore, EDTA, a chelating agent of divalent cations, completely inhibited the dithiothreitol-induced activation of adenylate cyclase while EDTA did not inhibit the cAMP-induced activation. The inhibition was nullified by addition of polyamines or divalent cations. These results suggest that extracellular polyamines and divalent cations play a specific role in the dithiothreitol-induced activation of adenylate cyclase.     

cAMP regulation of early gene expression in signal transduction mutants of Dictyostelium

We have examined the regulation of three early developmentally regulated genes in Dictyostelium. Two of these genes (D2 and M3) are induced by pulses of cAMP and the other (K5) is repressed. Expression of these genes has been examined in a number of developmental mutants that are specifically blocked in various aspects of the signal transduction/cAMP relay system involved in aggregation and control of early development. The mutant strains include Synag mutants, which are blocked in receptor-mediated activation of adenylate cyclase and do not relay cAMP pulses; FrigidA mutants, which are blocked in receptor-mediated activation of both adenylate cyclase and the putative phosphoinositol bisphosphate (PIP2) turnover pathway and appear to be mutations in the gene encoding one of the G alpha protein subunits; and a StreamerF allele, which lacks cGMP-specific cGMP phosphodiesterase. From the analysis of the developmental expression of these genes under a variety of conditions in these mutant strains, we have drawn a number of conclusions concerning the modes of regulation of these genes. Full induction of D2 and M3 genes requires cAMP interaction with the cell surface receptor and an "oscillation" of the receptor between active and adapted forms. Induction of these genes does not require activation of the signal transduction pathway that leads to adenylate cyclase activation and cAMP relay, but does require activation of other receptor-mediated intracellular signal transduction pathways, possibly that involving PIP2 turnover. Likewise, repression of the K5 gene requires pulses of cAMP. Expression of this gene is insensitive to cAMP pulses in FrigidA mutants, suggesting that a signal transduction pathway is necessary for its repression. Results using the StreamerF mutant suggest that the rise in cGMP in response to cAMP/receptor interactions may not be directly related to control of the pulse-induced genes. In addition, we have examined the effect of caffeine, which M. Brenner and S.D. Thomas (1984, Dev. Biol., 101, 136-146) showed preferentially blocks the cAMP relay system by blocking receptor-mediated activation of adenylate cyclase. We show that in many of the mutants and in an axenic wild-type strain, caffeine causes the induction of pulse-induced gene expression to almost wild-type levels or in some cases to higher than wild-type levels. Our data suggest that caffeine works by activating some step in the signal transduction pathway that must lie downstream from both the receptor and at least one of the G proteins and thus has effects other than simply blocking the receptor-mediated cAMP relay system.     

Molecular genetic analysis of two G alpha protein subunits in Dictyostelium.

In Dictyostelium, chemotaxis to folate during growth and cAMP during aggregation is controlled via cell surface receptors. To study the role of two G alpha proteins (G alpha 1 and G alpha 2) in these responses, we examined the physiological and biochemical effects of null mutations caused by antisense mutagenesis and gene disruptions. Disruption of G alpha 2 results in an aggregation-deficient phenotype and a loss of cAMP receptor-mediated functions, including activation of adenylate cyclase, guanylate cyclase, and gene expression and in a loss of GTP-mediated decrease in receptor affinity for cAMP, but it has no effect on chemotaxis to folate or folate activation of guanylate cyclase. These phenotypes can be rescued by a vector expressing G alpha 2, suggesting G alpha 2 is coupled to a cAMP receptor but not to folate receptors. Loss of G alpha 1 expression resulted in no visible growth or developmental phenotype, including cAMP- and folate-stimulated responses, suggesting G alpha 1 function is either not essential under standard laboratory conditions or is encoded by multiple genes. Availability of null mutations provides suitable genetic backgrounds for expressing mutant G alpha protein subunits which can then be used to examine the physiological roles of G alpha 1 and G alpha 2. Construction of these gene disruptions was facilitated by using the auxotrophic marker THY1, which allowed for selection of single-copy insertions into the genome.     

Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium.

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.     

Protein kinase C inhibits BKCa channel activity in pulmonary arterial smooth muscle

Signaling mechanisms that elevate cyclic AMP (cAMP) activate large-conductance, calcium- and voltage-activated potassium (BKCa) channels in pulmonary vascular smooth muscle and cause pulmonary vasodilatation. BKCa channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BKCa channel has been implicated in the development of pulmonary vasoconstriction. Protein kinase C (PKC) causes pulmonary vasoconstriction, but little is known about the effect of PKC on BKCa channel activity. Accordingly, studies were done to determine the effect of PKC activation on cAMP-induced BKCa channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), a recognized animal model of pulmonary hypertension. Forskolin (10 microM), a stimulator of adenylate cyclase and an activator of cAMP, opened BKCa channels in single FHR PASMC, which were blocked by the PKC activators phorbol 12-myristate 13-acetate (100 nM) and thymeleatoxin (100 nM). The inhibitory response by thymeleatoxin on forskolin-induced BKCa channel activity was blocked by G?-6983, which selectively blocks the alpha, beta, delta, gamma, and zeta PKC isozymes, and G?-6976, which selectively inhibits PKC-alpha, PKC-beta, and PKC-mu, but not by rottlerin, which selectively inhibits PKC-delta. Collectively, these results indicate that activation of specific PKC isozymes inhibits cAMP-induced activation of the BKCa channel in pulmonary arterial smooth muscle, which suggests a unique signaling pathway to modulate BKCa channels and subsequently cAMP-induced pulmonary vasodilatation.     

The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates,a positive effect of caffeine and potential role of microtubules

To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2βγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing.     

Dissection of the signaling mechanism for capsule detachment of lipid droplets in rat adrenocortical cells

In a previous study, we used a monoclonal antibody, A2, to demonstrate the presence of the lipid droplet-specific capsule in adrenocortical cells and the stimulation of steroid secretion with adrenocorticotrophic hormone (ACTH), resulting in the detachment of this capsule from the droplet surface into the cytosol. To investigate the signaling pathway for this event, we tested the role of adenylate cyclase, cAMP, and protein kinases A and C (PKA and PKC) in this response. ACTH-induced decapsulation of lipid droplets was blocked by either adenylate cyclase inhibitor or PKA inhibitor and stimulated by Bt₂cAMP. We conclude that the signaling mechanism involved in lipid droplet decapsulation is the cascade consisting of adenylate cyclase activation, cAMP elevation, and subsequent PKA activation. Furthermore, the cytosolic detached capsular protein was able to relocate to the lipid droplet surface on cessation of ACTH or Bt₂cAMP stimulation. In addition to PKA-mediated decapsulation, inhibition of PKC by calphostin C alone was enough to induce decapsulation, a process that was independent of PKA activity, whereas activation of PKC could prevent Bt₂cAMP-induced decapsulation. A cAMP radioimmunoassay also confirmed that ACTH caused a marked increase in intracellular levels of cAMP, while PMA or calphostin C caused no significant changes. We conclude that PKA and PKC are reciprocally operated to regulate the decapsulation of lipid droplets, the same mechanism adopted in steroidogenesis. A time-course study also indicates that decapsulation of lipid droplets was accompanied by detectable changes in the size and the area of lipid droplets upon the stimulation of Bt₂cAMP or calphostin C, implying a possible coupling between the capsule detachment and steroidogenesis. J. Cell. Biochem. 65:67–74. © 1997 Wiley-Liss, Inc.     

Activation of platelet-activating factor receptor-coupled G alpha q leads to stimulation of Src and focal adhesion kinase via two separate pathways in human umbilical vein endothelial cells

Platelet-activating factor (PAF), a phospholipid second messenger, has diverse physiological functions, including responses in differentiated endothelial cells to external stimuli. We used human umbilical vein endothelial cells (HUVECs) as a model system. We show that PAF activated pertussis toxin-insensitive G alpha(q) protein upon binding to its seven transmembrane receptor. Elevated cAMP levels were observed via activation of adenylate cyclase, which activated protein kinase A (PKA) and was attenuated by a PAF receptor antagonist, blocking downstream activity. Phosphorylation of Src by PAF required G alpha(q) protein and adenylate cyclase activation; there was an absolute requirement of PKA for PAF-induced Src phosphorylation. Immediate (1 min) PAF-induced STAT-3 phosphorylation required the activation of G alpha(q) protein, adenylate cyclase, and PKA, and was independent of these intermediates at delayed (30 min) and prolonged (60 min) PAF exposure. PAF activated PLC beta 3 through its G alpha(q) protein-coupled receptor, whereas activation of phospholipase C gamma 1 (PLC gamma 1) by PAF was independent of G proteins but required the involvement of Src at prolonged PAF exposure (60 min). We demonstrate for the first time in vascular endothelial cells: (i) the involvement of signaling intermediates in the PAF-PAF receptor system in the induction of TIMP2 and MT1-MMP expression, resulting in the coordinated proteolytic activation of MMP2, and (ii) a receptor-mediated signal transduction cascade for the tyrosine phosphorylation of FAK by PAF. PAF exposure induced binding of p130(Cas), Src, SHC, and paxillin to FAK. Clearly, PAF-mediated signaling in differentiated endothelial cells is critical to endothelial cell functions, including cell migration and proteolytic activation of MMP2.     

Surface receptor-mediated activation of adenylate cyclase in Dictyostelium. Regulation by guanine nucleotides in wild-type cells and aggregation deficient mutants

GTP and GTP analogs produced significant (up to 17-fold) and persistent activation of adenylate cyclase in lysates of Dictyostelium discoideum amoeba. The activation was enhanced 2- to 4-fold by cAMP (the agonist for receptor-mediated adenylate cyclase activation), was specific for guanine nucleoside triphosphates, and was inhibited by guanosine 5'-(O-2-thio)diphosphate. The order of potency of guanine nucleotides was guanosine 5'-(O-3-thio)triphosphate greater than guanyl-5'-yl imidodiphosphate greater than GTP; half-maximal activation was observed with 1-10 microM guanine nucleotide. Maximal activation occurred when the guanine nucleotide was added within seconds after cell lysis and the lysate was preincubated for 5 min prior to assay. Under these optimal in vitro conditions, the capacity of guanine nucleotides to activate decreased, closely correlating with adaptation or desensitization induced by exposure of intact cells to cAMP during a period of 10 min. These data strongly support that regulation of adenylate cyclase in Dictyostelium occurs via a receptor-linked GTP/GDP exchange protein. Two mutants, designated synag 7 and 49 were isolated in which cAMP and/or guanine nucleotides were not sufficient to activate adenylate cyclase. The wild-type pattern of guanine nucleotide regulation was restored to synag 7 lysates by the addition of a high-speed supernatant from wild-type cells. Characterization of these mutants demonstrates that activation of adenylate cyclase is not required for growth or cell-type specific differentiation but is essential for cellular aggregation and influences morphogenesis and pattern formation. This suggests that Dictyostelium may provide a model suitable for detailed genetic analysis of surface receptor-guanine nucleotide-binding regulatory protein linked adenylate cyclase systems and for determining the role of these systems in development.     

The regulation of adenylate cyclase by guanine nucleotides in Dictyostelium discoideum membranes

Extracellular cAMP induces the activation of adenylate cyclase in Dictyostelium discoideum cells. Conditions for both stimulation and inhibition of adenylate cyclase by guanine nucleotides in membranes are reported. Stimulation and inhibition were induced by GTP and non-hydrolysable guanosine triphosphates. GDP and non-hydrolysable guanosine diphosphates were antagonists. Stimulation was maximally twofold, required a cytosolic factor and was observed only at temperatures below 10 degrees C. An agonist of the cAMP-receptor-activated basal and GTP-stimulated adenylate cyclase 1.3-fold. Adenylate cyclase in mutant N7 could not be activated by cAMP in vivo; in vitro adenylate cyclase was activated by guanine nucleotides in the presence of the cytosolic factor of wild-type but of not mutant cells. Preincubation of membranes under phosphorylation conditions has been shown to alter the interaction between cAMP receptor and G protein [Van Haastert (1986) J. Biol. Chem. in the press]. These phosphorylation conditions converted stimulation to inhibition of adenylate cyclase by guanine nucleotides. Inhibition was maximally 30% and was not affected by the cytosolic factor involved in stimulation. In membranes obtained from cells that were treated with pertussis toxin, adenylate cyclase stimulation by guanine nucleotides was as in control cells, whereas inhibition by guanine nucleotides was lost. When cells were desensitized by exposure to cAMP agonists for 15 min, and adenylate cyclase was measured in isolated membranes, stimulation by guanine nucleotides was lost while inhibition was retained. These results suggest that Dictyostelium discoideum adenylate cyclase may be regulated by Gs-like and Gi-like activities, and that the action of Gs but not Gi is lost during desensitization in vivo and by phosphorylation conditions in vitro.     

Down-regulation of cell surface cyclic AMP receptors and desensitization of cyclic AMP-stimulated adenylate cyclase by cyclic AMP in Dictyostelium discoideum. Kinetics and concentration dependence

cAMP binds to Dictyostelium discoideum surface receptors and induces a transient activation of adenylatecyclase, which is followed by desensitization. cAMP also induces a loss of detectable surface receptors (down-regulation). Cells were incubated with constant cAMP concentrations, washed free of cAMP, and cAMP binding to surface receptors and cAMP-induced activation of adenylate cyclase were measured. cAMP could induce maximally 65% loss of binding activity and complete desensitization of cAMP-stimulated adenylate cyclase activity. Half-maximal effects for down-regulation were observed at 50 nM cAMP and for desensitization at 5 nM cAMP. Down-regulation was rapid with half-times of 4, 2.5, and 1 min at 0.1, 1, and 10 microM cAMP, respectively. Similar kinetic data have been reported for desensitization (Dinauer, M.C., Steck, T.L., and Devreotes, P.N. (1980) J. Cell Biol. 86, 554-561). Down-regulation and desensitization were not reversible at 0 degrees C. Down-regulation reversed slowly at 20 degrees C with a half-time of about 1 h. Resensitization of adenylate cyclase was biphasic showing half-times of 4 min and about 1 h, respectively; the contribution of the rapidly resensitizing component was diminished when down-regulation of receptors was enhanced. These results suggest that cAMP-induced down-regulation of receptors and desensitization of adenylate cyclase stimulation proceed by at least two steps. One step is rapidly reversible, occurs at low cAMP concentrations, and induces desensitization without down-regulation, while the second step is slowly reversible, requires higher cAMP concentrations, and also induces down-regulation.     

Regulation of adenylate cyclase in electropermeabilized Dictyostelium discoideum cells.

In Dictyostelium discoideum cells the enzyme adenylate cyclase is functionally coupled to cell surface receptors for cAMP. Coupling is known to involve one or more G-proteins. Receptor-mediated activation of adenylate cyclase is subject to adaptation. In this study we employ an electropermeabilized cell system to investigate regulation of D. discoideum adenylate cyclase. Conditions for selective permeabilization of the plasma membrane have been described by C.D. Schoen, J. C. Arents, T. Bruin, and R. Van Driel (1989, Exp. Cell Res. 181, 51-62). Only small pores are created in the membrane, allowing exchange of exclusively low molecular weight substances like nucleotides, and preventing the loss of macromolecules. Under these conditions functional protein-protein interactions are likely to remain intact. Adenylate cyclase in permeabilized cells was activated by the cAMP receptor agonist 2'-deoxy cAMP and by the nonhydrolyzable GTP-analogue GTP gamma S, which activates G-proteins. The time course of the adenylate cyclase reaction in permeabilized cells was similar to that of intact cells. Maximal adenylate cyclase activity was observed if cAMP receptor agonist or GTP-analogue was added just before cell permeabilization. If these activators were added after permeabilization adenylate cyclase was stimulated in a suboptimal way. The sensitivity of adenylate cyclase activity for receptor occupation was found to decay more rapidly than that for G-protein activation. Importantly, the adenylate cyclase reaction in permeabilized cells was subject to an adaptation-like process that was characterized by a time course similar to adaptation in vivo. In vitro adaptation was not affected by cAMP receptor agonists or by G-protein activation. Evidently electropermeabilized cells constitute an excellent system for investigating the positive and negative regulation of D. discoideum adenylate cyclase.     

Estrogen can regulate the cell cycle in the early G1 phase of yeast by increasing the amount of adenylate cyclase mRNA

The effects of beta-estradiol (estrogen; a minor component of yeast cells) on S. cerevisiae cells in the G0 and G1 phases were examined. Results showed that estrogen stimulated the recovery of growth from G0 arrest induced by nutrient limitation or ts mutation of cdc35 (adenylate cyclase) in the early G1 phase, and inhibited entry into the resting G0 phase by increasing the intracellular cAMP level. However, estrogen had no effect on late G1 arrest induced by the alpha factor or ts mutation of cdc36. Estrogen was found to lead to higher steady-state levels of adenylate cyclase mRNA but not to affect the expression of the RAS1 and RAS2 genes, although these can also alter the intracellular cAMP level. These results suggest that estrogen influences the cell cycle of yeast in the early G1 phase by controlling the level of cAMP through the increase of adenylate cyclase mRNA.