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The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics., Competing Interests: The authors declare no conflict of interest.

In many animal species, learning and memory have been found to play important roles in regulating intra- and interspecific behavioral interactions in varying environments. In such contexts, aggression is commonly used to obtain desired resources. Previous defeats or victories during aggressive interactions have been shown to influence the outcome of later contests, revealing loser and winner effects. In this study, we asked whether short- and/or long-term behavioral consequences accompany victories and defeats in dyadic pairings between male Drosophila melanogaster and how long those effects remain. The results demonstrated that single fights induced important behavioral changes in both combatants and resulted in the formation of short-term loser and winner effects. These decayed over several hours, with the duration depending on the level of familiarity of the opponents. Repeated defeats induced a long-lasting loser effect that was dependent on de novo protein synthesis, whereas repeated victories had no long-term behavioral consequences. This suggests that separate mechanisms govern the formation of loser and winner effects. These studies aim to lay a foundation for future investigations exploring the molecular mechanisms and circuitry underlying the nervous system changes induced by winning and losing bouts during agonistic encounters.

The tandem GAF domains from the cyanobacterium Anabaena PCC7120 cyaB2 adenylyl cyclase form an antiparallel dimer with cAMP bound to all four binding sites. cAMP binding causes highly cooperative allosteric enzyme activation (>500-fold; EC(50) = 1 microM; Hill coefficient >2.0). The cyaB2 GAF domains, like those of the cyclic nucleotide phosphodiesterases (PDEs), contain conserved NKFDE motifs that when mutated in the PDEs abrogate cyclic nucleotide binding. We mutated the aspartic acids within this motif in cyaB2 to determine which domains were required for signaling. Constructs containing an Asp/Ala mutation in either GAF domain still showed positive cooperative cAMP stimulation but with reduced Hill coefficients. The cyaB2 GAF domain NKFDE motifs contain inserts of 14 (GAF-A) and 19 (GAF-B) amino acids not present in PDE2 or cyaB1. Constructs having these inserts deleted could still be activated by cAMP (23- to 100-fold) but lost all positive cooperative activation, suggesting that the inserts play an important role in domain interaction and/or stabilization of the cAMP-binding pockets. In the shortened constructs, even those with a single Asp/Ala mutation in the NKFDE motifs could still be activated by cAMP. However, in a double Asp/Ala mutant of the shortened construct, stimulation by cAMP was almost completely lost, and the EC(50) shifted far to the right. Overall, the data suggest that in GAF domains without these inserts, only the canonical lysine:aspartate salt bridge keeps the alpha4-helix and the alpha4-beta5 linker that close over the cyclic nucleotide properly oriented, thereby stabilizing the binding pocket. The cyaB2 GAF ensemble appears to be an evolutionary intermediate where both GAF domains still participate in allosteric activation by cAMP.

Transformation of fibroblasts to myofibroblasts, characterized by expression of alpha-smooth muscle actin (alpha-SMA) and production of extracellular matrix (ECM) components, is a key event in connective tissue remodeling. Approaches to inhibit this transformation are needed in tissues, such as the heart, where excessive ECM production by cardiac fibroblasts (CFs) causes fibrosis, myocardial stiffening, and cardiac dysfunction. We tested whether adenylyl cyclase (AC) activation (increased cAMP levels) modulates the transformation of adult rat CF to myofibroblasts, as assessed by immunofluorescent microscopy, immunoblotting, and collagen synthesis. A 24-h incubation of CF with TGF-beta or angiotensin II increased alpha-SMA expression, which was inhibited by the AC agonist forskolin and a cAMP analog that activates protein kinase A. Treatment with forskolin blunted serum-, TGF-beta-, and angiotensin II-stimulated collagen synthesis. CFs engineered to overexpress type 6 AC had enhanced forskolin-promoted cAMP formation, greater inhibition by forskolin of TGF-beta-stimulated alpha-SMA expression, and a decrease in the EC(50) of forskolin to reduce serum-stimulated collagen synthesis. The AC stimulatory agonist adrenomedullin inhibited collagen synthesis in CF that overexpressed AC6 but not in controls. Thus, AC stimulation blunts collagen synthesis and, in parallel, the transformation of adult rat CF to myofibroblasts. AC overexpression enhances these effects, "uncovering" an inhibition by adrenomedullin. These findings implicate cAMP as an inhibitor of ECM formation by means of blockade of the transformation of CF to myofibroblasts and suggest that increasing AC expression, thereby enhancing cAMP generation through stimulation of receptors expressed on CF, could provide a means to attenuate and prevent cardiac fibrosis and its sequelae.

The GAL4-based Gene-Switch system has been engineered to regulate transgene expression in Drosophila in both time and space. We constructed a Gene-Switch transgene in which Gene-Switch expression is restricted spatially by a defined mushroom body enhancer. This system allows Gene-Switch to be active only in the mushroom bodies and only on administration of the pharmacological Gene-Switch ligand RU486. This line was used to drive the expression of a rutabaga cDNA in otherwise rutabaga mutant flies. Induction of the rutabaga cDNA in the mushroom bodies only during adulthood, or during adulthood along with the larval and pupal developmental stages, corrects the olfactory memory impairment found in rutabaga mutants. Induction of the cDNA only during the larval and pupal stages was inconsequential to performance in olfactory memory tasks. These data indicate that normal rutabaga function must be expressed in adulthood for normal memory and conclusively delimit the time and space expression requirements for correcting the rutabaga memory impairment. Such combined pharmacogenetic regulation of transgene expression now allows this time and space dissection to be made for other behavioral mutants.

The sympathetic nervous system is designed to respond to stress. Adenylyl cyclase (AC) is the keystone of sympathetic transmission, yet its role in response to acute overload in the heart or in the pathogenesis of heart failure is controversial. We examined the effects of pressure overload, induced by thoracic aortic banding, in mice in which type 5 AC, a major cardiac AC isoform, was disrupted (AC5-/-). Left ventricular weight/tibial length ratio (LVW/TL) was not different between the WT and AC5-/- at baseline and increased progressively and similarly in both groups at 1 and 3 wk after aortic banding. However, LV ejection fraction (LVEF) fell in WT at 3 wk after banding (from 70 +/- 2.8 to 57 +/- 3.9%, P < 0.05), and this decrease was associated with LV dilatation, indicating incipient cardiac failure. In contrast, AC5-/- mice did not exhibit a fall in LVEF from 74 +/- 2.2%. The number of apoptotic myocytes was similar at baseline, but it increased roughly 4-fold in WT at both 1 and 3 wk after banding, and significantly less, P < 0.05, in AC5-/-. Importantly, the increase in apoptosis occurred before the decline in LVEF in WT. The protective mechanism seems to involve Bcl-2, which was up-regulated significantly more in AC5-/- mice with pressure overload. Our findings suggest that limiting type 5 AC plays a protective role in response to pressure overload and the development of heart failure, potentially through limiting the incidence of myocardial apoptosis.

The thyrotropin (TSH) receptor (TSHR) is a member of the heterotrimeric G protein-coupled family of receptors whose main function is to regulate thyroid cell proliferation as well as thyroid hormone synthesis and release. In this study, we generated a TSHR knockout (TSHR-KO) mouse by homologous recombination for use as a model to study TSHR function. TSHR-KO mice presented with developmental and growth delays and were profoundly hypothyroid, with no detectable thyroid hormone and elevated TSH. Heterozygotes were apparently unaffected. Knockout mice died within 1 week of weaning unless fed a diet supplemented with thyroid powder. Mature mice were fertile on the thyroid-supplemented diet. Thyroid glands of TSHR-KO mice produced uniodinated thyroglobulin, but the ability to concentrate and organify iodide could be restored to TSHR-KO thyroids when cultured in the presence of the adenylate cyclase agonist forskolin. Consistent with this observation was the lack of detectable sodium-iodide symporter expression in TSHR-KO thyroid glands. Hence, by using the TSHR-KO mouse, we provided in vivo evidence, demonstrating that TSHR expression was required for expression of sodium-iodide symporter but was not required for thyroglobulin expression, suggesting that the thyroid hormone synthetic pathway of the mouse could be dissociated into TSHR-dependent and -independent steps.

Pollen tube growth and reorientation is a prerequisite for fertilization and seed formation. Here we report imaging of cAMP distribution in living pollen tubes microinjected with the protein kinase A-derived fluorosensor. Growing tubes revealed a uniform distribution of cAMP with a resting concentration of approximately 100-150 nM. Modulators of adenylyl cyclase (AC), forskolin, and dideoxyadenosine could alter these values. Transient elevations in the apical region could be correlated with changes in the tube-growth axis, suggesting a role for cAMP in polarized growth. Changes in cAMP arise through the activity of a putative AC identified in pollen. This signaling protein shows homology to functional motifs in fungal AC. Expression of the cDNA in Escherichia coli resulted in cAMP increase and complemented a catabolic defect in the fermentation of carbohydrates caused by the absence of cAMP in a cyaA mutant. Antisense assays performed with oligodeoxynucleotide probes directed against conserved motifs perturbed tip growth, suggesting that modulation of cAMP concentration is vital for tip growth.

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.

The gamma-aminobutyric acid type A (GABAA) receptor is the predominant Cl(-)-channel protein mediating inhibition in the retina and elsewhere in the mammalian brain. We have observed a time-dependent increase of GABA-induced whole-cell currents when dopamine was applied externally to rat retinal amacrine cells. After 20 min, the peak current was increased to 208% +/- 10% of its initial value. A comparable effect was observed with the dopamine D1 receptor agonist (+)-1-phenyl-2,3,4,5-tetrahydro(1H)-3-benzazepine-7,8-diol hydrochloride (SKF-38393) but not with the D2 agonist bromocryptine. The action of dopamine involved phosphorylation of GABAA receptors by protein kinase A, as evident from intracellular application of protein kinase A, cAMP, and forskolin. Both guanosine 5'-[gamma-thio]triphosphate and cholera toxin augmented the GABA response, indicating a role for the guanosine 5'-triphosphate-binding protein Gs in the transduction cascade. Phosphorylation of GABAA receptors shifted the half-maximally effective GABA concentration from 71 microM to 47 microM without affecting the maximal response amplitude. The elevated binding affinity for GABA was caused by an increase of the open probability of the channels from 0.09 to 0.33 (2 microM GABA); conductance and mean open time did not change. Several other receptor agonists such as adenosine, histamine, somatostatin, enkephalin, and vasoactive intestinal peptide were found to couple to the same intracellular phosphorylation pathway. Since some of these cotransmitters colocalize with GABA in amacrine cells, they may fine-tune GABAergic inhibition in the retina.

In the mouse, the light-sensitive pool of cAMP can be eliminated in the dark by application of the dopamine D2-like receptor agonists LY 171555 (quinpirole), (+)-N0437 (2-[N-(n-propyl)-N-2-(thienylethylamino)-5-hydroxytetralin]) , or (+)-3-PPP [3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride]. The rank-order affinity of the ability of the D2-like antagonists to block the action of LY 171555 matched that of the rat D4 receptor. Reverse transcription of retina mRNA followed by DNA amplification using D4-specific nucleotides demonstrates the presence of D4 mRNA in retina. In situ hybridization studies using D4-specific digoxygenin-labeled oligonucleotides or 35S-labeled UTP RNA probes demonstrate the presence of D4 mRNA in the photoreceptor cell layer and in the inner nuclear and ganglion cell layers. The modulation by D4 ligands of the dark level of light-sensitive cAMP in photoreceptors demonstrates the physiological coupling of the D4 receptor subtype.

The accumulation of many postaggregative mRNA species in Dictyostelium discoideum is dependent upon the continuous presence of elevated levels of cAMP. We have analyzed the cyclic nucleotide specificity of this requirement and show that it is similar to that of the cell-surface receptor and distinct from the specificity displayed by the cAMP-dependent protein kinase. The same specificity is displayed for the accumulation of two classes of prespore mRNAs (class I, early; class II, late) and a prestalk mRNA and for the shutoff of a growth-phase mRNA. Under conditions in which cAMP phosphodiesterase activity is competitively inhibited, half-maximal accumulation of prestalk mRNA can be obtained at cAMP concentrations of 320-520 nM, whereas a higher concentration, 1-2 microM, is required for half-maximal accumulation of the prespore mRNAs and shutoff of the growth-phase mRNA. These effects of cAMP and its analogues on gene expression have been obtained under conditions in which cAMP-mediated activation of adenylate cyclase is completely inhibited. We conclude that cAMP acts to stimulate postaggregative gene expression by interacting at the cell-surface receptor.

An odor-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] is thought to mediate olfactory transduction in vertebrates. However, it is not known whether the adenylate cyclase serves this function for all odorants or for only certain classes of odorants. To investigate this question, we have compared the abilities of 35 odorants to stimulate the adenylate cyclase and to elicit an electrophysiological response. We report a strong positive correlation between the magnitude of adenylate cyclase stimulation and the summated electrical response of the olfactory epithelium (electro-olfactogram) evoked by individual odorants. We also show that the adenylate cyclase stimulator forskolin equally attenuates the electro-olfactogram response for all odorants tested. These data provide evidence that the adenylate cyclase mediates transduction for a wide variety of odorants.

During short-term sensitization, a simple form of nonassociative learning in Aplysia, the presentation of a single brief noxious stimulus results in enhancement of the defensive withdrawal reflex lasting minutes to tens of minutes. This behavioral plasticity involves presynaptic facilitation of synaptic transmission from the mechanosensory neurons that mediate the reflex to their central target cells. This facilitation is due to cAMP-dependent protein phosphorylation. To determine whether the time course of presynaptic facilitation might be due to a persistent increase in activity of adenylate cyclase (EC 4.6.1.1) itself, persistence of the transmitter, or yet other processes, we developed a perfused-membrane method to analyze the time course of activation of adenylate cyclase by transient stimuli. After stimulation by a pulse of stimulatory transmitter, activation of adenylate cyclase decayed within 60 sec. This finding indicates that the enzyme does not remain persistently active in the absence of transmitter and suggests that short-term retention is likely to be due to other mechanisms. Possible additional mechanisms include continued activation of the cyclase by transmitter, cellular factors extrinsic to the cyclase that prolong the time course of its activation, and persistence of processes downstream from the cyclase.

The morphological effects on human endothelial cells of phorbol 12-myristate 13-acetate (PMA) and of agents that increase intracellular cAMP concentration were studied. The adenylate cyclase activator forskolin (10 microM), the cyclic nucleotide phosphodiesterase inhibitor methylisobutylxanthine (100 microM), dibutyryl-cAMP (10 microM), histamine (10 microM), and PMA (0.1 microM) significantly altered the morphology of human aortic and umbilical vein endothelial cells in primary cultures. These effects reached a maximum 40-80 min after the effector addition and became negligible 30-60 min after its removal. PMA and forskolin were strongly synergistic in altering endothelial cell morphology. All the effects of cAMP-elevating compounds and of PMA were abolished completely by 1 microM colchicine. In explants taken from human adult or child aortas, forskolin and PMA produced alterations in endothelial morphology qualitatively identical to those observed in endothelial cell cultures. Endothelium in these preparations closely resembled that found in zones of expected altered hemodynamic stresses of human aorta. Our data suggest that the morphology of endothelium in vivo may be regulated by separate or synergistic action of hormone-dependent adenylate cyclase and of inositol phospholipid turnover systems and might be important for maintenance of endothelial monolayer integrity under normal physiological and pathological conditions.