Showing papers on "Cyclase published in 2002"
TL;DR: Growth of the vte1 mutant, chlorophyll content, and photosynthetic quantum yield were similar to wild type under optimal growth conditions, suggesting that other antioxidants can compensate for the loss of tocopherol.
Abstract: Tocopherol (vitamin E) is a plant chloroplast lipid presumed to be involved in the response to oxidative stress. A tocopherol-deficient mutant (vte1) was isolated from Arabidopsis thaliana by using a TLC-based screening approach. Mutant plants lacked all four tocopherol forms and were deficient in tocopherol cyclase activity. Genetic mapping of vte1 and a genomics-based approach led to the identification of the ORF At4g32770 as a candidate gene for tocopherol cyclase. In vte1, At4g32770 contains a splicing site mutation and the corresponding mRNA expression is reduced. Expression of VTE1 in Escherichia coli resulted in the production of a protein with high tocopherol cyclase and tocotrienol cyclase activity. The VTE1 sequence shows no similarities to genes with known function, but is similar to that of SXD1, a gene that was recently isolated based on the availability of the sucrose export defective1 maize mutant (sxd1). Growth of the vte1 mutant, chlorophyll content, and photosynthetic quantum yield were similar to wild type under optimal growth conditions. Therefore, absence of tocopherol has no large impact on photosynthesis or plant viability, suggesting that other antioxidants can compensate for the loss of tocopherol. During photo-oxidative stress, chlorophyll content and photosynthetic quantum yield were slightly reduced in vte1 as compared with wild type indicating a potential role for tocopherol in maintaining an optimal photosynthesis rate under high-light stress.
311 citations
TL;DR: Structures of complexes with aza analogues of substrate and carbocation intermediates, as well as complexes with pyrophosphate and bornyl diphosphate, provide “snapshots” of the terpene cyclization cascade.
Abstract: The x-ray crystal structure of dimeric (+)-bornyl diphosphate synthase, a metal-requiring monoterpene cyclase from Salvia officinalis, is reported at 2.0-Å resolution. Each monomer contains two α-helical domains: the C-terminal domain catalyzes the cyclization of geranyl diphosphate, orienting and stabilizing multiple reactive carbocation intermediates; the N-terminal domain has no clearly defined function, although its N terminus caps the active site in the C-terminal domain during catalysis. Structures of complexes with aza analogues of substrate and carbocation intermediates, as well as complexes with pyrophosphate and bornyl diphosphate, provide “snapshots” of the terpene cyclization cascade.
285 citations
TL;DR: Nitration of lipids by NO- derived reactive species yields products with antiinflammatory properties, revealing a novel mechanism by which NO-derived nitrated biomolecules can influence the progression of vascular disease.
Abstract: Nitration of unsaturated fatty acids such as linoleate by NO-derived reactive species forms novel derivatives (including nitrolinoleate [LNO2]) that can stimulate smooth muscle relaxation and block platelet activation by either NO/cGMP or cAMP-dependent mechanisms. Here, LNO2 was observed to inhibit human neutrophil function. LNO2, but not linoleic acid or the nitrated amino acid 3-nitrotyrosine, dose-dependently (0.2 to 1 µmol/L) inhibited superoxide (O2·-) generation, Ca2+ influx, elastase release, and CD11b expression in response to either phorbol 12-myristate 13-acetate or N-formyl-Met-Leu-Phe. LNO2 did not elevate cGMP, and inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one did not restore neutrophil responses, ruling out a role for NO. In contrast, LNO2 caused elevations in intracellular cAMP in the presence and absence of phosphodiesterase inhibition, suggesting activation of adenylate cyclase. Compared with phorbol 12-myristate 13-acetate–activated neutrophils, N-formyl-Met-Leu-Phe–activated neutrophils were more susceptible to the inhibitory effects of LNO2, indicating that LNO2 may inhibit signaling both upstream and downstream of protein kinase C. These data suggest novel signaling actions for LNO2 in mediating its potent inhibitory actions. Thus, nitration of lipids by NO-derived reactive species yields products with antiinflammatory properties, revealing a novel mechanism by which NO-derived nitrated biomolecules can influence the progression of vascular disease.
161 citations
TL;DR: In this paper, a series of site-directed mutation experiments and some squalene analogues have provided deep insight into the polycyclization mechanism and catalytic sites in conjunction with the information from X-ray crystal data.
159 citations
TL;DR: It is shown that infection of pepper plants by Xanthomonas campestris pv.
Abstract: The calmodulin-dependent adenylate cyclase domain (Cya) of the Bordetella pertussis cyclolysin was used as a reporter protein to study the direct translocation of the Xanthomonas effector protein, AvrBs2, into the plant host cell. Adenylate cyclase activity (production of cAMP) depends on the presence of eukaryotic plant calmodulin and is only active after translocation from the prokaryotic cell into the eukaryotic plant cell. Here, we show that infection of pepper plants by Xanthomonas campestris pv. vesicatoria strains expressing the AvrBs2:Cya fusion protein results in detectable increases of cAMP levels in plant cells as early as 3 h after inoculation. Adenylate cyclase activity was shown to be type III secretion-dependent as the Xanthomonas hrp mutations, hrcV or hrpF, failed to produce detectable levels of cAMP in infected pepper plants. Furthermore, the N-terminal secretion and translocation signals of AvrBs2 were shown to be required for activity of the fusion protein in the plant. A single genomic copy of the avrBs2:cya fusion gene expressed under the control of the wild-type avrBs2 promoter was used to compare the effect of a susceptible and resistant plant interaction on the kinetics of effector protein delivery. Implications of these results and additional applications of this reporter construct are discussed.
152 citations
TL;DR: Heterologous sensitization of adenylate cyclase is characterized by an enhanced responsiveness to drug-stimulated cAMP accumulation following persistent activation of Galpha(i/o)-coupled receptors, which may represent cellular adaptive responses following prolonged receptor activation.
Abstract: The nine membrane-bound isoforms of the enzyme adenylate cyclase (EC [4.6.1.1][1]) are highly regulated by neurotransmitters and drugs acting through G protein-coupled receptors to modulate intracellular cAMP levels. In general, acute activation of Gαs-coupled receptors stimulates cAMP accumulation, whereas acute activation of Gαi/o-coupled receptors typically inhibits cAMP accumulation. It is also well established that persistent activation of G-protein coupled receptors will alter subsequent drug-modulated cAMP accumulation. These alterations are thought to represent cellular adaptive responses following prolonged receptor activation. One phenomenon commonly observed, heterologous sensitization of adenylate cyclase, is characterized by an enhanced responsiveness to drug-stimulated cAMP accumulation following persistent activation of Gαi/o-coupled receptors. Heterologous sensitization of adenylate cyclase was originally proposed to explain tolerance and withdrawal following chronic opiate administration and may be a mechanism by which cells adapt to prolonged activation of inhibitory receptors. Such an adaptive mechanism has been suggested to play a role in the processes of addiction to and withdrawal from many drugs of abuse and in psychiatric disorders including schizophrenia and depression. Although the precise mechanisms remain unknown, research over the last decade has led to advances toward understanding the molecular events associated with heterologous sensitization of recombinant and endogenous adenylate cyclases in cellular models. These events include the pertussis toxin-sensitive events that are associated with the development of heterologous sensitization and the more recently identified Gαs-dependent events that are involved in the expression of heterologous sensitization.
[1]: pending:yes
128 citations
TL;DR: The myristoyl group controls the Ca2+-sensitivity ofGCAP-1 (not that of GCAP-2) by an allosteric mechanism, but this control step does not involve a myristoysl switch.
Abstract: In visual transduction, guanylate cyclase-activating proteins (GCAPs) activate the membrane- bound guanylate cyclase 1 (ROS-GC1) to synthesize cGMP under conditions of low cytoplasmic (Ca 2+ )free. GCAPs are neuronal Ca 2+ -binding proteins with three functional EF-hands and a consensus site for N-terminal myristoylation. GCAP-1 and GCAP-2 regulated ROS-GC1 activities differently. The myristoyl group in GCAP-1 had a strong influence on the Ca 2+ -dependent regulation of ROS-GC1 (shift in IC50). In contrast, myristoylation of GCAP-2 did not change the cyclase activation profile (no shift in IC 50). Thus, the myristoyl group controlled the Ca 2+ -sensitivity of GCAP-1, but not that of GCAP-2. The myristoyl group restricted the accessibility of one cysteine in GCAP-1 and GCAP-2 observed by measuring the time-dependent thiol reactivity of cysteines. This shielding effect was not relieved when Ca 2+ was buffered by EGTA. We applied surface plasmon resonance (SPR) spectroscopy to monitor the Ca 2+ -dependent binding of myristoylated and nonmyristoylated GCAP-1 and GCAP-2 to immobilized phospholipid membranes. None of the GCAPs exhibited a Ca 2+ -myristoyl switch as observed for recoverin. Thus, the myristoyl group controls the Ca 2+ -sensitivity of GCAP-1 (not that of GCAP-2) by an allosteric mechanism,
98 citations
TL;DR: A much stronger effect of bcg1 mutation on pathogenicity in comparison to the effects of bac mutations suggests that BCG1 controls at least one more signalling component other than adenylate cyclase, and that the cAMP signalling pathway is not the only one responsible for pathogenicicity.
Abstract: SUMMARY The grey mould Botrytis cinerea is an economically important plant pathogen. Previously we found that null mutants of bcg1 encoding one of the two Galpha subunits of heterotrimeric GTP-binding proteins differed in colony morphology and showed reduced pathogenicity. To further understand the mechanisms involved in infection, we cloned the bac gene encoding adenylate cyclase, the enzyme that catalyses production of cAMP from ATP. The deduced protein sequence consists of 2300 amino acids, the ORF is interrupted by three conserved introns, and there is a high degree of similarity with the catalytic domains of other fungal adenylate cyclases. Gene replacement resulted in reduced vegetative growth and a morphology similar to that of bcg1 mutants. The wild-type (WT) colony morphology was partially restored by feeding exogenous cAMP. These bac mutants still had a low but constant level of cAMP, despite deletion of the complete catalytic domain of the enzyme. Conidia from bac mutants germinated, penetrated the leaves of Phaseolus vulgaris and caused spreading soft rot lesions (in contrast to bcg1 mutants), although these were slower to develop than in WT controls. Compared to the latter, the most striking difference was that no sporulation occurred on leaves inoculated with bac mutant conidia. These results confirm that the cAMP signalling pathway plays an important role in vegetative growth and pathogenicity in B. cinerea. On the other hand, a much stronger effect of bcg1 mutation on pathogenicity in comparison to the effects of bac mutations suggests that BCG1 controls at least one more signalling component other than adenylate cyclase, and that the cAMP signalling pathway is not the only one responsible for pathogenicity.
98 citations
TL;DR: In this paper, the authors determined the crystal structures of the extracellular region of human BST-1 at atomic resolution in the free form and in complexes with five substrate analogues: nicotinamide, NMN, ATPgammaS, ethenoNADP, and ethenonNAD.
94 citations
TL;DR: Findings show that the NO‐guanylate cyclase signal transduction pathway is strongly altered in brain in patients with chronic liver failure and that the effects are different in different brain areas.
78 citations
TL;DR: The present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells, and favor the concept thatPACAP may play important roles in the control of proliferation and/or differentiation of Cerebellar neuroblasts.
Abstract: The presence of receptors for the novel neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been recently demonstrated in the external granule cell layer of the cerebellum, a germinative matrix that generates the majority of cerebellar interneurons. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the effect of PACAP on the adenylyl cyclase and phospholipase C transduction pathways. The two molecular forms of PACAP, i.e., 27-(PACAP27) and 38-(PACAP38) amino-acid forms of PACAP, induced a dose-dependent stimulation of cyclic AMP production in granule cells. The potencies of PACAP27 and PACAP38 were similar (ED50 = 0.12 +/- 0.01 and 0.23 +/- 0.07 nM, respectively), whereas vasoactive intestinal polypeptide (VIP) was approximately 100 times less potent. PACAP27 and PACAP38 also induced a dose-dependent stimulation of polyphosphoinositide breakdown (ED50 = 19.1 +/- 6.3 and 13.4 +/- 6.0 nM, respectively), whereas VIP had no effect on polyphosphoinositide metabolism. The effect of PACAP38 on inositol phosphate formation was significantly reduced by U-73122 and by pertussis toxin, indicating that activation of PACAP receptors causes stimulation of a phospholipase C through a pertussis toxin-sensitive G protein. In contrast, forskolin and dibutyryl cyclic AMP did not affect PACAP-induced stimulation of inositol phosphates. Taken together, the present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells. These data favor the concept that PACAP may play important roles in the control of proliferation and/or differentiation of cerebellar neuroblasts.
TL;DR: It is reported that in addition to CD38 ecto‐catalysis, intracellularly expressed CD38 may catalyze NAD+→cADPr conversion to cause cytosolic Ca2+ release, indicating the downstream activation of ryanodine receptors by cADPr.
Abstract: CD38 is an ectocyclase that converts NAD+ to the Ca2+-releasing second messenger cyclic ADP-ribose (cADPr). Here we report that in addition to CD38 ecto-catalysis, intracellularly expressed CD38 may catalyze NAD+→cADPr conversion to cause cytosolic Ca2+ release. High levels of CD38 were found in the plasma membranes, endoplasmic reticulum, and nuclear membranes of osteoblastic MC3T3-E1 cells. More important, intracellular CD38 was colocalized with target ryanodine receptors. The cyclase also converted a NAD+ surrogate, NGD+, to its fluorescent product, cGDPr (Km ∼5.13 μM). NAD+ also triggered a cytosolic Ca2+ signal. Similar results were obtained with NIH3T3 cells, which overexpressed a CD38-EGFP fusion protein. The Δ−49-CD38-EGFP mutant with a deleted amino-terminal tail and transmembrane domain appeared mainly in the mitochondria with an expected loss of its membrane localization, but the NAD+-induced cytosolic Ca2+ signal was preserved. Likewise, Ca2+ release persisted in cells transfected with the Myr...
TL;DR: The function of carRA is determined by gene disruption, suggesting the existence of a feedback regulatory mechanism in the filamentous fungus Fusarium fujikuroi.
Abstract: Phytoene synthase, phytoene dehydrogenase and carotene cyclase are three of the four enzyme activities needed to produce the acidic carotenoid neurosporaxanthin from the precursor geranylgeranyl pyrophosphate. In the filamentous fungus Fusarium fujikuroi, these three enzyme activities are encoded by two closely linked genes, carRA and carB, oriented in the same direction in the genome. The two genes are separated by 548 bp and code for two polypeptides of 612 and 541 amino acids, respectively, which are highly similar to the homologous proteins from other filamentous fungi. The ORF of carRA contains a 96-bp insertion that is absent in the other fungal homologues. The 32 additional residues are located in one of the two repeated domains responsible for the cyclase activity in the homologous fungal proteins. We have determined the function of carRA by gene disruption. The resulting mutants were albino and had lost the ability to produce phytoene, as expected from the simultaneous loss of phytoene synthase and carotene cyclase. In the same experiments, we also found transformants in which carB had been deleted. These mutants accumulate phytoene, confirming the function of the gene previously shown by gene-targeted mutagenesis. Expression of carRA and carB is strongly induced by light. Loss of carB or disruption of the carRA ORF led to enhanced expression of the carRA gene, suggesting the existence of a feedback regulatory mechanism.
TL;DR: The existence of a single gene responsible for phytoene synthase and carotene cyclase seems to be a widespread trait among filamentous fungi, as shown by the examples now known in a basidiomycete, two zygomycetes and one ascomycete.
Abstract: Phytoene synthase and carotene cyclase, two key enzymes in carotenoid biosynthesis, are encoded by two separate genes in bacteria and plants, but by a single bifunctional gene in fungi. The cyclase function has been demonstrated for the products of the genes crtYB from the basidiomycete Xanthophyllomyces dendrorhous, and for carRA and carRP from the zygomycetes Phycomyces blakesleeanus and Mucor circinelloides, respectively. These three genes are highly similar to al-2 from Neurospora crassa. Taking advantage of the high proportion of the final product of the carotenoid pathway that accumulates Neurospora when mycelium is illuminated at low temperature, we have isolated two mutants with a pale reddish pigmentation. Both mutants are complemented by the wild-type al-2 gene, and carry mutations in the al-2 domain to which cyclase activity has been attributed in other fungi. The mutants lack neurosporaxanthin and accumulate an unidentified reddish carotenoid, as shown by column chromatography and HPLC. The chemical and spectrophotometrical properties of this carotenoid are consistent with the absence of carotenoid cyclization, and indicate that the product of al-2 is bifunctional. The existence of a single gene responsible for phytoene synthase and carotene cyclase thus seems to be a widespread trait among filamentous fungi, as shown by the examples now known in a basidiomycete, two zygomycetes and one ascomycete.
TL;DR: A nonradioactive homogeneous HTS assay using HitHunter™ enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Gαs-coupled receptor, which results in a positive signal unlike other technologies.
Abstract: In the new high-throughput screening (HTS) campaign, receptor functional assays, 3',5'-cyclic adenosine monophosphate (cAMP), intracellular [Ca(2)+](i), phosphatidylinositol turnover, and reporter-based assays are being used as primary screens as they are now developed as homogeneous and automation-friendly assays. FlashPlate assay and scintillation proximity assay using radiolabeled cAMP have been used for measuring cAMP. A nonradioactive homogeneous HTS assay using HitHunter trade mark enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Galpha(s)-coupled receptor. In the EFC-cAMP assay, the beta-galactosidase (beta-gal) donor fragment-cAMP (ED-cAMP) conjugate complements with the beta-gal enzyme acceptor (EA) fragment to form an active beta-gal enzyme. Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form an active enzyme. Cyclic AMP in the samples compete with ED-cAMP to bind to the anti-cAMP antibody, thus increasing the free ED-cAMP that can complement with the EA fragment to form an active enzyme that is assayed with a luminescent substrate. Thus, this assay results in a positive signal unlike other technologies, wherein the signal is completed by cAMP in the sample. Glucagon-like peptide (GLP)-1 binds to GLP-1 receptor (with a Kd of 0.2 nM) signals through Galpha(s) to activate adenylate cyclase, which results in an increase of intracellular cAMP (EC(50) of 0.3 nM). GLP-1 stimulation of cAMP levels measured by the EFC method was similar in both adherent and suspension cell formats (EC(50)~0.3 nM) at different cell numbers. The assay was further validated with forskolin, exendin, and several active GLP-1 peptide analogues. The stimulation of cAMP by GLP-1 and forskolin was effectively inhibited by the adenylate cyclase inhibitors MDL-12330A and SQ-22536, confirming that the increased cAMP is through the AC pathway. The assay tolerates dimethyl sulfoxide (DMSO) up to 10%, and tartrazine does not interfere with the assay with the adherent cells up to 1 mM and affects minimally up to 10 microM in suspension cells. The assay is very robust, with a Z' value of 0.7 to 0.8. The assay was validated with several plates of low molecular weight nonpeptide compounds and peptide agonists with different potencies. The suspension cell protocol is a robust homogeneous assay that involves fewer steps than the adherent cell protocol and is suitable for HTS. The cAMP assay using EFC technology is advantageous in that it has a greater dynamic range of detection; is nonradioactive, very sensitive, robust; has minimal interference from DMSO and colored compounds; and is amenable for automation. An added advantage of this assay is that the cAMP is measured as a positive signal, thereby reducing the incidence of false positives.
TL;DR: It is concluded that the stimulation of adenylate cyclase by phosphorylated EIIA(Glc) is controlled by G3P and otherosphorylated sugars such as D-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds.
Abstract: The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIAGlc, as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIAGlc phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIAGlc. Isopropyl-β-d-thiogalactopyranoside-induced overexpression of EIIAGlc did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIAGlc. A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIAGlc is controlled by G3P and other phosphorylated sugars such as d-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and d-tagatose-1,6-bisphosphate aldolase.
TL;DR: The findings provide evidence for a new cyclic GMP transduction system in synaptic layers and thereby extend existing concepts of how a membrane‐bound guanylate cyclase is regulated by small Ca2+‐sensor proteins.
Abstract: This study documents the identity of a calcium- regulated membrane guanylate cyclase transduction system in the photoreceptor-bipolar synaptic region. The guanylate cyclase is the previously characterized ROS-GC1 from the rod outer segments and its modulator is S100β. S100β senses increments in free Ca2+ and stimulates the cyclase. Specificity of photoreceptor guanylate cyclase activation by S100β is validated by the identification of two S100β-regulatory sites. A combination of peptide competition, surface plasmon resonance binding and deletion mutation studies has been used to show that these sites are specific for S100β and not for another regulator of ROS-GC1, guanylate cyclase-activating protein 1. One site comprises amino acids (aa) Gly962–Asn981, the other, aa Ile1030–Gln1041. The former represents the binding site. The latter binds S100β only marginally, yet it is critical for control of maximal cyclase activity. The findings provide evidence for a new cyclic GMP transduction system in synaptic layers and thereby extend existing concepts of how a membrane-bound guanylate cyclase is regulated by small Ca2+-sensor proteins.
TL;DR: Results indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleaving of APP.
Abstract: alpha-Secretase cleaves the full-length Alzheimer's amyloid precursor protein (APP) within the amyloid beta peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP751 to examine whether the alpha-secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester-stimulated secretion of nexin II (NXII), the secreted product of the alpha-secretase cleavage of APP751. At 1 microM, forskolin inhibited secretion of NXII by approximately 50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9-dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the alpha-secretase cleavage of APP751. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.
TL;DR: Soluble AC was poorly fixed during evolution, whereas membrane-bound AC has expanded to form the subgroups of prevailing adenylyl and guanylyl cyclases.
Abstract: The family of eukaryotic adenylyl cyclases consists of a very large group of 12 transmembrane adenylyl cyclases and a very small group of soluble adenylyl cyclase (sAC). Orthologs of human sAC are present in rat Diclyostelium and bacteria but absent from the completely sequenced genomes of Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Saccharomyces cereviciae. sAC consists of two cyclase domains and a long similar to 1,000 amino acid C-terminal (sCKH) region. This sCKH region and one cyclase domain have been found in only four bacterial genes; the sCKH region was also detected in bacterial Lux-transcription factors and in complex bacterial and fungal kinases. The phylogenies of the kinase and cyclase domains are identical to the phylogeny of the corresponding sCKH domain, suggesting that the sCKH region fused with the other domains early during evolution in bacteria. The amino acid sequences of sAC proteins yield divergence times from the human lineage for rat and Diclyostelium that are close to the reported divergence times of many other proteins in these species. The combined results suggest that the sCKH region was fused with one cyclase domain in bacteria, and a second cyclase domain was added in bacteria or early eukaryotes. The sAC was retained in a few bacteria and throughout the entire evolution of the human lineage but lost independently from many bacteria and from the lineages of plants, yeast, worms, and flies. We conclude that within the family of adenylyl cyclases, soluble AC was poorly fixed during evolution, whereas membrane-bound AC has expanded to form the subgroups of prevailing adenylyl and guanylyl cyclases.
TL;DR: Results indicate that pinealocytes express predominantly α1D L‐channels, and the β‐adrenergic agonist isoproterenol and PACAP inhibit the L‐channel currents through elevation of cyclic AMP, however, an α‐ adrenergic‐mediated mechanism also appears to be involved in the effect of norepinephrine on the L-channel currents.
Abstract: In this study the subunits of the dihydropyridine-sensitive L-type Ca2+ channels (L-channels) expressed in rat pinealocytes were characterized using reverse transcription (RT)-PCR analysis, and the modulation of these channels by adrenergic agonists and by pituitary adenylate cyclase-activating polypeptide (PACAP) was studied using the patch-clamp technique. RT-PCR analysis showed that rat pinealocytes expressed alpha 1D, alpha 2b, beta 2, and beta 4 Ca(2+)-channel subunit mRNAs. Other alpha 1 subunit transcripts were either not expressed or present at very low levels, indicating that the pinealocytes express predominantly alpha 1D L-channels. Electrophysiological studies confirmed that the pineal expressed a single population of L-channels. The L-channel currents were inhibited by two agonists that elevate cyclic AMP: the beta-adrenergic agonist isoproterenol and PACAP. Similar inhibition was observed with a cyclic AMP analogue, 8-bromo-cyclic AMP. The presence of a cyclic AMP antagonist, Rp-adenosine 3',5'-cyclic monophosphorothioate, blocked the inhibition by isoproterenol and PACAP. Norepinephrine, a mixed alpha- and beta-adrenergic agonist, also inhibited the L-channel currents, but the inhibition was smaller. The smaller inhibition by norepinephrine was secondary to the simultaneous activation of alpha- and beta-adrenergic receptors. These results indicate that (a) pinealocytes express predominantly alpha 1D L-channels, and (b) the beta-adrenergic agonist isoproterenol and PACAP inhibit the L-channel currents through elevation of cyclic AMP. However, an alpha-adrenergic-mediated mechanism also appears to be involved in the effect of norepinephrine on the L-channel currents.
TL;DR: Results show that cAMP and Ca2+ signaling characteristics of the A3 adenosine receptor are comparable to the ones found for the A1 subtype.
TL;DR: The receptors for the neuropeptides vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptin can elicit a number of additional intracellular signals, and it is now clear that others reflect novel primary coupling events of the receptors.
Abstract: The receptors for the neuropeptides vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptide are strong activators of adenylate cyclase, but recent evidence suggests that they can elicit a number of additional intracellular signals. Some of these are likely to be downstream of the conventional adenylate cyclase pathway, but it is now clear that others reflect novel primary coupling events of the receptors.
TL;DR: The crystal structure of SHC complexed with Ro48-8071, a potent inhibitor of OSC and therefore of cholesterol biosynthesis, is established and the knowledge of the inhibitor binding mode in SHC is likely to help develop more potent inhibitors for OSC.
TL;DR: Two splice variants of the frog receptor that display additional amino acid cassettes in the third intracellular loop were characterized and a third splice variant of PAC1-R, exhibiting a completely different intrACEllular C-terminal domain, named PAC 1-Rmc has also been identified.
Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts its various effects through activation of two types of G protein-coupled receptors, a receptor with high affinity for PACAP named PAC1-R and two receptors exhibiting similar affinity for both PACAP and vasoactive intestinal polypeptide named VPAC1-R and VPAC2-R. Here, we report the characterization of PAC1-R and novel splice variants in the frog Rana ridibunda. The frog PAC1-R has 78% homology with human PAC1-R and is highly expressed in the central nervous system. Two splice variants of the frog receptor that display additional amino acid cassettes in the third intracellular loop were characterized. PAC1-R25 carries a 25-amino acid insertion that matches the hop cassette of the mammalian receptor, whereas PAC1-R41 carries a cassette with no homology to any mammalian PAC1-R variant. A third splice variant of PAC1-R, exhibiting a completely different intracellular C-terminal domain, named PAC1-Rmc has also been identified. Determination of c...
TL;DR: Results clearly show that the insulinotropic effect of PACAP involved both VPAC and PAC1, and the PAC1 variants expressed in rat and mouse pancreatic islets seem to be coupled to adenylate cyclase but not to PLC.
Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potentiator of glucose-induced insulin secretion. PACAP binds to a PACAP-specific receptor (PAC1) and to VPAC receptors (VPAC1 and VPAC2), which share high affinity for vasoactive intestinal polypeptide (VIP). In the present study, the molecular expression of PACAP receptor isoforms and the signaling pathways involved in the insulin secretory effect of PACAP were investigated in isolated rat and mouse pancreatic islets. mRNA encoding PAC1-short, -hop, and -very short variants, as well as VPAC1 and VPAC2, were expressed in pancreatic islets. PACAP and VIP were equipotent in potentiating glucose-induced insulin release. Both peptides were also equipotent in increasing cAMP production, but PACAP was more efficient than VIP. Unlike carbachol, PACAP and VIP had no effect on inositol phosphate production. In the PAC1-deficient mouse, the insulinotropic effect of PACAP was reduced, and its differential effect on cAMP production was abolished, whereas the effects of VIP remained unchanged. These results clearly show that the insulinotropic effect of PACAP involved both VPAC and PAC1. The PAC1 variants expressed in rat and mouse pancreatic islets seem to be coupled to adenylate cyclase but not to PLC. (Endocrinology 143: 1253–1259, 2002)
TL;DR: Sequence and structural homology modeling of CAS indicate that the observed product specificity-altering mutations occur both within (Tyr410Cys, Ile481Thr, and Tyr532His) and outside of (Ala469Val and His477Tyr) the cyclase active site.
Abstract: A random mutagenesis/in vivo selection approach was applied to generate and identify mutations that alter the product specificity of oxidosqualene-cycloartenol synthase (CAS) from Arabidopsis thaliana This work complements previous studies of triterpene cyclase enzymes and was undertaken to provide knowledge of the frequency and locations at which point mutations can alter cyclase product specificity Random mutations were introduced by treatment with hydroxylamine or passage through a mutator strain of bacteria Libraries of mutated plasmids carrying the cas1 gene were transformed into a cyclase-deficient strain of Saccharomyces cereVisiae (CBY57) bearing a complementing plasmid (pZS11) carrying an Erg7 gene that encodes wild-type yeast oxidosqualene-lanosterol cyclase and a URA3 marker that could be counterselected by growth in media containing 5-fluoroorotic acid (5-FOA) This allowed use of a plasmid shuffle to select for cas1 mutants that could substitute for ERG7 activity Five of 73000 transformants were observed to grow in media containing 5-FOA but lacking ergosterol pTKP5-derived plasmids isolated from these transformants were sequenced, revealing five distinct and unique point mutations: Tyr410Cys, Ala469Val, His477Tyr, Ile481Thr, and Tyr532His Analysis of the nonsaponifiable lipids from CBY57 cells expressing these mutants suggests that the Tyr410Cys and His477Tyr mutants produce lanosterol as the dominant product, whereas the Ala469Val, Ile481Thr, and Tyr532His mutants produce a mixture of lanosterol and achilleol A, a product of monocyclization Sequence and structural homology modeling of CAS indicate that the observed product specificity-altering mutations occur both within (Tyr410Cys, Ile481Thr, and Tyr532His) and outside of (Ala469Val and His477Tyr) the cyclase active site
TL;DR: Pituitary adenylate cyclase-activating polypeptide functions as a GH-releasing factor in common carp by activating pituitary receptors resembling mammalian PAC-1 receptors, which is suggested to be responsible for growth hormone secretion in carp species.
Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon/secretin peptide family and its molecular structure is highly conserved among vertebrates. In this study, the rol
TL;DR: It is indicated that PACAP is the first ligand identified that increases cyclic AMP accumulation in the pineal gland without increasing cyclic GMP accumulation and that the effects of PACAP on cyclicAMP and MT production can be potentiated by an α1‐adrenergic → PKC mechanism.
Abstract: In this study, the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cyclic nucleotide accumulation and melatonin (MT) production in dispersed rat pinealocytes were measured. Treatment with PACAP (10(-7) M) increased MT production 2.5-fold. PACAP (10(-7) M) also increased cyclic AMP accumulation four- to fivefold; this effect was potentiated two- to three-fold by alpha 1-adrenergic activation. This potentiation appears to involve protein kinase C (PKC) because alpha 1-adrenergic activation is known to translocate PKC and the PACAP-stimulated cyclic AMP accumulation was potentiated ninefold by a PKC activator, 4 beta-phorbol 12-myristate 13-acetate (PMA). Phenylephrine and PMA also potentiated the PACAP-stimulated MT accumulation. These results indicate that cyclic AMP is one second messenger of PACAP in the pineal gland and that the effects of PACAP on cyclic AMP and MT production can be potentiated by an alpha 1-adrenergic-->PKC mechanism. In addition to these findings, it was observed that PACAP treatment with or without phenylephrine or PMA did not alter cyclic GMP accumulation. This indicates that PACAP is the first ligand identified that increases cyclic AMP accumulation in the pineal gland without increasing cyclic GMP accumulation. That PACAP fails to activate the vasoactive intestinal peptide/cyclic GMP pathway suggests that the vasoactive intestinal peptide receptors present in the pineal may be distinct from the type II PACAP receptors.
TL;DR: PC12 cells are used to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment and it is concluded that P2y12 receptors are expressed in neuronal cells and inhibit adenyll cyclase activity.
Abstract: P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells In platelets and glioma cells, these receptors were identified as P2Y12 Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment
ADP and ATP (01 – 100 μM) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A2A or recombinant β2 receptors Forskolin-dependent cyclic AMP production was reduced by ⩽1 μM and enhanced by 10 – 100 μM ADP; this latter effect was turned into an inhibition when A2A receptors were blocked
The nucleotide inhibition of cyclic AMP synthesis was not altered when P2X receptors were blocked, but abolished by pertussis toxin
The rank order of agonist potencies for the reduction of cyclic AMP was (IC50 values): 2-methylthio-ADP (012 nM)=2-methylthio-ATP (013 nM)>ADPβS (71 nM)>ATP (164 nM)=ADP (244 nM) The inhibition by ADP was not antagonized by suramin, pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid, or adenosine-3′-phosphate-5′-phosphate, but attenuated by reactive blue 2, ATPαS, and 2-methylthio-AMP
RT – PCR demonstrated the expression of P2Y2, P2Y4, P2Y6, and P2Y12, but not P2Y1, receptors in PC12 cells In Northern blots, only P2Y2 and P2Y12 were detectable Differentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged
We conclude that P2Y12 receptors are expressed in neuronal cells and inhibit adenylyl cyclase activity
Keywords: Adenylyl cyclase, ADP, ATP, neuronal P2Y receptor, PC12, pertussis toxin
Introduction
Adenine and uridine nucleotides exert their actions on either neurons or non-neural cells via membrane receptors known as P2 receptors This family comprises two groups: P2X receptors are ATP-gated ion channels, whereas P2Y receptors belong to the superfamily of G protein-coupled receptors (Ralevic & Burnstock, 1998) At least six DNA sequences coding for putative P2Y receptors have been identified in mammalian species (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12; Ralevic & Burnstock, 1998; Hollopeter et al, 2001; Zhang et al, 2001) Amongst these, P2Y1 (Tokuyama et al, 1995), P2Y11 (Communi et al, 1997), and P2Y12 (Hollopeter et al, 2001; Zhang et al, 2001) are activated by adenine nucleotides, and P2Y6 by uridine nucleotides (Chang et al, 1995) P2Y2 (Lustig et al, 1993) and P2Y4 (Communi et al, 1995) receptors, in contrast, are sensitive to both adenine and uridine nucleotides At P2Y2 receptors, UTP and ATP are equipotent agonists (Lustig et al, 1993), whereas at P2Y4 receptors, the actions of ATP are species-dependent: ATP is an agonist at rat P2Y4, but an antagonist at human P2Y4 receptors (Kennedy et al, 2000) Five of these receptors, namely P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12, have been detected in rats (Ralevic & Burnstock, 1998; Hollopeter et al, 2001) In heterologous expression systems, all P2Y receptor subtypes of the rat, with the exception of P2Y12, couple to phospholipase C and mediate nucleotide-dependent increases in intracellular inositol phosphates (Ralevic & Burnstock, 1998) Activation of the P2Y12 receptor, in contrast, mediates an inhibition of adenylyl cyclase activity (Hollopeter et al, 2001) Recombinant rat P2Y1, P2Y2 and P2Y6 receptors also inhibit M-type K+ (KM) channels as well as voltage-gated N-type Ca2+ channels, when expressed in neurons (Filippov et al, 2000, and references therein)
Aside of cloned P2Y receptors, several native nucleotide receptors have been detected in functional assays and have been characterized by pharmacological means Most of these receptors were reported to be coupled to phospholipase C and to mediate increases in inositol phosphates In addition, several reports described P2Y receptors which reduced cyclic AMP (Harden et al, 1995), and such receptors have been characterized most extensively in human platelets (Daniel et al, 1998) and in rat C6 glioma cells (Boyer et al, 1993) Most recently, the platelet receptor has been identified as P2Y12, and this receptor subtype is also expressed in the glioma cells (Hollopeter et al, 2001; Jin et al, 2001) An agonist profile similar to that of the P2Y12 receptor was reported for a P2Y receptor which also reduced cyclic AMP in a rat brain endothelial cell line (B10), but the authors suggested this receptor to be a P2Y1 subtype (Webb et al, 1996) More recently, a detailed pharmacological analysis of the cyclase inhibiting P2Y receptor in B10 endothelial cells indicated that this receptor was not P2Y1, but rather identical to the platelet P2Y receptor which mediates the reduction in cyclic AMP (Simon et al, 2001) Taken together, it appears likely that it is P2Y12, but not P2Y1 receptors, which mediate adenine nucleotide-dependent inhibition of adenylyl cyclase activity in rat tissues
In neurons, the activation of endogenous P2Y receptors frequently modulates the functions of voltage-gated ion channels For instance, in rat sympathetic neurons, uridine nucleotides were found to inhibit M type K+ channels (Boehm, 1998) In rat hippocampal neurons (Dave & Mogul, 1996), ATP has been reported to augment voltage-dependent Ca2+ currents, whereas in frog sympathetic neurons (Elmslie, 1992) and in neuroblastoma×glioma hybrid cells (NG108-15; Filippov et al, 1996), ATP and other nucleotides diminished Ca2+ currents In the latter cell line, adenine nucleotides were also found to stimulate the synthesis of cyclic AMP (Ohkubo et al, 2000) However, neuronal nucleotide receptors that inhibit adenylyl cyclase in order to reduce cyclic AMP have not been described Here, we used the rat pheochromocytoma cell line PC12 which is ontogenetically related to sympathetic neurons (Greene & Tischler, 1976) to search for such receptors Our results indicate that these cells express P2Y12 receptors which mediate an inhibition of adenylyl cyclase activity
TL;DR: It is concluded that myristoylation ofGCAP-1 and GCAP-2 is important for fine tuning of guanylate cyclase activity.