Differential regulation of adenylyl cyclases by Galphas

Regulation of adenylyl cyclases 1, 2, and 6 by Galphas was studied. All three mammalian adenylyl cyclases were expressed in insect (Sf9 or Hi-5) cells by baculovirus infection. Membranes containing the different adenylyl cyclases were stimulated by varying concentrations of mutant (Q227L) activated Galphas expressed in reticulocyte lysates. Galphas stimulation of AC1 involved a single site and had an apparent Kact of 0.9 nM. Galphas stimulation of AC2 was best explained by a non-interactive two site model with a "high affinity" site at 0.9 nM and a "low affinity" site at 15 nM. Occupancy of the high affinity site appears to be sufficient for Gbetagamma stimulation of AC2. Galphas stimulation of AC6 was also best explained by a two-site model with a high affinity site at 0. 6-0.8 nM and a low affinity site at 8-22 nM; however, in contrast to AC2, only a model that assumed interactions between the two sites best fit the AC6 data. With 100 microM forskolin, Galphas stimulation of all three adenylyl cyclases showed very similar profiles. Galphas stimulation in the presence of forskolin involved a single site with apparent Kact of 0.1-0.4 nM. These observations indicate a conserved mechanism by which forskolin regulates Galphas coupling to the different adenylyl cyclases. However, there are fundamental differences in the mechanism of Galphas stimulation of the different adenylyl cyclases with AC2 and AC6 having multiple interconvertible sites. These mechanistic differences may provide an explanation for the varied responses by different cells and tissues to hormones that elevate cAMP levels.     

Spinal nitric oxide mediates antinociception from intravenous morphine

Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. Stimulation of rat adipocytes with isoproterenol results in phosphorylation of HSL and a 50-fold increase in the rate of lipolysis. In this study, we used site-directed mutagenesis and two-dimensional phosphopeptide mapping to show that phosphorylation sites other than the previously identified Ser-563 are phosphorylated in HSL in response to isoproterenol stimulation of 32P-labeled rat adipocytes. Phosphorylation of HSL in adipocytes in response to isoproterenol and in vitro phosphorylation of HSL containing Ser --> Ala mutations in residues 563 and 565 (S563A, S565A) with protein kinase A (PKA), followed by tryptic phosphopeptide mapping resulted in two tryptic phosphopeptides. These tryptic phosphopeptides co-migrated with the phosphopeptides released by the same treatment of F654HPRRSSQGVLHMPLYSSPIVK675 phosphorylated with PKA. Analysis of the phosphorylation site mutants, S659A, S660A, and S659A,S660A disclosed that mutagenesis of both Ser-659 and Ser-660 was necessary to abolish the activation of HSL toward a triolein substrate after phosphorylation with PKA. Mutation of Ser-563 to alanine did not cause significant change of activation compared with wild-type HSL. Hence, our results demonstrate that in addition to the previously identified Ser-563, two other PKA phosphorylation sites, Ser-659 and Ser-660, are present in HSL and, furthermore, that Ser-659 and Ser-660 are the major activity controlling sites in vitro.     

Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.     

Type I adenylyl cyclase mutant mice have impaired mossy fiber long-term potentiation

Long-term potentiation (LTP) at the mossy fiber-->CA3 pyramidal cell synapse in the hippocampus is an NMDA-independent form of LTP that requires cAMP-dependent protein kinase (PKA) activity and can be induced by forskolin, a general activator of adenylyl cyclases. Presynaptic Ca2+ influx and elevated cAMP may be obligatory for mossy fiber LTP. Because the Ca2+-stimulated type 1 adenylyl cyclase (AC1) is expressed in the dentate gyrus and CA3 pyramidal cells, it is hypothesized that AC1 may be critical for mossy fiber LTP. To test this hypothesis, we examined several forms of hippocampal LTP in wild-type and AC1 mutant mice. Wild-type and AC1 mutant mice exhibited comparable perforant path LTP recorded in the dentate gyrus as well as decremental LTP at the Schaffer collateral-->CA1 pyramidal cell synapse. Although the mutant mice exhibited normal paired pulse facilitation, mossy fiber LTP was impaired significantly in AC1 mutants. High concentrations of forskolin induced mossy fiber LTP to comparable levels in wild-type and AC1 mutant mice, indicating that signaling components downstream from the adenylyl cyclase, including PKA, ion channels, and secretory machinery, were not affected by disruption of the AC1 gene. These data indicate that coupling of Ca2+ to activation of AC1 is crucial for mossy fiber LTP, most likely via activation of PKA and enhancement of excitatory amino acid secretion.     

Stimulation of type 1 and type 8 Ca2+/calmodulin-sensitive adenylyl cyclases by the Gs-coupled 5-hydroxytryptamine subtype 5-HT7A receptor

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays an important regulatory role in developing and adult nervous systems. With the exception of the 5-HT3 receptor, all of the cloned serotonin receptors belong to the G protein-coupled receptor superfamily. Subtypes 5-HT6 and 5-HT7 couple to stimulation of adenylyl cyclases through Gs and display high affinities for antipsychotic and antidepressant drugs. In the brain, mRNA for 5-HT6 is found at high levels in the hippocampus, striatum, and nucleus accumbens. 5-HT7 mRNA is most abundant in the hippocampus, neocortex, and hypothalamus. To better understand how serotonin might control cAMP levels in the brain, we coexpressed 5-HT6 or 5-HT7A receptors with specific isoforms of adenylyl cyclase in HEK 293 cells. The 5-HT6 receptor functioned as a typical Gs-coupled receptor in that it stimulated AC5, a Gs-sensitive adenylyl cyclase, but not AC1 or AC8, calmodulin (CaM)-stimulated adenylyl cyclases that are not activated by Gs-coupled receptors in vivo. Surprisingly, serotonin activation of 5-HT7A stimulated AC1 and AC8 by increasing intracellular Ca2+. 5-HT also increased intracellular Ca2+ in primary neuron cultures. These data define a novel mechanism for the regulation of intracellular cAMP by serotonin.     

Selective effects of ethanol on the generation of cAMP by particular members of the adenylyl cyclase family

A selective action of ethanol on major signal transduction proteins, such as adenylyl cyclase, has been considered to be important for certain actions of ethanol, and alcoholics have been demonstrated to differ from controls in measures of platelet adenylyl cyclase activity. Recent advances in identification and characterization of isoforms of adenylyl cyclase have demonstrated that there exists at least eight different forms of this enzyme. To examine whether the effect of ethanol on generation of cAMP is modified by the presence of particular isoforms of adenylyl cyclase within a cell, we transiently expressed each of six adenylyl cyclases in human embryonic kidney (HEK293) cells and measured cAMP accumulation in whole cells in the presence and absence of ethanol. The treatment of cells expressing the various adenylyl cyclases with ethanol alone did not enhance cAMP generation. In the presence of prostaglandin E1, cAMP generation by type I and type III adenylyl cyclases was insensitive to ethanol. cAMP accumulation generated by the other adenylyl cyclases was, however, increased by incubation of cells with ethanol in the presence of stimulatory agonists (e.g., prostaglandin E1). Stimulation by ethanol of cAMP generation by type VII adenylyl cyclase was 2- to 3-fold greater than that seen with the other tested adenylyl cyclases. The noted stimulation of cAMP generation by ethanol was dose-dependent and required concurrent activation of adenylyl cyclase through the stimulatory G protein. The effects of ethanol were reversible and mimicked by butanol but not by chloroform.     

Ca2+/calcineurin-inhibited adenylyl cyclase, highly abundant in forebrain regions, is important for learning and memory

Activation of cAMP synthesis by intracellular Ca2+ is thought to be the main mode of cAMP generation in the brain. Accordingly, the Ca2+-activated adenylyl cyclases I and VIII are expressed prominently in forebrain neurons. The present study shows that the novel adenylyl cyclase type IX is inhibited by Ca2+ and that this effect is blocked selectively by inhibitors of calcineurin such as FK506 and cyclosporin A. Moreover, adenylyl cyclase IX is inhibited by the same range of intracellular free Ca2+ concentrations that stimulate adenylyl cyclase I. Adenylyl cyclase IX is expressed prominently in the forebrain. Substantial arrays of neurons positive for AC9 mRNA were found in the olfactory lobe, in limbic and neocortical areas, in the striatum, and in the cerebellar system. These data show that the initiation of the cAMP signal by adenylyl cyclase may be controlled by Ca2+/calcineurin and thus provide evidence for a novel mode of tuning the cAMP signal by protein phosphorylation/dephosphorylation cascades.     

D1/D5 dopamine receptors inhibit depotentiation at CA1 synapses via cAMP-dependent mechanism

Recent work has shown that D1/D5 dopamine receptors can enhance long-term potentiation (LTP). We investigated whether D1/D5 receptors also affect depotentiation, the reversal of LTP by low-frequency stimulation. D1/D5 agonists greatly reduced depotentiation, an effect that was inhibited by a D1/D5 antagonist. The D1/D5 effect appears to be mediated by adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA), because it was mimicked by the AC activator forskolin and was inhibited by the AC and PKA inhibitors. In vivo studies show that dopamine is released when a reward occurs. Our results raise the possibility that the memory of events before reward might be retained selectively, because dopamine blocks their erasure.     

Identity of adenylyl cyclase isoform determines the rate of cell cycle progression in NIH 3T3 cells

Cell cycle progression is regulated by cAMP in several cell types. Cellular cAMP levels depend on the activity of different adenylyl cyclases (ACs), which have varied signal-receiving capabilities. The role of individual ACs in regulating proliferative responses was investigated. Native NIH 3T3 cells contain AC6, an isoform that is inhibited by a variety of signals. Proliferation of exogenous AC6-expressing cells was the same as in control cells. In contrast, expression of AC2, an isoform stimulated by protein kinase C (PKC), resulted in inhibition of cell cycle progression and increased doubling time. In AC2-expressing cells, platelet-derived growth factor (PDGF) elevated cAMP levels in a PKC-dependent manner. PDGF stimulation of mitogen-activated protein kinases 1 and 2 (MAPK 1,2), DNA synthesis, and cyclin D1 expression was reduced in AC2-expressing cells as compared with control cells. Dominant negative protein kinase A relieved the AC2 inhibition of PDGF-induced DNA synthesis. Expression of AC2 also blocked H-ras-induced transformation of NIH 3T3 cells. These observations indicate that, because AC2 is stimulated by PKC, it can be activated by PDGF concurrently with the stimulation of MAPK 1,2. The elevation in cAMP results in inhibition of signal flow from the PDGF receptor to MAPK 1,2 and a significant reduction in the proliferative response to PDGF. Thus, the molecular identity and signal receiving capability of the AC isoforms in a cell could be important for proliferative homeostasis.     

Phosphorylation at conserved carboxyl-terminal hydrophobic motif regulates the catalytic and regulatory domains of protein kinase C

Mature protein kinase C is phosphorylated at a conserved carboxyl-terminal motif that contains a Ser (or Thr) bracketed by two hydrophobic residues; in protein kinase C betaII, this residue is Ser-660 (Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403). This contribution examines how negative charge at this position regulates the function of protein kinase C. Specifically, Ser-660 in protein kinase C betaII was mutated to Ala or Glu and the enzyme's stability, membrane interaction, Ca2+ regulation, and kinetic parameters were compared with those of wild-type protein phosphorylated at residue 660. Negative charge at this position had no significant effect on the enzyme's diacylglycerol-stimulated membrane interaction nor the conformational change accompanying membrane binding. In contrast, phosphate caused a 10-fold increase in the enzyme's affinity for Ca2+ and a comparable increase in its affinity for phosphatidylserine, two interactions that are mediated by the C2 domain. Negative charge also increased the protein's thermal stability and decreased its Km for ATP and peptide substrate. These data indicate that phosphorylation at the extreme carboxyl terminus of protein kinase C structures the active site so that it binds ATP and substrate with higher affinity and structures determinants in the regulatory region enabling higher affinity binding of Ca2+. The motif surrounding Ser-660 in protein kinase C betaII is found in a number of other kinases, suggesting interactions promoted by phosphorylation of the carboxyl terminus may provide a general mechanism for stabilizing kinase structure.     

G alphas-induced neurodegeneration in Caenorhabditis elegans

We describe a genetic model for neurodegeneration in the nematode Caenorhabditis elegans. Constitutive activation of the GTP-binding protein Galphas induces neurodegeneration. Neuron loss occurs in two phases whereby affected cells undergo a swelling response in young larvae and subsequently die sometime during larval development. Different neural cell types vary greatly in their susceptibility to Galphas-induced cytotoxicity, ranging from 0 to 88% of cells affected. Mutations that prevent programmed cell death do not prevent Galphas-induced killing, suggesting that these deaths do not occur by apoptosis. Mutations in three genes protect against Galphas-induced cell deaths. The acy-1 gene is absolutely required for neurodegeneration, and the predicted ACY-1 protein is highly similar (40% identical) to mammalian adenylyl cyclases. Thus, Gs-induced neurodegeneration is mediated by the second messenger cAMP. Mutations in the unc-36 and eat-4 genes are partially neuroprotective, which indicates that endogenous signaling modulates the severity of the neurotoxic effects of Galphas. These experiments define an intracellular signaling cascade that triggers a necrotic form of neurodegeneration.     

Acute inhibition of Na/H exchanger NHE-3 by cAMP. Role of protein kinase a and NHE-3 phosphoserines 552 and 605

Regulation of the renal Na/H exchanger NHE-3 by protein kinase A (PKA) is a key intermediate step in the hormonal regulation of acid-base and salt balance. We studied the role of NHE-3 phosphorylation in this process in NHE-deficient AP-1 cells transfected with NHE-3 and in OKP cells expressing native NHE-3. A dominant-negative PKA-regulatory subunit completely abolished the effect of cAMP on NHE-3 activity demonstrating a role of PKA in the functional regulation of NHE-3 by cAMP. NHE-3 isolated from cAMP-treated cells showed lower phosphorylation by purified PKA in vitro suggesting that NHE-3 is a PKA substrate in vivo. Although changes in NHE-3 whole protein phosphorylation is difficult to detect in response to cAMP addition, the tryptic phosphopeptide map of in vivo phosphorylated NHE-3 showed a complex pattern of constitutive and cAMP-induced phosphopeptides. To test the causal relationship between phosphorylation and activity, we mutated eight serines in the cytoplasmic domain to glycine or alanine. Single or multiple mutants harboring S552A or S605G showed no PKA activation or reduced regulation by PKA activation. Ser-552 and Ser-605 were phosphorylated in vivo. However, multiple mutations of serines other than Ser-552 or Ser-605 also reduced the functional PKA regulation. We conclude that regulation of NHE-3 by PKA in vivo involves complex mechanisms, which include phosphorylation of Ser-552 and Ser-605.     

Gonadotropin-releasing hormone and pituitary adenylate cyclase-activating polypeptide affect levels of cyclic adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) subunits in the clonal gonadotrope alphaT3-1 cells: evidence for cross-talk between PKA and protein kinase C pathways

We have shown previously that protein kinase A (PKA) subunit levels are regulated by activation of PKA or protein kinase C (PKC) in anterior pituitary cells. GnRH also influenced PKA subunit levels, suggesting that hormonal regulation occurs in gonadotrophs, and therefore, we have reexamined this question using the clonal gonadotrope-derived cell line (alphaT3-1 cells). Western blot analysis, using specific immunoaffinity purified immunoglobulins, revealed expression of catalytic (Cat) and regulatory type I (RI) and type II (RII) subunits of PKA in these cells. Activation of adenylyl cyclase (AC) with forskolin, or of PKC with tetradecanoyl phorbol acetate (TPA), caused a rapid (detectable at 0.5-1 h) and concentration-dependent loss of all PKA subunits. Forskolin (10-100 microM) reduced Cat and RI by 60% and RII by 30%, whereas TPA (0.1-1 microM) reduced Cat and RII by 50% and RI by 40%. Simultaneous activation of PKA and PKC caused the expected dose-dependent reductions in Cat, and the effects of forskolin or TPA were nearly additive. RI and RII were reduced similarly by 10 nM TPA, whereas 100 nM TPA tended to prevent the reduction of RI or RII caused by forskolin. GnRH, which activates phosphoinositidase C and not AC in these cells, caused a clear loss of Cat or RII at all concentrations tested and of RI at 0.1 nM. Pituitary adenylate cyclase-activating polypeptide 38, which acts via PVR-1 receptors to stimulate both phosphoinositidase C and AC in these cells, also caused a clear dose-dependent decrease in Cat, RI, and RII, although higher concentrations were needed for the latter effects. Together, the data demonstrate that catalytic and regulatory subunits of PKA are subject to both hormonal and receptor-independent regulation in alphaT3-1 cells, reinforcing the possibility that such effects occur in nonimmortalized gonadotropes. Whereas the effects of PKA activators very likely involve proteolytic degradation of the dissociated PKA holoenzyme, the effects of TPA and GnRH occur in the absence of cAMP elevation by unknown mechanisms. Whatever the mechanisms involved, the data reveal a mechanism for cross-talk between phosphoinositidase C and AC-mediated hormonal signals, in which PKC activation seems to play a pivotal role.     

Signal recognition and integration by Gs-stimulated adenylyl cyclases

Six Gs-stimulated adenylyl cyclases have been cloned. Two additional forms have been identified as partial cDNAs. These adenylyl cyclases have distinct functional properties and are differentially regulated by protein kinases. The adenylyl cyclases have distinct patterns of distribution in peripheral tissues and various brain regions. The unique functional characteristics of the members of this effector family may allow each cell type and/or brain region to customize the signal-recognition and integrative properties of its cAMP-generation system by varying the ratios of the various adenylyl cyclases.     

Cloning and expression of an adenylyl cyclase localized to the corpus striatum

The neurotransmitter dopamine acts through various receptor subtypes that are largely associated with enhancement or inhibition of adenylyl cyclases. These dopamine-sensitive adenylyl cyclases are highly concentrated in the corpus stratum and associated limbic structures of the brain, where their levels exceed by orders of magnitude those in other areas of the brain. Here we use in situ hybridization to show that messenger RNA for three of these adenylyl cyclases is not found in the corpus striatum. We have isolated and expressed a complementary DNA encoding new adenylyl cyclase whose selective concentration in the corpus striatum indicates that it may be responsible for the synaptic actions of dopamine.     

Type I and type VIII adenylyl cyclases constitute a family whose activation is coupled to cellular deformation through the action of calcium-calmodulin

In certain tissues and cells, increases in concentrations of the second messenger cAMP are seen in response to mechanical or deformational stimuli. Type I and type VIII adenylyl cyclases, representing members of a family of calcium-calmodulin-stimulated adenylyl cyclases, and type VII adenylyl cyclase were each stably expressed in human embryonal kidney (HEK) 293 cells. HEK 293 cells exogenously expressing either type I adenylyl cyclase or any one of three type VIII adenylyl cyclase splice variants respond to swelling with increases in cAMP, requiring the presence of calcium in the extracellular medium for such responsiveness. Type VII expressing HEK 293 cells failed to respond to swelling with increased cAMP but demonstrated potentiation of isoproterenol-stimulated activity. This is characteristic of the influence of protein kinase C on the activity of the type VII protein. The relative swelling responsiveness of HEK 293 cells expressing splice variants of the type VIII adenylyl cyclase is consistent with the relative EC50 values for calcium-calmodulin stimulation of these splice variants. This is consistent with the involvement of calmodulin and the requirement for increases in intracellular calcium in mediating swelling-induced acceleration of type VIII adenylyl cyclase activity.     

Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases

cDNA encoding a hormone- and guanine nucleotide-stimulated adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] (type 6) from rat liver and kidney has been cloned and expressed. This enzyme is stimulated by forskolin, guanosine 5'-[gamma-thio]triphosphate, and isoproterenol plus GTP but is not stimulated by beta gamma subunits of guanine nucleotide-binding proteins. A second form (type 5), which is 75% similar to type 6, has also been cloned. Both types 5 and 6 cDNAs have multiple messages. PCR-based detection of the mRNA for the type 5 and 6 enzymes indicates that both are widely distributed. Homology analyses indicate at least four distinct subfamilies of guanine nucleotide stimulatory protein-regulated adenylyl cyclases. Types 5 and 6 enzymes define one distinct subfamily of mammalian adenylyl cyclases. Diversity of one guanine nucleotide-binding protein-regulated effector may allow different modes of regulation of cell-surface signal transmission.     

Differential regulation of cardiac actomyosin S-1 MgATPase by protein kinase C isozyme-specific phosphorylation of specific sites in cardiac troponin I and its phosphorylation site mutants

The significance of site-specific phosphorylation by protein kinase C (PKC) isozymes alpha and delta and protein kinase A (PKA) of troponin I (TnI) and its phosphorylation site mutants in the regulation of Ca(2+)-stimulated MgATPase activity of reconstituted actomyosin S-1 was investigated. The genetically defined TnI mutants used were T144A, S43A/S45A, S43A/S45A/T144A (in which the PKC phosphorylation sites Thr-144 and Ser-43/Ser-45 were respectively substituted by Ala) and N32 (in which the first 32 amino acids in the NH2-terminal sequence containing Ser-23/Ser-24 were deleted). Although the PKC isozymes displayed different substrate phosphorylation kinetics, PKC-alpha phosphorylated equally well TnI wild type and all mutants, whereas N32 was a much poorer substrate for PKC-delta. Furthermore, the two PKC isozymes exhibited discrete specificities in phosphorylating distinct sites in TnI and its mutants, either as individual subunits or as components of the reconstituted troponin complex. Unlike PKC-alpha, PKC-delta favorably phosphorylated the PKA-preferred site Ser-23/Ser-24 and hence, like PKA, reduced the Ca2+ sensitivity of the reconstituted actomyosin S-1 MgATPase. In contrast, PKC-alpha preferred to phosphorylate Ser-43/Ser-45 (common sites for all isozymes) and thus reduced the maximal Ca(2+)-stimulated activity of the MgATPase. In this respect, PKC-delta, by cross-phosphorylating the PKA sites, functioned as a hybrid of PKC-alpha and PKA. The site specificities and hence functional differences between PKC-alpha and -delta were most evident at low phosphorylation (1 mol of phosphate/mol) of TnI wild type and were magnified when S43A/S45A and N32 were used as substrates. The present study has demonstrated, for the first time, that distinct functional consequences could arise from the site-selective preferences of PKC-alpha and -delta for phosphorylating a single substrate in the myocardium, i.e., TnI.     

Inhibition of cyclic AMP-dependent kinase by expression of a protein kinase inhibitor/enhanced green fluorescent fusion protein attenuates angiotensin II-induced type 1 AT1 receptor mRNA down-regulation in vascular smooth muscle cells

Expression of the angiotensin II type 1 receptor (AT1-R) mRNA in vascular smooth muscle cells (VSMC) is down-regulated by a variety of agonists, including growth factors, agonists of Galphaq protein-coupled receptors, and activators of adenylyl cyclase. To determine whether cAMP-dependent protein kinases (PKA) participates in AT1-R mRNA down-regulation controlled by multiple classes of receptors, a PKA inhibitor peptide (PKIalpha) was developed and expressed in rat VSMC as a fusion with the enhanced green fluorescent protein (eGFP). PKA activity elicited both by forskolin and angiotensin II is suppressed in cells expressing this fusion protein (PKIalpha-eGFP), but platelet-derived growth factor-BB does not stimulate PKA activity in this preparation. PKIalpha-eGFP expression fully inhibits the forskolin-stimulated down-regulation of AT1-R mRNA levels and blocks 50% of the effect elicited by angiotensin II. This indicates that PKA plays a substantial role in angiotensin II-stimulated AT1-R mRNA down-regulation. However, inhibition of PKA has no effect on AT1-R mRNA down-regulation caused by platelet-derived growth factor-BB. These findings show how agonists such as angiotensin II that are not normally considered as activators of PKA can use PKA-dependent processes to modulate gene expression. These findings also provide definitive evidence that PKA-dependent pathways are involved in modulation of AT1-R mRNA levels in VSMC.