Identification of RGS2 and type V adenylyl cyclase interaction sites

The production of cAMP is controlled on many levels, notably at the level of cAMP synthesis by the enzyme adenylyl cyclase. We have recently identified a new regulator of adenylyl cyclase activity, RGS2, which decreases cAMP accumulation when overexpressed in HEK293 cells and inhibits the in vitro activity of types III, V, and VI adenylyl cyclase. In addition, RGS2 blocking antibodies lead to elevated cAMP levels in olfactory neurons. Here we examine the nature of the interaction between RGS2 and type V adenylyl cyclase. In HEK293 cells expressing type V adenylyl cyclase, RGS2 inhibited Galpha(s)-Q227L- or beta(2)-adrenergic receptor-stimulated cAMP accumulation. Deletion of the N-terminal 19 amino acids of RGS2 abolished its ability to inhibit cAMP accumulation and to bind adenylyl cyclase. Further mutational analysis indicated that neither the C terminus, RGS GAP activity, nor the RGS box domain is required for inhibition of adenylyl cyclase. Alanine scanning of the N-terminal amino acids of RGS2 identified three residues responsible for the inhibitory function of RGS2. Furthermore, we show that RGS2 interacts directly with the C(1) but not the C(2) domain of type V adenylyl cyclase and that the inhibition by RGS2 is independent of inhibition by Galpha(i). These results provide clear evidence for functional effects of RGS2 on adenylyl cyclase activity that adds a new dimension to an intricate signaling network.     

Protein kinase C activation reduces microglial cyclic AMP response to prostaglandin E2 by interfering with EP2 receptors

We studied the modulation by protein kinase C (PKC) of the cyclic AMP (cAMP) accumulation induced by prostaglandin (PG) E2 in rat neonatal microglial cultures. Short pretreatment of microglia with phorbol 12-myristate 13-acetate (PMA) or 4beta-phorbol 12,13-didecanoate, which activate PKC, but not with the inactive 4alpha-phorbol 12,13-didecanoate, substantially reduced cAMP accumulation induced by 1 microM PGE2. The action of PMA was dose and time dependent, and the maximal inhibition (approximately 85%) was obtained after 10-min preincubation with 100 nM PMA. The inhibitory effect of PMA was mimicked by diacylglycerol and was prevented by the PKC inhibitor calphostin C. As PMA did not affect isoproterenol- or forskolin-stimulated cAMP accumulation, we investigated whether activation of PKC decreased cAMP production by acting directly at PGE2 EP receptors. Neither sulprostone (10(-9)-10(-5) M), a potent agonist at EP3 receptors (coupled to adenylyl cyclase inhibition), nor 17-phenyl-PGE2 (10(-6)-10(-5) M), an agonist of EP1 receptors, modified cAMP accumulation induced by forskolin. On the contrary, 11-deoxy-16,16-dimethyl PGE2, which does not discriminate between EP2 and EP4 receptors, both coupled to the activation of adenylyl cyclase, and butaprost, a selective EP2 agonist, induced a dose-dependent elevation of cAMP that was largely reduced by PMA pretreatment, as in the case of PGE2. These results indicated EP2 receptors as a possible target of PKC and suggest that PKC-activating agents present in the pathological brain may prevent the cAMP-mediated microglia-deactivating function of PGE2.     

Regulation by butyrate of the cAMP response to cholera toxin and forskolin in pituitary GH1 cells

In pituitary GH1 cells, a rat growth hormone-producing cell line, butyrate elicited a dose-dependent increase in cholera toxin receptors as measured by an increased binding of 125I-labeled cholera toxin to the intact cells. Butyrate did not alter the affinity of cholera toxin binding, the dissociation constant being 0.4 nM for both control and butyrate-treated cells. Despite the increased binding, the cAMP response to cholera toxin was strongly reduced after exposure to butyrate. This reduction was dose-dependent and with butyrate 1--5 mM, intracellular and extracellular (medium) cAMP levels were decreased by more than 70% in cells incubated for 24 h with 1 nM cholera toxin. Forskolin (30 microM) elicited a cAMP response similar to that found with the toxin, and a similar inhibition of cAMP was also found after incubation of GH1 cells with butyrate. Butyrate also affected basal cAMP levels which were reduced by 40--60% in cells cultured for 24--48 h with the fatty acid. In order to study whether butyrate influenced cAMP synthesis and/or cAMP degradation, adenylyl cyclase and phosphodiesterase activities were determined in control cells and in cells incubated for 24 h with cholera toxin or forskolin. Butyrate had a dual effect since, besides activating phosphodiesterase by more than twofold, it also inhibited the cyclase by 40--50% in all groups. The in vitro response of adenylyl cyclase to stimulatory (NaF) and inhibitory (carbachol and adenosine) effectors was also examined. The absolute activity of the cyclase was always 40--50% lower in the cells incubated with butyrate, but the percentage change of activity obtained in butyrate-treated and untreated cells was unaltered. In addition, ADP-ribosylation of the guanine nucleotide stimulatory component of the cyclase (Gs) was not affected in the cells incubated with butyrate. These results suggest that the catalytic (C) subunit of adenylyl cyclase and/or its interaction with the regulatory components might be altered in butyrate-treated GH1 cells. The inhibition of the cAMP response in GH1 cells was accompanied by an inhibition of a biological action of the nucleotide, namely growth hormone (somatotropin) production which is primarily controlled by thyroid hormones in these cells. Forskolin alone did not affect the somatotropin levels but potentiated the growth hormone response to triiodothyronine. Butyrate produced a dose-dependent inhibition of this response, which was totally abolished at concentrations of butyrate higher than 1 mM.     

Benzyloxybenzaldehyde analogues as novel adenylyl cyclase activators.

Several benzyloxybenzaldehyde analogues were prepared and found to have significant inhibitory activity toward neutrophil superoxide formation. Consequently, these compounds were evaluated for cAMP-elevating capability. Among them, benzyloxybenzaldehyde (7), exhibiting activity equivalent to forskolin, was determined as an adenylyl cyclase activator since it elevates cAMP levels by activation of adenylyl cyclase but not by inhibition of phosphodiesterase. Having a chemical structure very different from known adenylyl cyclase activators, compound 7 is recommended by us for use as a new lead compound in the future development of adenylyl cyclase activators.     

Prostaglandin E2 can bimodally inhibit and stimulate the epididymal adipocyte adenylyl cyclase activity.

Measurements of prostaglandin E2 (PGE2)-induced adenylyl cyclase activity in membranes isolated from epididymal rat adipocytes revealed inhibition of cAMP production at low concentrations of PGE2 (less than 10 mM) and stimulation at higher concentrations. This biphasic effect of PGE2 was obtained when adenylyl cyclase was stimulated with GTP or NaF. In the presence of forskolin only the inhibitory phase by PGE2 was observed. Sulprostone, a PGE2 analogue, did not affect cAMP synthesis in the presence of either GTP or NaF; however, in the presence of forskolin, it inhibited cAMP production similarly to PGE2. Treatment of the membranes with cholera or pertussis toxin did not alter the biphasic effect of PGE2 on cAMP production. These findings raise the possibility that PGE2 acts through several receptor subtypes which are coupled to GTP binding proteins different from the classical Gi or Gs proteins.     

Differential targeting of beta -adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway

Differential modes for beta(1)- and beta(2)-adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdomains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in cardiomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in beta(2)-ARs, Galpha(i), protein kinase A RIIalpha subunits, caveolin-3, and flotillins (caveolin functional homologues); beta(1)-ARs, m(2)-muscarinic cholinergic receptors, Galpha(s), and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RIalpha subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface beta(2)-ARs localize to caveolae in cardiomyocytes and cardiac fibroblasts (with markedly different beta(2)-AR expression levels), indicating that the fidelity of beta(2)-AR targeting to caveolae is maintained over a physiologic range of beta(2)-AR expression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of beta(2)-ARs (but not beta(1)-ARs) in caveolae. Other studies show co-immunoprecipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. However, caveolin is not required for adenylyl cyclase targeting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Nevertheless, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accumulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domain-specific differences in the stoichiometry of individual elements in the beta-AR signaling cascade represent important modifiers of cAMP-dependent signaling in the heart.     

Positive coupling of atypical adenosine A3 receptors on human eosinophils to adenylyl cyclase

Adenosine A(3) receptors are reported to couple negatively to adenylyl cyclase (AC) but their mediation of anti-inflammatory effects in human eosinophils prompted us to investigate their coupling to AC. The A(3)-selective agonists IB-MECA and Cl-IB-MECA evoked a concentration-dependent generation of cAMP (EC(50), 3.2 and 1.8 microM, respectively) and were more potent than the A(2A) agonist CGS 21680 (EC(50)=15.4 microM) and adenosine (EC(50)=19.2 microM). The cAMP response was additive to that produced by forskolin (10 microM). The effect of IB-MECA was insensitive to A(1) and A(2A) receptor antagonists, but was antagonized by the A(3)-selective antagonist MRS 1220 (0.1-2.5 microM) in a competitive manner. The estimated K(B) of 190 nM was, however, atypical. The cyclo-oxygenase inhibitor, indomethacin, had no effect on the cAMP response. A general inverse relationship between cAMP generation and inhibition of degranulation was seen. We conclude that in human eosinophils, an atypical form of A(3) receptors positively coupled to AC may exist. The resulting cAMP generation may underlie the anti-inflammatory actions of A(3) agonists in eosinophils.     

Cyclic AMP-Independent Inhibition of Voltage-Sensitive Calcium Channels by Forskolin in PC12 Cells

Abstract: Forskolin has been used to stimulate adenylyl cyclase. However, we found that forskolin inhibited voltage-sensitive Ca²⁺ channels (VSCCs) in a cyclic AMP (cAMP)-independent manner in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ was inhibited when cells were preincubated with 10 µ M forskolin. Almost maximum inhibitory effect on Ca²⁺ influx without any significant increase in cellular cAMP level was observed in PC12 cells exposed to forskolin for 1 min. In addition, the forskolin effect on Ca²⁺ influx was not affected by the presence of 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase that reduces dramatically forskolin-induced cAMP production. 1,9-Dideoxyforskolin, an inactive analogue of forskolin, also inhibited ∼80% of Ca²⁺ influx induced by 70 m M K⁺ without any increase in cAMP. The data suggest that forskolin and its analogue inhibit VSCCs in PC12 cells and that the inhibition is independent of cAMP generation.     

Interaction between brain natriuretic peptide and atrial natriuretic peptide in caecal circular smooth muscle cells

Guinea pig caecal circular smooth muscle cells were used to determine whether brain natriuretic peptide (BNP) can inhibit the contractile response produced by cholecystokinin-octapeptide (CCK-8). In addition, we examined the effect of an inhibitor of cAMP-dependent protein kinase, an inhibitor of particulate or soluble guanylate cyclase, an atrial natriuretic peptide (ANP) antagonist (ANP 1-11), and selective receptor protection on the BNP-induced relaxation of these muscle cells. The effect of BNP on cAMP formation was also examined. BNP inhibited the contractile response produced by CCK-8 in a dose-response manner, with an IC50 value of 8.5 nM, and stimulated the production of cAMP. The inhibitor of cAMP-dependent protein kinase and the inhibitor of soluble guanylate cyclase significantly inhibited the relaxation produced by BNP. In contrast, the inhibitor of particulate guanylate cyclase did not have any significant effect on the relaxation produced by BNP. ANP 1-11 significantly but partially inhibited the relaxation produced by BNP. The muscle cells where CCK-8 and ANP binding sites were protected completely preserved the inhibitory response to ANP, but partially preserved the inhibitory response to BNP. The muscle cells where CCK-8 and BNP binding sites were protected completely preserved the inhibitory response to both ANP and BNP. This study demonstrates that BNP induces relaxation of these muscle cells via both ANP binding sites coupled to soluble guanylate cyclase and distinct BNP binding sites coupled to adenylate cyclase.     

Hormone inhibition of adenylyl cyclase. Differences in the mechanisms for inhibition by hormones and G protein beta gamma

S49 mouse lymphoma cells contain a beta-adrenergic receptor coupled to Gs that stimulates adenylyl cyclase and a somatostatin receptor coupled to Gi that inhibits adenylyl cyclase. Membranes from these cells were used to compare the inhibitory effects of somatostatin and G protein beta gamma complex to determine under what conditions beta gamma is likely to be a mediator of somatostatin action. Somatostatin was equally effective at inhibiting basal adenylyl cyclase activity in the presence of GTP, forskolin-stimulated activity, and hormone-stimulated activity. G protein beta gamma was more effective at inhibiting basal activity than was somatostatin, and these effects were partially additive. In the presence of forskolin, the two inhibitors were equally effective and not additive. In the presence of isoproterenol, beta gamma was much less effective than somatostatin, and the two inhibitors did not have additive or synergistic effects. At very high concentrations beta gamma did inhibit isoproterenol stimulation of adenylyl cyclase, although its effects were not saturating even at 100 micrograms/ml. Under conditions where beta gamma did inhibit hormone stimulation, beta gamma was a mixed inhibitor of isoproterenol stimulation, proportionally decreasing the maximum effect of the hormone and increasing the half-maximally effective concentration. Somatostatin, on the other hand, was a simple noncompetitive inhibitor of isoproterenol stimulation. These results indicate that beta gamma and somatostatin inhibit adenylyl cyclase by different mechanisms, at least in the presence of hormones that stimulate the enzyme. It is proposed that alpha i is the primary mediator of hormone inhibition of adenylyl cyclase when Gs is activated by a hormone, but that beta gamma may have a role as a mediator of inhibition of basal activity.     

Acetylcholine and cholecystokinin receptors functionally couple by different G-proteins to phospholipase C in pancreatic acinar cells

We have studied the involvement of GTP-binding proteins in the stimulation of phospholipase C from rat pancreatic acinar cells. Pretreatment of permeabilized cells with activated cholera toxin inhibited both cholecystokinin-octapeptide (CCK-OP) and GTPγS but not carbachol (CCh)-induced production of inositol trisphosphate. Pertussis toxin had no effect. Neither vasoactive intestinal polypeptide, a stimulator of adenylyl cyclase, nor the cAMP-analogue, 8-bromo cAMP, mimicked the inhibitory effect of cholera toxin on agonist-induced phospholipase C activation. This indicates that inhibition by cholera toxin could not be attributed to a direct interaction of cholera toxin activated G_s with phospholipase C or to an elevation of cAMP. In isolated rat pancreatic plasma membranes cholera toxin ADP-ribosylated a 40 kDa protein, which was inhibited by CCK-OP but not by CCh. We conclude from these data that both CCK- and muscarinic acetylcholine receptors functionally couple to phospholipase C by two different GTP-binding proteins.     

Removal of G(ialpha1) constraints on adenylyl cyclase in the hippocampus enhances LTP and impairs memory formation

Stimulation of adenylyl cyclase in the hippocampus is critical for memory formation. However, generation of cAMP signals within an optimal range for memory may require a balance between stimulatory and inhibitory mechanisms. The role of adenylyl cyclase inhibitory mechanisms for memory has not been addressed. One of the mechanisms for inhibition of adenylyl cyclase is through activation of G(i)-coupled receptors, a mechanism that could serve as a constraint on memory formation. Here we report that ablation of G(ialpha1) by gene disruption increases hippocampal adenylyl cyclase activity and enhances LTP in area CA1. Furthermore, gene ablation of G(ialpha1) or antisense oligonucleotide-mediated depletion of G(ialpha1) disrupted hippocampus-dependent memory. We conclude that G(ialpha1) provides a critical mechanism for tonic inhibition of adenylyl cyclase activity in the hippocampus. We hypothesize that loss of G(ialpha1) amplifies the responsiveness of CA1 postsynaptic neurons to stimuli that strengthen synaptic efficacy, thereby diminishing synapse-specific plasticity required for new memory formation.     

Pertussis-toxin insensitive enkephalin inhibition of adenylyl cyclase in lacrimal acinar cells

The mechanism by which the inhibitory effect of d-ala²-met-enkephalinamide (DALA) on lacrimal acinar adenylyl cyclase is exerted was assessed in membrane preparations by a cAMP protein binding assay. Inhibition by the analogue was GTP-dependent with a significant enhancement of the inhibitory effect by GTP. While pretreatment of membranes with either cholera or pertussis toxin resulted in stimulation of adenylyl cyclase activity, modification of the G_iα subunit by pertussis-toxin catalyzed ADP-ribosylation did not effect the hormonal inhibition of adenylyl cyclase. Incubation of membranes with manganese, however, prevented the inhibitory action of DALA in addition to enhancing basal and forskolin-stimulated adenylyl cyclase activity. The results suggest that the inhibitory effect of DALA in lacrimal acinar cells is exerted via a mechanism other than pertussis-toxin sensitive coupling of the receptor to adenylyl cyclase through G_i. The mechanism may be effected through a pertussis-toxin insensitive G protein, through an interaction with G_i that is pertussis-toxin insensitive, or through an interaction with the catalytic subunit of adenylyl cyclase.     

Inhibition of fetal rat pancreatic beta-cell replication by interleukin-1 beta in vitro is not mediated through pertussis toxin-sensitive G-proteins, a decrease in cyclic AMP, or protease activation.

It has been proposed that the cytokine interleukin-1 beta (IL-1 beta), secreted by islet-infiltrating macrophages, may be involved in the pathogenesis of insulin-dependent diabetes mellitus by participation in beta-cell destruction. Addition of IL-1 beta to isolated pancreatic islets in vitro results in cytotoxic effects on beta-cell function, but there is little information on the intracellular events that convey the actions of the cytokine. In the present study, fetal rat pancreatic islets containing a high fraction of beta-cells were exposed in culture to IL-1 beta. It was found that IL-1 beta markedly decreased beta-cell DNA synthesis, insulin secretion and cyclic AMP content. In order to explore whether the decrease in cAMP resulted from IL-1 beta interaction with GTP-binding proteins coupled to adenylyl cyclase, islets were treated for 24 h with pertussis toxin prior to addition of cytokine. While this treatment restored the decrease in cAMP, the reduced DNA synthesis and insulin secretion persisted. Pertussis toxin treatment without the addition of IL-1 beta resulted in increases in cAMP, DNA synthesis and insulin secretion. Addition of the stimulatory cAMP analog Sp-cAMPS also increase DNA synthesis and insulin secretion, but failed to affect the decrease in these functions evoked by IL-1 beta. The protease inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone, recently shown to protect completely against IL-1 beta-induced suppression of insulin production and secretion, was found to markedly reduce DNA synthesis without affecting insulin secretion. When the protease inhibitor was combined with IL-1 beta, the suppressed secretion was counteracted while DNA synthesis inhibition was not. It is concluded that cAMP stimulates DNA synthesis and insulin secretion in beta-cells, but that the inhibitory effect of IL-1 beta on these functions cannot be ascribed to the decrease in cAMP evoked by the cytokine. However, the repressive effect of the cytokine on insulin secretion, but not DNA synthesis, may be prevented by protease inhibition.     

The thrombin receptor in adrenal medullary microvascular endothelial cells is negatively coupled to adenylyl cyclase through a Gi protein

The effects of thrombin on adenylyl cyclase activity were examined in rat adrenal medullary microvascular endothelial cells (RAMEC). Confluent RAMEC monolayers were stimulated for 5 min with cAMP-generating agents in the absence and presence of thrombin, and intracellular cAMP was measured with a radioligand binding assay. Thrombin (0.001–0.25 U/ml) dose-dependently inhibited IBMX-, isoproterenol- and forskolin-stimulated cAMP accumulation. A peptide agonist of the thrombin receptor, γ-thrombin, and the serine proteases trypsin and plasmin, also inhibited agonist-stimulated cAMP levels, while proteolytically inactive PPACK- or DIP-α-thrombins were without effect. Moreover, the thrombin inhibitor hirudin abolished the inhibitory effect of thrombin but not of the peptide agonist. These results suggest that the inhibitory action of thrombin on cAMP accumulation is mediated by a proteolytically-activated thrombin receptor. The inhibitor of G_i-proteins pertussis toxin abolished the inhibitory effect of thrombin on isoproterenol- or IBMX-stimulated cAMP production, while the phorbol ester PMA partly impaired it. The protein kinase C inhibitors staurosporine or H7 and the intracellular Ca²⁺ chelator BAPTA-AM were without effect. Collectively, our data suggest that the thrombin receptor in RAMEC is negatively coupled to adenylyl cyclase through a pertussis toxin-sensitive G_i-protein.     

Effector coupling mechanisms of the cloned 5-HT1A receptor

The signal transduction pathways of the cloned human 5-HT1A receptor have been examined in two mammalian cell lines transiently (COS-7) or permanently (HeLa) expressing this receptor gene. In both systems, 5-hydroxytryptamine (5-HT, serotonin) mediated a marked inhibition of beta 2-adrenergic agonist-stimulated (80% inhibition in COS-7 cells) or forskolin-stimulated cAMP formation (up to 90% inhibition in HeLa cells). This serotonin effect (EC50 = 20 nM) could be competitively antagonized by metitepine and spiperone (Ki = 81 and 31 nM, respectively) and could also be blocked by pretreatment of cells with pertussis toxin. In both cell types, 5-HT failed to stimulate adenylyl cyclase through the expressed receptors. In HeLa cells, 5-HT also stimulated phospholipase C (approximately 40-75% stimulation of formation of inositol phosphates). Again, this effect was inhibited by metitepine. However, the EC50 of 5-HT was considerably higher (approximately 3.2 microM) than that found for inhibition of adenylyl cyclase. Both pathways were demonstrated to be similarly affected by pertussis toxin. These findings indicate that like the M2 and M3 muscarinic cholinergic receptors, the 5-HT1A receptor can couple to multiple transduction pathways with varying efficiencies via pertussis toxin-sensitive G-proteins. The lack of stimulation of cAMP formation by this 5-HT1A receptor may suggest the existence of another pharmacologically closely related receptor.