Interactive Fly, Drosophila

Insect PKA genes

The Drosophila DC2 gene was isolated on the basis of sequence similarity to Pka-C1, the major Drosophila protein kinase A catalytic subunit gene. The 67-kD DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in the mesodermal anlagen of early embryos. This expression depends on dorsal but not on the activity of eithertwist or snail. DC2 is also expressed in the wing disc, leg discs and subsets of cells in the optic lamina of third instar larvae. Mutants are viable and fertile. The absence of DC2 does not affect the viability or phenotype of imaginal disc cells lacking Pka-C1 activity or embryonic hatching of animals with reduced Pka-C1 activity. Transgenes expressing DC2 from a Pka-C1 promoter does not efficiently rescue a variety of Pka-C1 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with Pka-C1 (Meléndez, 1995).

In Manduca sexta, levels of basal and PTTH-stimulated secretion of ecdysteroids by prothoracic glands in vitro increase with time from day 1 to day 4 of the fifth larval stage (see Bombyx and Manduca prothoracicotropic hormone). Glandular content of cAMP-dependent protein kinase was analyzed to determine if the enzyme changes in concert with increased secretory response. Photoaffinity labeling with radioactively labeled cAMP reveals a 55-kDa cAMP-binding protein characteristic of the regulatory subunit of type-II cAMP-dependent protein kinase (RII). It appears that RII is one of a limited number of cellular proteins that is phosphorylated in the presence of [gamma-35S]ATP: the thiophosphorylated protein and the photoaffinity-labeled regulatory subunit possess the same M(r) and pI, and thiophosphorylation is blocked by mammalian cAMP-dependent protein kinase inhibitor. From day 1 to day 4 of the fifth instar, glandular content of RII increases in conjunction with increased ecdysteroid secretory capacity. Application of JH analog on day 1 significantly inhibits the observed increase in RII. Catalytic subunit activity does not change from days 1 to 4 of the fifth instar, nor does cellular content of a 34-kDa protein previously shown to be phosphorylated in response to PTTH. While it is unlikely that increased content of RII is solely responsible for enhanced ecdysteroid secretion by the prothoracic glands, it may serve as a convenient marker for investigating the mechanism by which steroidogenic capacity is regulated (Smith, 1993).

Prothoracicotropic hormone (PTTH), a peptide produced by the insect brain, stimulates the prothoracic glands to secrete ecdysteroids. The big form of this peptide (25.5 kDa) has been postulated to act through cyclic AMP in larval Manduca sexta, but the role of the cyclic nucleotide in the action of PTTH in pupal glands has been less clear. PTTH-stimulated ecdysteroid secretion and protein phosphorylation by glands removed from pupal Manduca sexta are blocked by two inhibitors of cAMP-dependent protein kinase: Rp-cAMPS, an antagonist of cAMP binding to the regulatory subunit of the kinase, and H-89, an inhibitor of the catalytic subunit of the kinase. Further, PTTH stimulates significant accumulation of cAMP in pupal glands, although less than that previously seen in PTTH-stimulated larval glands. Cyclic AMP-dependent protein kinase is found in cytoplasmic and membrane-associated glandular subfractions, as measured by incorporation of radioactively labeled cAMP into the regulatory subunit of the kinase. PTTH enhances cytoplasmic cAMP content and appears to increase the amount of cAMP bound to a cytoplasmic type II regulatory subunit of cAMP-dependent protein kinase. The results indicate that cAMP plays a requisite role in PTTH action in pupal glands, thus arguing in favor of a uniform mechanism of action for the peptide during Manduca development (Smith, 1996).

Conservation of PKA involvement in Hedgehog signaling

Zebrafish embryos injected with RNAs encoding Sonic hedgehog (Shh), Indian hedgehog (Ihh), or a dominant-negative regulatory subunit of PKA, PKI, have equivalent phenotypes. These include the expansion of proximal fates in the eye, ventral fates in the brain, and adaxial fates in somites and head mesenchyme. Moreover, ectopic expression of PKI partially rescues somite and optic stalk defects in no tail and cyclops mutants that lack midline structures that normally synthesize Shh. Conversely, all cell types promoted by ectopic expression of hhs and PKI are suppressed in embryos injected with RNA encoding a constitutively active catalytic subunit of PKA (PKA*). These results, together with epistasis studies on the block of ectopic Hh signaling by PKA*, indicate that PKA acts in target cells as a common negative regulator of Hedgehog signaling (Hammerschmidt, 1996).

A long-range signal encoded by the Sonic hedgehog (Shh) gene has been implicated as the ventral patterning influence from the notochord that induces sclerotome and represses dermomyotome in somite differentiation. The long-range somite patterning effects of SHH are instead mediated by a direct action of the amino-terminal cleavage product of Shh. Pharmacological manipulations to increase the activity of cyclic AMP-dependent protein kinase A can selectively antagonize the effects of the amino-terminal cleavage product. These results support the operation of a single evolutionarily conserved signaling pathway for both local and direct long-range inductive actions of HH family members (Fan, 1995).

Hedgehog (Hh) signaling plays a significant role in defining the polarity of a variety of tissue types along the anterior/posterior and dorsal/ventral axes in both vertebrate and invertebrate organisms. The pathway through which Hh transduces its signal is still obscure, however, recent data have implicated the cyclic AMP-dependent protein kinase A as a negative regulator of the Hh signal transduction pathway. One of the vertebrate Hh family members, Sonic hedgehog (Shh), can induce ventral neural cell types both in vivo and in vitro; high concentrations induce floor plate and lower concentrations motor neurons. To investigate whether PKA plays an active role in the suppression of ventral neural differentiation, transgenic embryos were generated expressing a dominant negative form of PKA (dnPKA) in primarily dorsal aspects of the mouse CNS. Induction of floor plate and motor neuron markers were observed in embryos expressing the dominant negative PKA transgene and the loss of dorsal gene expression at rostral levels. Thus suppression of PKA activity is sufficient to activate targets of the Shh signaling pathway in the vertebrate CNS suggesting that induction of ventral cell types occurs via the antagonistic action of Shh on PKA activity. Two mammalian target genes that are strongly expressed in ectopic dorsal locations in response to dnPKA are Ptc and Gli. As both of these are targets of Drosophila Hh signaling, these data point to an evolutionary conservation in both the mechanisms of signaling and the effectors of the signaling pathway (Epstein, 1996).

In vertebrate skin, sonic hedgehog is expressed specifically in the feather bud epithelia. Cyclic AMP, a protein kinase A activator, suppresses the expression of Sonic hedgehog, (SHH) and continuous feather growth. The results suggest that SHH and PKA also have antagonistic action during vertebrate skin morphogenesis, similar to the interaction between Hedgehog and PKA in the fly (Noveen, 1996).

Drosophila transcription factor cubitus interruptus (Ci) and its co-activator CRE (cAMP response element)-binding protein (CBP) activate a group of target genes on the anterior-posterior border in response to Hedgehog protein (Hh) signaling. In contrast, in the anterior region, the carboxyl-truncated form of Ci generated by protein processing represses Hh expression. In vertebrates, three Ci-related transcription factors (glioblastoma gene products [GLIs] 1, 2, and 3) have been identified, but their functional difference in Hh signal transduction is unknown. Distinct roles are reported for GLI1 and GLI3 in Sonic hedgehog (Shh) signaling. GLI3, which contains both repression and activation domains, acts both as an activator and a repressor, as does Ci, whereas GLI1 contains only the activation domain. Consistent with this, GLI3, but not GLI1, is processed to generate the repressor form. Transcriptional co-activator CBP binds to GLI3, but not to GLI1. The trans-activating capacity of GLI3 is positively and negatively regulated by Shh and cAMP-dependent protein kinase, respectively, through a specific region of GLI3, which contains the CBP-binding domain and the phosphorylation sites of cAMP-dependent protein kinase. GLI3 directly binds to the Gli1 promoter and induces Gli1 transcription in response to Shh. Thus, GLI3 may act as a mediator of Shh signaling in the activation of the target gene Gli1 (Dai, 1999).

Gli protein activity is controlled by multisite phosphorylation in vertebrate Hedgehog signaling

Gli proteins are transcriptional effectors of the Hedgehog (Hh) pathway in both normal development and cancer. This paper describes a program of multisite phosphorylation that regulates the conversion of Gli proteins into transcriptional activators. In the absence of Hh ligands, Gli activity is restrained by the direct phosphorylation of six conserved serine residues by protein kinase A (PKA), a master negative regulator of the Hh pathway. Activation of signaling leads to a global remodeling of the Gli phosphorylation landscape: the PKA target sites become dephosphorylated, while a second cluster of sites undergoes phosphorylation. The pattern of Gli phosphorylation can regulate Gli transcriptional activity in a graded fashion, suggesting a phosphorylation-based mechanism for how a gradient of Hh signaling in a morphogenetic field can be converted into a gradient of transcriptional activity (Niewiadomski, 2014).

Signaling upstream of PKA

Neural tube patterning in vertebrates is controlled in part by locally secreted factors that act in a paracrine manner on nearby cells to regulate proliferation and gene expression. Genes for the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and one of its high-affinity receptors (PAC1) are widely expressed in the mouse neural tube on embryonic day (E) 10.5. Transcripts for the ligand are present in differentiating neurons in much of the neural tube, whereas the receptor gene is expressed in the underlying ventricular zone, most prominently in the alar region and floor plate. PACAP potently increased cAMP levels more than 20-fold in cultured E10.5 hindbrain neuroepithelial cells, suggesting that PACAP activates protein kinase A (PKA) in the neural tube and might act in the process of patterning. Consistent with this possibility, PACAP down-regulates expression of the sonic hedgehog- and PKA-dependent target gene gli-1 in cultured neuroepithelial cells, concomitant with a decrease in DNA synthesis. PACAP is thus an early inducer of cAMP levels in the embryo and may act in the neural tube during patterning to control cell proliferation and gene expression (Waschek, 1998).

PACAP potentially interacts with other genes involved in neurogenesis and patterning. For example, in Drosophila, a DNA sequence conforming to a cAMP-responsive consensus element (CRE) is necessary for the combinatorial activation (by wingless and decapentaplegic) of midgut Ubx gene expression. Corresponding mammalian homologs (wnt gene products and bone morphogenic proteins, respectively), like Shh, are thought to have important roles in early neural patterning. PACAP activation of a CRE-binding protein thus could affect signaling in these alternative pathways (Waschek, 1998 and references).

The effects of the pituitary adenylase cyclate-activating peptides (PACAP) 27 and 38 on proenkephalin (PENK) gene transcription were examined in PC12 (rat pheochromocytoma) cells using transient transfection assays. Both ligands stimulate PENK gene transcription in a dose-dependent manner, with an apparent ED50 close to 5 x 10(-11) M. Inactivation of cAMP dependent-protein kinase (PKA) with a dominant inhibitory mutant strongly reduces PACAP-stimulated PENK transcription. Using reporter genes driven by either the minimal TPA-responsive element (TRE: TGACTCA) or cAMP-responsive element (CRE: TGACGTCA), it has been shown that the two PACAPs activate transcription through both regulatory sequences. These effects could result from direct post-translational activation of Jun and CREB, as shown using GAL4-Jun or GAL4-CREB fusion proteins. Expression of a dominant inhibitory mutant of CREB decreases by 60% the response to PACAP, suggesting that CREB is implicated in PENK transactivation. Similarly, expression of c-fos antisense RNA reduces by 80% the stimulatory effects of PACAP. Taken together, these results indicate that PACAP stimulates PENK transcription by members of both the AP1 and the CREB families. However, AP1 by itself is not sufficient to increase PENK transcription, as insulin-like growth factor 1 (IGF1), which stimulates AP1 activity but not cAMP production, is unable to stimulate PENK transcription. These results indicate a cooperative effect of AP1 and CREB on PENK transcription (Monnier, 1998).

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to stimulate melanotroph secretion, and PACAP-like immunoreactivity and expression of PACAP type I receptor messenger RNA have been identified in the pituitary pars intermedia (PI). The present study has shown that PACAP messenger RNA is also expressed in the PI. To examine the mechanism of PACAP action in the PI, cytosolic Ca2+ concentrations ([Ca2+]i) and ionic currents were measured in acutely dissociated rat melanotrophs. In about 40% of the melanotrophs studied, PACAP induces an increase in [Ca2+]i, which is suppressed each of the following: extracellular Ca2+ removal, extracellular Na+ replacement, the blocker of L-type Ca2+ channels (nicardipine), and the secreto-inhibitory neurotransmitter dopamine. The PACAP-induced [Ca2+]i increase is mimicked by activators of protein kinase A (PKA) and protein kinase C (PKC) and is reduced by inhibitors of PKA and PKC. Patch-clamp analysis reveals that PACAP causes inward currents with a reversal potential of -0.8 mV and facilitates voltage-dependent Ba2+ currents. These results suggest that PACAP potentiates Ca2+ entry mechanisms of rat melanotrophs by activation of nonselective cation channels via PKC and facilitation of voltage-dependent Ca2+ channels via PKA (Tanaka, 1997).

Although phosphorylation of Thr-197 in the activation loop of the catalytic subunit of cAMP-dependent protein kinase (PKA) is an essential step for its proper biological function, the kinase responsible for this reaction in vivo has remained elusive. Using nonphosphorylated recombinant catalytic subunit as a substrate, it has been shown that the phosphoinositide-dependent protein kinase, PDK1, expressed in 293 cells, phosphorylates and activates the catalytic subunit of PKA. The phosphorylation of PKA by PDK1 is rapid and is insensitive to PKI, the highly specific heat-stable protein kinase inhibitor. A mutant form of the catalytic subunit where Thr-197 is replaced with Asp is not a substrate for PDK1. In addition, phosphorylation of the catalytic subunit can be monitored immunochemically by using antibodies that recognize Thr-197 phosphorylated enzyme but not unphosphorylated enzyme or the Thr197Asp mutant. PDK1, or one of its homologs, is thus a likely candidate for the in vivo PKA kinase that phosphorylates Thr-197. This finding opens a new dimension in thinking about this ubiquitous protein kinase and how it is regulated in the cell (Cheng, 1998).

RalGDS is a GDP/GTP exchange protein for ral p24, a member of the small GTP-binding protein superfamily. RalGDS interacts directly with the GTP-bound active form of ras p21 through the effector loop of ras p21 in vitro, in insect cells and in the yeast two-hybrid system. These results suggest that RalGDS functions as an effector protein of ras p21. RalGDS interacts with ras p21 in mammalian cells in response to an extracellular signal. Epidermal growth factor (EGF) induces the interaction of c-ras p21 and RalGDS in COS cells expressing both proteins, but not in the cells expressing RalGDS and c-ras p21T35A (an effector loop mutant of ras p21). Cyclic AMP-dependent protein kinase (protein kinase A) regulates the selectivity of ras p21-binding to either RalGDS or Raf-1. Protein kinase A phosphorylates RalGDS as well as (1-149)Raf (amino acid residues 1-149). Although the phosphorylated (1-149)Raf has a lower affinity for ras p21 than the unphosphorylated (1-149)Raf, both the phosphorylated and unphosphorylated RalGDS have the similar affinities for ras p21. The phosphorylation of RalGDS does not affect its ability to stimulate the GDP/GTP exchange of ral p24. Pretreatment of COS cells with forskolin further stimulates the interaction of ras p21 and RalGDS induced by EGF under conditions in which EGF-dependent Raf-1 activity is inhibited. These results indicate that ras p21 distinguishes between RalGDS and Raf-1 according to their phosphorylation by protein kinase A (Kikuchi, 1996).

Cyclic adenosine monophosphate (cAMP) produces tissue-specific effects involving growth, differentiation, and gene expression. cAMP can activate the transcription factor Elk-1 and induce neuronal differentiation of PC12 cells via its activation of the MAP kinase cascade. These cell type-specific actions of cAMP require the expression of the serine/threonine kinase B-Raf and activation of the small G protein Rap1. Rap1, activated by mutation or by the cAMP-dependent protein kinase PKA, is a selective activator of B-Raf and an inhibitor of Raf-1. In PC12 cells, Rap1 and B-Raf are localized to the cell membrane and cytosol, respectively. 8-CPT stimulates the association of B-Raf with Rap1 within membranes. This action is specific for both cAMP and Rap1; no association of B-Raf with Rap1 as detected within membranes following treatment with EGF or in untreated cells, nor is B-Raf detected in immunoprecipitates using Ras antibody Y13-238. The dependence on GTP of this interaction was examined in COS-7 cells transfected with B-Raf and histidine-tagged Rap1b (His-Rap) or His-RapV12. B-Raf and its kinase activity are detected in eluates. The small amount of B-Raf associating with His-Rap1 is increased in cells cotransfected with PKA. The highest level of B-Raf is detected in cells cotransfected with His-RapV12. Only eluates from B-Raf-transfected cells contain B-Raf activity, as measured by immune complex assay using B-Raf antisera. B-Raf activity associated with His-Rap1 is greatly stimulated by PKA, to a level similar to that associated with His-RapV12. The expression of equal amounts of His-Rap was confirmed by immunoblotting with Rap1 antisera. These data suggest that the association of activated B-Raf protein with Rap1 is increased upon GTP loading, stimulated by PKA or by a V12 mutation. Therefore, in B-Raf-expressing cells, the activation of Rap1 provides a mechanism for tissue-specific regulation of cell growth and differentiation via MAP kinase (Vossler, 1998).

Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells. ACA is highly active during aggregation, with negligible activity in the slug stage. ACG activity is not present at significant levels until mature spores have formed. ACB activity increases strongly after slugs have formed with optimal activity at early fruiting body formation. The same high activity is observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it is not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions that these mutants can develop into fruiting bodies in the complete absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. Both REGA inhibition and PKA overexpression cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation (Meima, 1999).

Cullins function as scaffolds that, along with F-box/WD40-repeat-containing proteins, mediate the ubiquitination of proteins to target them for degradation by the proteasome. The cullin CulA is required at several stages during Dictyostelium development. culA null cells are defective in inducing cell-type-specific gene expression and exhibit defects during aggregation, including reduced chemotaxis. PKA is an important regulator of Dictyostelium development. The levels of intracellular cAMP and PKA activity are controlled by the rate of synthesis of cAMP and its degradation by the cAMP-specific phosphodiesterase RegA. Overexpression of the PKA catalytic subunit (PKAcat) rescues many of the culA null defects and those of cells lacking FbxA/ChtA, a previously described F-box/WD40-repeat-containing protein, suggesting CulA and FbxA proteins are involved in regulating PKA function. Whereas RegA protein levels drop as the multicellular organism forms in the wild-type strain, they remain high in culA null and fbxA null cells. Although PKA can suppress the culA and fbxA null developmental phenotypes, it does not suppress the altered RegA degradation, suggesting that PKA lies downstream of RegA, CulA, and FbxA. CulA, FbxA, and RegA are found in a complex in vivo, and formation of this complex is dependent on the MAP kinase ERK2, which is also required for PKA function. It is proposed that CulA and FbxA regulate multicellular development by targeting RegA for degradation via a pathway that requires ERK2 function, leading to an increase in cAMP and PKA activity (Mohanty, 2001).

Dopamine release is activated by ethanol and addicting drugs, but molecular mechanisms linking dopaminergic signaling to neuronal responses and drinking behavior are poorly understood. Dopamine-D2 receptors induce PKA Calpha translocation and increase CRE-regulated gene expression. Ethanol also activates PKA signaling. Subthreshold concentrations of the D2 agonist NPA and ethanol, without effect alone, together cause synergistic PKA translocation and CRE-mediated gene transcription. D2 or adenosine A2 receptor blockade, pertussis toxin, Rp-cAMPS, or overexpression of dominant-negative peptides that sequester ßgamma dimers prevent synergy. Importantly, overexpression of a ßgamma inhibitor peptide in the nucleus accumbens (NAc) strikingly reduces sustained alcohol consumption. It is proposed that synergy of D2 and A2 confers ethanol hypersensitivity and that ßgamma dimers are required for voluntary drinking (Yao, 2002).

Dopaminergic signaling in NAc is activated by all addicting drugs and appears to be an integral component of the neural circuitry of addiction. Release of dopamine in anticipation of drinking or during exposure to ethanol is thought to contribute to incentive processes and ethanol consumption. The importance of NAc dopamine to ethanol intake was confirmed by microinjection of dopaminergic agents into the NAc: dopamine D2 receptor (D2) agonists increase and antagonists decrease ethanol self-administration. Furthermore, ethanol preference, sensitivity, and ethanol-conditioned place preference are reduced in mice lacking D2. This may be related to an overall reduction in their motivated response. In contrast, overexpression of D2 is reported to reduce ethanol preference and intake (Yao, 2002 and references therein).

D2 activation inhibits adenylyl cyclase activity, reducing cAMP levels in tissues, cell lines, and brain. D2 activates the trimeric GTP binding proteins Gi and/or Go, consisting of alphai or alphao subunits and ßgamma dimers; alphai subunits directly inhibit adenylyl cyclase activity. However, ßgamma dimers have diverse biological effects, which are probably responsible for other intracellular events observed on D2 activation. Despite the importance of dopamine in responding to ethanol, the molecular mechanism linking D2 signaling to neuronal activation and drinking behavior is not clear (Yao, 2002 and references therein).

NG108-15 is a clonal neuroblastoma-glioma hybrid cell line that expresses many neuronal properties. These cells have been used as a model system to investigate ethanol-induced changes in cAMP production and cAMP-dependent protein kinase (PKA) regulation of intracellular signaling. Short-term exposure to ethanol increases extracellular adenosine, which activates adenosine A2 receptors (A2) and increases cAMP levels. Receptors that increase cAMP activate the trimeric GTP binding protein Gs to release alphas and ßgamma dimers; alphas directly activate adenylyl cyclase. cAMP then binds to the two regulatory subunits in the inactive PKA tetrameric holoenzyme to release two active catalytic (C) subunits. In turn, C subunits phosphorylate diverse proteins to regulate their activity. C subunits can also translocate to the nucleus and regulate gene expression. One substrate of PKA is the cAMP element regulatory binding protein (CREB), which binds to the cAMP regulatory element (CRE) in the regulatory region of certain genes. Phosphorylation of CREB leads to CRE-mediated gene expression. Using NG108-15 cells, it has been shown that ethanol induces translocation of the catalytic subunit of PKA (Calpha) to the nucleus, stimulates CREB phosphorylation, and increases CRE-mediated gene expression (Yao, 2002 and references therein).

To investigate the relationship of D2 to ethanol induced changes in cAMP-dependent signaling, an NG108-15/D2 cell line stably expressing D2 receptors was developed. It was first asked whether D2 can activate the PKA pathway because of evidence that D2 activation can increase cAMP levels. This study shows that the D2 agonist NPA increases cAMP levels, causes translocation of PKA Calpha, and increases CRE-mediated gene expression. Next, it was found that (1) subthreshold concentrations of NPA and ethanol synergistically induce PKA Calpha translocation and increase CRE-mediated gene expression, (2) free ßgamma subunits released from G proteins mediate these synergistic responses; (3) D2 and A2 activation is required for synergy; and (4) overexpression in the NAc of a dominant-negative ßgamma scavenger peptide, which sequesters free ßgamma dimers, strikingly reduces ethanol consumption in rats (Yao, 2002).

This study shows that a D2 agonist activates PKA signaling from increases in cAMP to CRE-mediated gene expression in a neural cell line. These results suggest that the mechanism involves D2 coupling to Gi/o, release of ßgamma dimers, activation of AC II and/or IV, translocation of PKA Calpha to the nucleus, and subsequent PKA-dependent increases in gene expression. Ethanol also activates PKA Calpha translocation and gene expression. There is a remarkable synergy between D2 and ethanol-induced activation. Subthreshold concentrations of NPA or ethanol, which have no effect alone, when added together induce maximal translocation of PKA Calpha and activation of CRE-mediated gene expression. Release of ßgamma dimers is required for synergy. Synergy appears to be due to ßgamma stimulation of AC II and/or IV concomitant with ethanol/A2 activation of AC via Galphas. The functional significance of PKA Calpha translocation induced by subthreshold levels of NPA and ethanol is suggested directly by synergistic increases in CRE mediated gene expression and indirectly by decreases in ethanol consumption caused by expression of a dominant-negative ßgamma scavenger peptide in the NAc (Yao, 2002).

There is extensive evidence that dopaminergic mechanisms contribute to ethanol consumption. In addition, PKA is also implicated in ethanol drinking. Mice heterozygous for Galphas with reduced AC activity and mice expressing a dominant-negative inhibitor of a PKA regulatory subunit with reduced PKA activity both exhibit increased sensitivity to ethanol sedation and reduced ethanol preference and consumption. Increased sensitivity to ethanol intoxication has also been reported in Drosophila mutants that lack the gene 'cheapdate,' an allele of amnesiac. This can be reversed by agents that increase cAMP/PKA signaling. In contrast, mice lacking the RIIß regulatory subunit of PKA, with reduced cAMP-dependent PKA activity, exhibit resistance to ethanol sedation and increased ethanol preference and consumption. Similarly, Drosophila that are also deficient in the same Type II cAMP-dependent protein kinase show decreased sensitivity to ethanol. It appears, therefore, that the relationship between PKA activity and drinking behavior is not well understood. This study reports that overexpression of dominant-negative ßgamma scavenger peptides block D2- and ethanol-induced PKA Calpha translocation, gene expression, and synergy, probably by preventing ßgamma activation of AC. Expression of a ßgamma inhibitor peptide in rat medial NAc reduces voluntary ethanol intake, while water consumption is not decreased. These data provide evidence to suggest that ßgamma dimers in a specific brain-reward location are required to sustain voluntary ethanol consumption. Moreover, this finding depends on direct intervention not affected by developmental compensatory changes, which limit interpretation of data derived from genetically engineered mice and Drosophila (Yao, 2002).

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