Adenylyl Cyclase Activity Research Articles

1563-P: Hepatic PHD3 Stimulates Gluconeogenesis and Aggravates Hyperglycemia and Type 2 Diabetes by Hydroxylating CRTC2 at Pro129 and Pro615

Hepatic gluconeogenesis is critical for metabolic adaption in response to fasting, and its abnormal elevation contributes to the pathogenesis of hyperglycemia and type 2 diabetes. However, whether and how protein hydroxylation regulates hepatic gluconeogenesis and glucose metabolism remains largely unknown. Here we report that PHD3 is identified as a fasting-induced factor and regulates hepatic gluconeogenesis through interaction and hydroxylation of CRTC2. Pro129 and Pro615 hydroxylation of CRTC2 following PHD3 activation is necessary for its nuclear translocation and association with CREB, and enhanced binding to promoters of gluconeogenic target genes in response to fasting or the cAMP activator forskolin. CRTC2 hydroxylation-stimulated gluconeogenic gene expression is independent of SIK-mediated phosphorylation of CRTC2. Liver-specific knockout of PHD3 (PHD3 LKO) mice or prolyl hydroxylase-deficient knockin mice with alanine mutation in PHD3 at Pro196 (PHD3 KI) show significant attenuated gluconeogenic genes, glycaemia and hepatic capacity to produce glucose during fasting or fed with high-fat, high-sucrose diet. The efficacies of glucagon or adenylyl cyclase activator forskolin on stimulating the expression of gluconeogenic enzymes, such as PEPCK and G6Pase, and on glucose output are abolished in PHD3-/- mouse primary hepatocytes. Importantly, Pro615 hydroxylation of CRTC2 by PHD3 is increased in livers of fasted mice, diet-induced insulin resistance or genetically obese ob/ob mice and humans with diabetes. Together, these findings increase our understanding of the molecular mechanisms linking protein hydroxylation to gluconeogenesis under physiological and insulin resistant conditions, and may offer therapeutic potential for treating sustained gluconeogenesis, hyperglycemia and type 2 diabetes. Disclosure Y.Xue: None. W.Su: None. J.Zhao: None. C.Gao: None. J.Wen: None. Y.Xu: None. W.Fu: None. J.C.Fang: None. Y.Li: None. A.Cui: None. S.Wei: None. F.Ma: None. Z.Liu: None. X.Fang: None. W.Li: None. Y.Liu: None. G.Cai: None. Funding National Key R&D Program of China (2019YFA0802502); National Natural Science Foundation of China (81925008, 82073061, 82170587, 32100943); Ministry of Science and Technology of China (2020YFA0803301); Open Project Program of Metabolic Vascular Diseases Key Laboratory of Sichuan Province (2022MVDKL-K2); Shandong Natural Science Foundation (ZR2020MH206); China Postdoctoral Science Foundation (2021M693273)

Platelet zinc status regulates prostaglandin-induced signaling, altering thrombus formation

BackgroundApproximately 17.3% of the global population exhibits an element of zinc (Zn2+) deficiency. One symptom of Zn2+ deficiency is increased bleeding through impaired hemostasis. Platelets are crucial to hemostasis and are inhibited by endothelial-derived prostacyclin (prostaglandin I2 [PGI2]), which signals via adenylyl cyclase (AC) and cyclic adenosine monophosphate signaling. In other cell types, Zn2+ modulates cyclic adenosine monophosphate concentrations by changing AC and/or phosphodiesterase activity. ObjectivesTo investigate if Zn2+ can modulate platelet PGI2 signaling. MethodsPlatelet aggregation, spreading, and western blotting assays with Zn2+ chelators and cyclic nucleotide elevating agents were performed in washed platelets and platelet-rich plasma conditions. In vitro thrombus formation with various Zn2+ chelators and PGI2 was assessed in whole blood. ResultsIncubation of whole blood or washed platelets with Zn2+ chelators caused either embolization of preformed thrombi or reversal of platelet spreading, respectively. To understand this effect, we analyzed resting platelets and identified that incubation with Zn2+ chelators elevated pVASPser157, a marker of PGI2 signaling. In agreement that Zn2+ affects PGI2 signaling, addition of the AC inhibitor SQ22536 blocked Zn2+ chelation–induced platelet spreading reversal, while addition of Zn2+ blocked PGI2-mediated platelet reversal. Moreover, Zn2+ specifically blocked forskolin-mediated AC reversal of platelet spreading. Finally, PGI2 inhibition of platelet aggregation and in vitro thrombus formation was potentiated in the presence of low doses of Zn2+ chelators, increasing its effectiveness in inducing platelet inhibition. ConclusionZn2+ chelation potentiates platelet PGI2 signaling, elevating PGI2’s ability to prevent effective platelet activation, aggregation, and thrombus formation.

Adenylyl cyclases (ACs) in GtoPdb v.2023.1

Adenylyl cyclase, E.C. 4.6.1.1, converts ATP to cyclic AMP and pyrophosphate. Mammalian membrane-delimited adenylyl cyclases (nomenclature as approved by the NC-IUPHAR Subcommittee on Adenylyl cyclases [11]) are typically made up of two clusters of six TM domains separating two intracellular, overlapping catalytic domains that are the target for the nonselective activators Gαs (the stimulatory G protein α subunit) and forskolin (except AC9, [28]). adenosine and its derivatives (e.g. 2',5'-dideoxyadenosine), acting through the P-site,are inhibitors of adenylyl cyclase activity [35]. Four families of membranous adenylyl cyclase are distinguishable: calmodulin-stimulated (AC1, AC3 and AC8), Ca2+- and Gβγ-inhibitable (AC5, AC6 and AC9), Gβγ-stimulated and Ca2+-insensitive (AC2, AC4 and AC7), and forskolin-insensitive (AC9) forms. A soluble adenylyl cyclase (AC10) lacks membrane spanning regions and is insensitive to G proteins.It functions as a cytoplasmic bicarbonate (pH-insensitive) sensor [7].

CAMP signaling affects age-associated deterioration of pacemaker beating interval dynamics.

Sinoatrial node (SAN) beating interval variability (BIV) and the average beating interval (BI) are regulated by a coupled-clock system, driven by Ca2+-calmodulin activated adenylyl cyclase, cAMP, and downstream PKA signaling. Reduced responsiveness of the BI and BIV to submaximal, [X]50, β-adrenergic receptor (β-AR) stimulation, and phosphodiesterase inhibition (PDEI) have been documented in aged SAN tissue, whereas the maximal responses, [X]max, do not differ by age. To determine whether age-associated dysfunction in cAMP signaling leads to altered responsiveness of BI and BIV, we measured cAMP levels and BI in adult (2-4months n = 27) and aged (22-26months n = 25) C57/BL6 mouse SAN tissue in control and in response to β-AR or PDEI at X50 and [X]max. Both cAMP and average BI in adult SAN were reduced at X50, whereas cAMP and BI at Xmax did not differ by age. cAMP levels and average BI were correlated both within and between adult and aged SAN. BIV parameters in long- and short-range terms were correlated with cAMP levels for adult SAN. However, due to reduced cAMP within aged tissues at [X]50, these correlations were diminished in advanced age. Thus, cAMP level generated by the coupled clock mechanisms is tightly linked to average BI. Reduced cAMP level at X50 in aged SAN explains the reduced responsiveness of the BI and BIV to β-AR stimulation and PDEI.

Characterization of Adenylyl Cyclase Isoform 6 Residues Interacting with Forskolin.

The adenylyl cyclase (AC) pathway, crucial for pulmonary vasodilation, is inhibited by hypoxia. Forskolin (FSK) binds allosterically to AC, stimulating ATP catalysis. As AC6 is the primary AC isoform in the pulmonary artery, selective reactivation of AC6 could provide targeted reinstatement of hypoxic AC activity. This requires elucidation of the FSK binding site in AC6. HEK293T cells stably overexpressing AC 5, 6, or 7 were incubated in normoxia (21% O2) or hypoxia (10% O2) or exposed to s-nitrosocysteine (CSNO). AC activity was measured using terbium norfloxacin assay; AC6 structure built by homology modeling; ligand docking to examine FSK-interacting amino acids; roles of selected residues determined by site-directed mutagenesis; FSK-dependent cAMP generation measured in wild-type and FSK-site mutants by biosensor-based live cell assay. Only AC6 is inhibited by hypoxia and nitrosylation. Homology modeling and docking revealed residues T500, N503, and S1035 interacting with FSK. Mutation of T500, N503, or S1035 decreased FSK-stimulated AC activity. FSK site mutants were not further inhibited by hypoxia or CSNO; however, mutation of any of these residues prevented AC6 activation by FSK following hypoxia or CSNO treatment. FSK-interacting amino acids are not involved in the hypoxic inhibition mechanism. This study provides direction to design FSK derivatives for selective activation of hypoxic AC6.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

Delineation of G Protein-Coupled Receptor Kinase Phosphorylation Sites within the D1 Dopamine Receptor and Their Roles in Modulating β-Arrestin Binding and Activation.

The D1 dopamine receptor (D1R) is a G protein-coupled receptor that signals through activating adenylyl cyclase and raising intracellular cAMP levels. When activated, the D1R also recruits the scaffolding protein β-arrestin, which promotes receptor desensitization and internalization, as well as additional downstream signaling pathways. These processes are triggered through receptor phosphorylation by G protein-coupled receptor kinases (GRKs), although the precise phosphorylation sites and their role in recruiting β-arrestin to the D1R remains incompletely described. In this study, we have used detailed mutational and in situ phosphorylation analyses to completely identify the GRK-mediated phosphorylation sites on the D1R. Our results indicate that GRKs can phosphorylate 14 serine and threonine residues within the C-terminus and the third intracellular loop (ICL3) of the receptor, and that this occurs in a hierarchical fashion, where phosphorylation of the C-terminus precedes that of the ICL3. Using β-arrestin recruitment assays, we identified a cluster of phosphorylation sites in the proximal region of the C-terminus that drive β-arrestin binding to the D1R. We further provide evidence that phosphorylation sites in the ICL3 are responsible for β-arrestin activation, leading to receptor internalization. Our results suggest that distinct D1R GRK phosphorylation sites are involved in β-arrestin binding and activation.

Nitric oxide augments signaling for contraction in hypoxic pulmonary arterial smooth muscle-Implications for hypoxic pulmonary hypertension.

Introduction: Hypoxic persistent pulmonary hypertension in the newborn (PPHN) is usually treated with oxygen and inhaled nitric oxide (NO), both pulmonary arterial relaxants. But treatment failure with NO occurs in 25% of cases. We previously demonstrated that 72h exposure to hypoxia, modeling PPHN, sensitized pulmonary artery smooth muscle cells (PASMC) to the contractile agonist thromboxane and inhibited relaxant adenylyl cyclase (AC) activity. Methods: In this study, we examined the effects of sodium nitroprusside (SNP), as NO donor, on the thromboxane-mediated contraction and NO-independent relaxation pathways and on reactive oxygen species (ROS) accumulation in PASMC. In addition, we examined the effect of the peroxynitrite scavenger 5,10,15,20-Tetrakis (4-sulfonatophenyl)porphyrinato Iron (III) (FeTPPS) on these processes. Results: Exposure of PASMC to 72h hypoxia increased total intracellular ROS compared to normoxic control cells and this was mitigated by treatment of cells with either SNP or FeTPPS. Total protein nitrosylation was increased in hypoxic PASMC compared to controls. Both normoxic and hypoxic cells treated with SNP exhibited increased total protein nitrosylation and intracellular nitrite; this was reduced by treatment with FeTPPS. While cell viability and mitochondrial number were unchanged by hypoxia, mitochondrial activity was decreased compared to controls; addition of FeTPPS did not alter this. Basal and maximal mitochondrial metabolism and ATP turnover were reduced in hypoxic PASMC compared to controls. Hypoxic PASMC had higher basal Ca2+, and a heightened peak Ca2+ response to thromboxane challenge compared to controls. Addition of SNP further elevated the peak Ca2+ response, while addition of FeTPPS brought peak Ca2+ response down to control levels. AC mediated relaxation was impaired in hypoxic PASMC compared to controls but was normalized following treatment with FeTPPS. Addition of SNP inhibited adenylyl cyclase activity in both normoxic and hypoxic PASMC. Moreover, addition of the Ca2+ chelator BAPTA improved AC activity, but the effect was minimal. Discussion: We conclude that NO independently augments contraction and inhibits relaxation pathways in hypoxic PASMC, in part by a mechanism involving nitrogen radical formation and protein nitrosylation. These observations may partially explain impaired effectiveness of NO when treating hypoxic pulmonary hypertension.

Quantitative phosphoproteomic analysis reveals unique cAMP signaling pools emanating from AC2 and AC6 in human airway smooth muscle cells.

Human airway smooth muscle (HASM) is the primary target of ßAR agonists used to control airway hypercontractility in asthma and chronic obstructive pulmonary disease (COPD). ßAR agonists induce the production of cAMP by adenylyl cyclases (ACs), activate PKA and cause bronchodilation. Several other G-protein coupled receptors (GPCR) expressed in human airway smooth muscle cells transduce extracellular signals through cAMP but these receptors elicit different cellular responses. Some G-protein coupled receptors couple to distinct adenylyl cyclases isoforms with different localization, partly explaining this compartmentation, but little is known about the downstream networks that result. We used quantitative phosphoproteomics to define the downstream signaling networks emanating from cAMP produced by two adenylyl cyclases isoforms with contrasting localization in uman airway smooth muscle. After a short stimulus of adenylyl cyclases activity using forskolin, phosphopeptides were analyzed by LC-MS/MS and differences between cells overexpressing AC2 (localized in non-raft membranes) or AC6 (localized in lipid raft membranes) were compared to control human airway smooth muscle. The degree of AC2 and AC6 overexpression was titrated to generate roughly equal forskolin-stimulated cAMP production. 14 Differentially phosphorylated proteins (DPPs) resulted from AC2 activity and 34 differentially phosphorylated proteins resulted from AC6 activity. Analysis of these hits with the STRING protein interaction tool showed that AC2 signaling is more associated with modifications in RNA/DNA binding proteins and microtubule/spindle body proteins while AC6 signaling is associated with proteins regulating autophagy, calcium-calmodulin (Ca2+/CaM) signaling, Rho GTPases and cytoskeletal regulation. One protein, OFD1, was regulated in opposite directions, with serine 899 phosphorylation increased in the AC6 condition 1.5-fold but decreased to 0.46-fold by AC2. In conclusion, quantitative phosphoproteomics is a powerful tool for deciphering the complex signaling networks resulting from discreet signaling events that occur in cAMP compartments. Our data show key differences in the cAMP pools generated from AC2 and AC6 activity and imply that distinct cellular responses are regulated by these two compartments.

8-Aminoguanine and Its Actions on Renal Excretory Function.

The endogenous purine 8-aminoguanine induces diuresis/natriuresis/glucosuria by inhibiting PNPase (purine nucleoside phosphorylase); however, mechanistic details are unknown. Here, we further explored in rats 8-aminoguanine's effects on renal excretory function by combining studies using intravenous 8-aminoguanine, intrarenal artery infusions of PNPase substrates (inosine and guanosine), renal microdialysis, mass spectrometry, selective adenosine receptor ligands, adenosine receptor knockout rats, laser doppler blood flow analysis, cultured renal microvascular smooth muscle cells, HEK293 cells expressing A2B receptors and homogeneous time resolved fluorescence assay for adenylyl cyclase activity. Intravenous 8-aminoguanine caused diuresis/natriuresis/glucosuria and increased renal microdialysate levels of inosine and guanosine. Intrarenal inosine, but not guanosine, exerted diuretic/natriuretic/glucosuric effects. In 8-aminoguanine-pretreated rats, intrarenal inosine did not induce additional diuresis/natriuresis/glucosuria. 8-Aminoguanine did not induce diuresis/natriuresis/glucosuria in A2B-receptor knockout rats, yet did so in A1- and A2A-receptor knockout rats. Inosine's effects on renal excretory function were abolished in A2B knockout rats. Intrarenal BAY 60-6583 (A2B agonist) induced diuresis/natriuresis/glucosuria and increased medullary blood flow. 8-Aminoguanine increased medullary blood flow, a response blocked by pharmacological inhibition of A2B, but not A2A, receptors. In HEK293 cells expressing A2B receptors, inosine activated adenylyl cyclase, and this was abolished by MRS 1754 (A2B antagonist). In renal microvascular smooth muscle cells, 8-aminoguanine and forodesine (PNPase inhibitor) increased inosine and 3',5'-cAMP; however, in cells from A2B knockout rats, 8-aminoguanine and forodesine did not augment 3',5'-cAMP yet increased inosine. 8-Aminoguanine induces diuresis/natriuresis/glucosuria by increasing renal interstitial levels of inosine which, via A2B receptor activation, increases renal excretory function, perhaps in part by increasing medullary blood flow.

Very Low Protein Diets Induce a Rapid Decrease in Hepatic cAMP-Dependent Protein Kinase Followed by a Slower Increase in Adenylyl Cyclase Activity in Rats

Recent evidence indicates that cAMP-mediated responses are desensitized in liver during malnutrition. While receptor-stimulated production of cAMP is increased in hepatocytes from rats fed very low protein diets for 14 d, activity of cAMP-dependent protein kinase (PKA) is decreased in liver cytosol. The present study investigated the time course for this desensitization. Weanling rats were fed either a 0.5 (malnourished) or 15% protein (control) diet for 1, 3, 7 or 14 d. Total PKA activity decreased after only 3 d of feeding the low protein diet. This decrease was confined to the cytosolic compartment and was associated with a lower quantity of immunoreactive RI regulatory subunit of PKA, with no difference in the quantity of immunoreactive RII regulatory subunit. In contrast, basal-, MnCl2- and guanine nucleotide regulatory protein-stimulated adenylyl cyclase activities were not greater in liver membranes of malnourished rats than in those of the control rats until the 2nd wk of feeding. Greater activity was paralleled by an increase in the quantity of the stimulatory guanine nucleotide regulatory protein at d 14. The inhibitory guanine nucleotide regulatory protein quantity did not differ between dietary groups. Greater cAMP production was not mediated by changes in PKA phosphorylation of adenylyl cyclase because preincubation of membranes with purified PKA catalytic subunit decreased MnCl2-stimulated cAMP production equally in liver membranes of both control and malnourished rats. Similarly, treatment with alkaline phosphatase decreased adenylyl cyclase activity but did not eliminate the difference in adenylyl cyclase activity between control and malnourished rats. These data demonstrate that loss of PKA activity is an early response to a low protein diet and that, subsequently, a number of molecular adaptations occur which increase cAMP production. These changes may be adaptive responses to malnutrition that maintain essential cAMP-dependent functions.

Hypoxia Sensing of β-Adrenergic Receptor Is Regulated by Endosomal PI3Kγ.

Impaired beta-adrenergic receptor (β1 and β2AR) function following hypoxia underlies ischemic heart failure/stroke. Activation of PI3Kγ (phosphoinositide 3-kinase γ) by beta-adrenergic receptor leads to feedback regulation of the receptor by hindering beta-adrenergic receptor dephosphorylation through inhibition of PP2A (protein phosphatase 2A). However, little is known about PI3Kγ feedback mechanism in regulating hypoxia-mediated β1 and β2AR dysfunction and cardiac remodeling. Human embryonic kidney 293 cells or mouse adult cardiomyocytes and C57BL/6 (WT) or PI3Kγ knockout (KO) mice were subjected to hypoxia. Cardiac plasma membranes and endosomes were isolated and evaluated for β1 and β2AR density and function, PI3Kγ activity and β1 and β2AR-associated PP2A activity. Metabolic labeling was performed to assess β1 and β2AR phosphorylation and epinephrine/norepinephrine levels measured post-hypoxia. Hypoxia increased β1 and β2AR phosphorylation, reduced cAMP, and led to endosomal accumulation of phosphorylated β2ARs in human embryonic kidney 293 cells and WT cardiomyocytes. Acute hypoxia in WT mice resulted in cardiac remodeling and loss of adenylyl cyclase activity associated with increased β1 and β2AR phosphorylation. This was agonist-independent as plasma and cardiac epinephrine and norepinephrine levels were unaltered. Unexpectedly, PI3Kγ activity was selectively increased in the endosomes of human embryonic kidney 293 cells and WT hearts post-hypoxia. Endosomal β1- and β2AR-associated PP2A activity was inhibited upon hypoxia in human embryonic kidney 293 cells and WT hearts showing regulation of beta-adrenergic receptors by PI3Kγ. This was accompanied with phosphorylation of endogenous inhibitor of protein phosphatase 2A whose phosphorylation by PI3Kγ inhibits PP2A. Increased β1 and β2AR-associated PP2A activity, decreased beta-adrenergic receptor phosphorylation, and normalized cardiac function was observed in PI3Kγ KO mice despite hypoxia. Compared to WT, PI3Kγ KO mice had preserved cardiac response to challenge with β1AR-selective agonist dobutamine post-hypoxia. Agonist-independent activation of PI3Kγ underlies hypoxia sensing as its ablation leads to reduction in β1- and β2AR phosphorylation and amelioration of cardiac dysfunction.

Innate and learned odor-guided behaviors utilize distinct molecular signaling pathways in a shared dopaminergic circuit.

Odor-based learning and innate odor-driven behavior have been hypothesized to require separate neuronal circuitry. Contrary to this notion, innate behavior and olfactory learning were recently shown to share circuitry that includes the Drosophila mushroom body (MB). But how a single circuit drives two discrete behaviors remains unknown. Here, we define an MB circuit responsible for both olfactory learning and innate odor avoidance and the distinct dDA1 dopamine receptor-dependent signaling pathways that mediate these behaviors. Associative learning and learning-induced MB plasticity require rutabaga-encoded adenylyl cyclase activity in the MB. In contrast, innate odor preferences driven by naive MB neurotransmission are rutabaga independent, requiring the adenylyl cyclase ACXD. Both learning and innate odor preferences converge on PKA and the downstream MBON-γ2α'1. Importantly, the utilization of this shared circuitry for innate behavior only becomes apparent with hunger, indicating that hardwired innate behavior becomes more flexible during states of stress.

Ethanol potentiates follicle-stimulating hormone action in ovarian granulosa cells.

There are many previous reports on the effects of ethanol on physiological function, including reports of elevated blood estrogen levels in women who drank alcohol. However, the mechanism of ethanol's effects on ovarian functions, such as follicle development and hormone secretion, has not been fully clarified. Therefore, in this study, we investigated the impacts of ethanol on these phenomena and their mechanisms using a primary culture system of rat ovarian granulosa cells (GCs). In the present experiment, groups were created in which follicle-stimulating hormone (FSH) or ethanol was added alone or FSH and ethanol were co-added, and mRNA and protein expression in each group was measured for luteinizing hormone receptor (LHR) and sex steroid hormone synthase, as well as for estradiol (E2) production, cAMP production, and FSH receptor (FSHR) internalization rate. The addition of FSH induced mRNA expression of LHR and aromatase, which led to membrane LHR expression and E2 production. The coexistence of ethanol enhanced all these responses. The action of FSH is exerted via cAMP, and the co-addition of ethanol enhanced this cAMP production. Ethanol alone did not induce cAMP production. The enhancing effect of ethanol was also observed for cAMP induced by cholera toxin. Ethanol had no significant effect on the internalization rate of FSHR. In conclusion, ethanol increased FSH-stimulated cAMP production by increasing the activity of adenylyl cyclase, which enhanced FSH actions in rat GCs. Alcohol is an exacerbating factor in several female hormone-related diseases, and the mechanism of ethanol-induced increase in estrogen secretion revealed in this study may be involved in the pathogenesis of these diseases.

An Arabidopsis Maternal Effect Embryo Arrest Protein is an Adenylyl Cyclase with Predicted Roles in Embryo Development and Response to Abiotic Stress

Background: Second messengers play a key role in linking environmental stimuli to cellular responses. One such messenger, 3′,5′-cyclic adenosine monophosphate (cAMP) generated by adenylyl cyclase (AC), has long been established as an essential signaling molecule in many physiological processes of higher plants, including growth, development, and stress response. Very few ACs have been identified in plants so far, so more must be sought. Objective: To test the probable AC activity of an Arabidopsis MEE (AtMEE) protein and infer its function bioinformatically. Methods: A truncated version of the AtMEE protein (encoded by At2g34780 gene) harboring the annotated AC catalytic center (AtMEE-AC) was cloned and expressed in BL21 Star pLysS Escherichia coli cells followed by its purification using the nickel-nitriloacetic acid (Ni-NTA) affinity system. The purified protein was tested for its probable in vitro AC activity by enzyme immunoassay. The AtMEE-AC protein was also expressed in the SP850 mutant E. coli strain, followed by an assessment (visually) of its ability to complement the AC-deficiency (cyaA mutation) in this mutant. Finally, the AtMEE protein was analyzed bioinformatically to infer its probable biological function(s). Results: AtMEE is an AC molecule whose in vitro activity is Mn2+-dependent and positively modulated by NaF. Moreover, AtMEE is capable of complementing the AC-deficiency (cyaA) mutation in the SP850 mutant strain. AtMEE is primarily involved in embryo development and also specifically expressed in response to abiotic stress via the MYB expression core motif signaled by cAMP. Conclusion: AtMEE is an AC protein whose functions are associated with embryo development and response to abiotic stress.

A Class IV Adenylate Cyclase, CyaB, Is Required for Capsule Polysaccharide Production and Biofilm Formation in Vibrio parahaemolyticus.

Cyclic AMP (cAMP) receptor protein (CRP), encoded by crp, is a global regulator that is activated by cAMP, a second messenger synthesized by a class I adenylate cyclase (AC-I) encoded by cyaA in Escherichia coli. cAMP-CRP is required for growth on nonpreferred carbon sources and is a global regulator. We constructed in-frame nonpolar deletions of the crp and cyaA homologs in Vibrio parahaemolyticus and found that the Δcrp mutant did not grow in minimal media supplemented with nonpreferred carbon sources, but the ΔcyaA mutant grew similarly to the wild type. Bioinformatics analysis of the V. parahaemolyticus genome identified a 181-amino-acid protein annotated as a class IV adenylate cyclase (AC-IV) named CyaB, a member of the CYTH protein superfamily. AC-IV phylogeny showed that CyaB was present in Gammaproteobacteria and Alphaproteobacteria as well as Planctomycetes and Archaea. Only the bacterial CyaB proteins contained an N-terminal motif, HFxxxxExExK, indicative of adenylyl cyclase activity. Both V. parahaemolyticus cyaA and cyaB genes functionally complemented an E. coli ΔcyaA mutant. The Δcrp and ΔcyaB ΔcyaA mutants showed defects in growth on nonpreferred carbon sources and in swimming and swarming motility, indicating that cAMP-CRP is an activator. The ΔcyaA and ΔcyaB single mutants had no defects in these phenotypes, indicating that AC-IV complements AC-I. Capsule polysaccharide and biofilm production assays showed significant defects in the Δcrp, ΔcyaBΔcyaA, and ΔcyaB mutants, whereas the ΔcyaA strain behaved similarly to the wild type. This is consistent with a role of cAMP-CRP as an activator of these phenotypes and establishes a cellular role for AC-IV in capsule and biofilm formation, which to date has been unestablished. IMPORTANCE Here, we characterized the roles of CRP and CyaA in V. parahaemolyticus, showing that cAMP-CRP is an activator of metabolism, motility, capsule production, and biofilm formation. These results are in contrast to cAMP-CRP in V. cholerae, which represses capsule and biofilm formation. Previously, only an AC-I CyaA had been identified in Vibrio species. Our data showed that an AC-IV CyaB homolog is present in V. parahaemolyticus and is required for optimal growth. The data demonstrated that CyaB is essential for capsule production and biofilm formation, uncovering a physiological role of AC-IV in bacteria. The data showed that the cyaB gene was widespread among Vibrionaceae species and several other Gammaproteobacteria, but in general, its phylogenetic distribution was limited. Our phylogenetic analysis also demonstrated that in some species the cyaB gene was acquired by horizontal gene transfer.

Unusual phototransduction via cross-motif signaling from Gq to adenylyl cyclase in intrinsically photosensitive retinalganglion cells

Nonimage-forming vision in mammals is mediated primarily by melanopsin (OPN4)-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs). In mouse M1-ipRGCs, melanopsin predominantly activates, via Gαq,11,14, phospholipase C-β4 to open transient receptor 6 (TRPC6) and TRPC7 channels. In M2- and M4-ipRGCs, however, a prominent phototransduction mechanism involves the opening of hyperpolarization- and cyclic nucleotide-gated channels via cyclic nucleotide, although the upstream steps remain uncertain. We report here experiments, primarily on M4-ipRGCs, with photo-uncaging of cyclic nucleotides and virally expressed CNGA2 channels to conclude that the second messenger is cyclic adenosine monophosphate (cAMP) - very surprising considering that cyclic guanosine monophosphate (cGMP) is used in almost all cyclic nucleotide-mediated phototransduction mechanisms across the animal kingdom. We further found that the upstream G protein is likewise Gq, which via its Gβγ subunits directly activates adenylyl cyclase (AC). Our findings are a demonstration in a native cell of a cross-motif GPCR signaling pathway from Gq directly to AC with a specific function.

Urinary ATP Levels Are Controlled by Nucleotidases Released from the Urothelium in a Regulated Manner.

Adenosine 5′-triphosphate (ATP) is released in the bladder lumen during filling. Urothelial ATP is presumed to regulate bladder excitability. Urinary ATP is suggested as a urinary biomarker of bladder dysfunctions since ATP is increased in the urine of patients with overactive bladder, interstitial cystitis or bladder pain syndrome. Altered urinary ATP might also be associated with voiding dysfunctions linked to disease states associated with metabolic syndrome. Extracellular ATP levels are determined by ATP release and ATP hydrolysis by membrane-bound and soluble nucleotidases (s-NTDs). It is currently unknown whether s-NTDs regulate urinary ATP. Using etheno-ATP substrate and HPLC-FLD detection techniques, we found that s-NTDs are released in the lumen of ex vivo mouse detrusor-free bladders. Capillary immunoelectrophoresis by ProteinSimple Wes determined that intraluminal solutions (ILS) collected at the end of filling contain ENTPD3 > ENPP1 > ENPP3 ≥ ENTPD2 = NT5E = ALPL/TNAP. Activation of adenylyl cyclase with forskolin increased luminal s-NTDs release whereas the AC inhibitor SQ22536 had no effect. In contrast, forskolin reduced and SQ22536 increased s-NTDs release in the lamina propria. Adenosine enhanced s-NTDs release and accelerated ATP hydrolysis in ILS and lamina propria. Therefore, there is a regulated release of s-NTDs in the bladder lumen during filling. Aberrant release or functions of urothelial s-NTDs might cause elevated urinary ATP in conditions with abnormal bladder excitability.

Predicting the response to GLP-1 receptor agonists: an unexpected role for β-arrestin-1

Drugs targeting glucagon-like peptide 1 (GLP-1) and its heterotrimeric G protein-coupled receptor (GPCR) are important therapies for type 2 diabetes.1 GLP-1 is an incretin hormone which potentiates glucose-stimulated insulin secretion after meals. After binding to its receptor on β cells (GLP1R), GLP-1 activates a cascade of intracellular regulators, including the Gαs subunit, which activates adenylyl cyclase and increases intracellular cyclic AMP concentrations;1 and β-arrestins, which play a more complex role in receptor trafficking and signal transduction.

Adenosine reduces sinoatrial node cell action potential firing rate by uncoupling its membrane and calcium clocks.

The spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca2+ and membrane ion current clocks driven by Ca2+-calmodulin-activated adenylyl cyclase-protein kinase-A signaling. The mean AP-cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine binds to adenosine receptors (A1, A3) that couple to Gi protein-coupled receptors, reducing spontaneous AP firing rate via Gβγ signaling that activates IKAch,Ado. Adenosine also inhibits adenylyl cyclase activity via Gαi signaling, impacting cAMP-mediated protein kinase-A-dependent protein phosphorylation. We hypothesize that in addition to IKAch,Ado activation, adenosine impacts also Ca2+ via Gαi signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca2+ and membrane clock coupling. To this end, we measured Ca2+ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 µM adenosine substantially increased both the mean APCL (on average by 43%, n = 10) and AP beat-to-beat variability from 5.1 ± 1.7% to 7.2 ± 2.0% (n = 10) measured via membrane potential and 5.0 ± 2.2% to 10.6 ± 5.9% (n = 40) measured via Ca2+ (assessed as the coefficient of variability = SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic local Ca2+releases (LCRs) (Ca2+-clock effect): as LCR size distributions shifted to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of adenosine "drifted" upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher adenosine concentrations (>100µM): small stochastic LCRs failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of adenosine to activate Gβγ and IKACh,Ado and to activate Gαi, suppressing adenylyl cyclase activity, both contribute to the adenosine-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.