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The mRNA encoding a high-affinity cAMP phosphodiesterase is regulated by hormones and cAMP.

ABSTRACT: To elucidate the mechanisms by which hormones regulate cAMP phosphodiesterases (PDEs), a group of cDNA clones that had been isolated from a rat Sertoli cell library were characterized. These cDNAs are derived from a single gene (ratPDE3). The deduced amino acid sequence of the ratPDE3 cDNA corresponds to a 66,200-Da protein homologous to other testicular PDEs, to the Drosophila melanogaster dunce-encoded cAMP PDE, and to bovine and yeast PDEs. Expression of ratPDE3 in eukaryotic and prokaryotic cells leads to the appearance of a cAMP PDE with properties identical to the cAMP PDE purified from Sertoli cells. Although of different size, transcripts corresponding to ratPDE3 were present in all organs studied. In the immature Sertoli cell in culture, the level of mRNA transcripts of ratPDE3 was increased more than 100-fold by follicle-stimulating hormone or N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate treatment. Stimulation of ratPDE3 mRNA by N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate was also observed in a C6 glioma cell line. These data demonstrate that cAMP regulates the expression of one of its own degrading enzymes by an intracellular feedback mechanism that involves changes in mRNA levels.

SUBMITTER: Swinnen JV

PROVIDER: S-EPMC298247 | biostudies-other | 1989 Nov

REPOSITORIES: biostudies-other

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The mRNA encoding a high-affinity cAMP phosphodiesterase is regulated by hormones and cAMP.

Swinnen J V JV Joseph D R DR Conti M M

Proceedings of the National Academy of Sciences of the United States of America 19891101 21

To elucidate the mechanisms by which hormones regulate cAMP phosphodiesterases (PDEs), a group of cDNA clones that had been isolated from a rat Sertoli cell library were characterized. These cDNAs are derived from a single gene (ratPDE3). The deduced amino acid sequence of the ratPDE3 cDNA corresponds to a 66,200-Da protein homologous to other testicular PDEs, to the Drosophila melanogaster dunce-encoded cAMP PDE, and to bovine and yeast PDEs. Expression of ratPDE3 in eukaryotic and prokaryotic ...[more]

PMID: 2554303

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Project description:Cyclic AMP-dependent pathways mediate the communication between external stimuli and the intracellular signaling machinery, thereby influencing important aspects of cellular growth, morphogenesis and differentiation. Crucial to proper function and robustness of these signaling cascades is the strict regulation and maintenance of intracellular levels of cAMP through a fine balance between biosynthesis (by adenylate cyclases) and hydrolysis (by cAMP phosphodiesterases). We functionally characterized gene-deletion mutants of a high-affinity (PdeH) and a low-affinity (PdeL) cAMP phosphodiesterase in order to gain insights into the spatial and temporal regulation of cAMP signaling in the rice-blast fungus Magnaporthe oryzae. In contrast to the expendable PdeL function, the PdeH activity was found to be a key regulator of asexual and pathogenic development in M. oryzae. Loss of PdeH led to increased accumulation of intracellular cAMP during vegetative and infectious growth. Furthermore, the pdeHDelta showed enhanced conidiation (2-3 fold), precocious appressorial development, loss of surface dependency during pathogenesis, and highly reduced in planta growth and host colonization. A pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 10 fold) cAMP levels relative to the wild type, and was completely defective in virulence. Exogenous addition of 8-Br-cAMP to the wild type simulated the pdeHDelta defects in conidiation as well as in planta growth and development. While a fully functional GFP-PdeH was cytosolic but associated dynamically with the plasma membrane and vesicular compartments, the GFP-PdeL localized predominantly to the nucleus. Based on data from cAMP measurements and Real-Time RTPCR, we uncover a PdeH-dependent biphasic regulation of cAMP levels during early and late stages of appressorial development in M. oryzae. We propose that PdeH-mediated sustenance and dynamic regulation of cAMP signaling during M. oryzae development is crucial for successful establishment and spread of the blast disease in rice.

Project description:BackgroundWe investigated the role of cyclic nucleotide phosphodiesterases (PDEs) in the spatiotemporal control of intracellular cAMP concentrations in rat aortic smooth muscle cells (RASMCs).Methodology/principal findingsThe rank order of PDE families contributing to global cAMP-PDE activity was PDE4> PDE3 ?=? PDE1. PDE7 mRNA expression but not activity was confirmed. The Fluorescence Resonance Energy Transfer (FRET)-based cAMP sensor, Epac1-camps, was used to monitor the time course of cytosolic cAMP changes. A pulse application of the ?-adrenoceptor (?-AR) agonist isoproterenol (Iso) induced a transient FRET signal. Both ?(1)- and ?(2)-AR antagonists decreased the signal amplitude without affecting its kinetics. The non-selective PDE inhibitor (IBMX) dramatically increased the amplitude and delayed the recovery phase of Iso response, in agreement with a role of PDEs in degrading cAMP produced by Iso. Whereas PDE1, PDE3 and PDE7 blockades [with MIMX, cilostamide (Cil) and BRL 50481 (BRL), respectively] had no or minor effect on Iso response, PDE4 inhibition [with Ro-20-1724 (Ro)] strongly increased its amplitude and delayed its recovery. When Ro was applied concomitantly with MIMX or Cil (but not with BRL), the Iso response was drastically further prolonged. PDE4 inhibition similarly prolonged both ?(1)- and ?(2)-AR-mediated responses. When a membrane-targeted FRET sensor was used, PDE3 and PDE4 acted in a synergistic manner to hydrolyze the submembrane cAMP produced either at baseline or after ?-AR stimulation.Conclusion/significanceOur study underlines the importance of cAMP-PDEs in the dynamic control of intracellular cAMP signals in RASMCs, and demonstrates the prominent role of PDE4 in limiting ?-AR responses. PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, and acts synergistically with PDE3 inhibition at the submembrane compartment. This suggests that mixed PDE4/PDE1 or PDE4/PDE3 inhibitors would be attractive to potentiate cAMP-related functions in vascular cells.

Project description:Trypanosoma cruzi, the causative agent of Chagas disease, encodes a number of different cAMP-specific PDE (phosphodiesterase) families. Here we report the identification and characterization of TcrPDEB1 and its comparison with the previously identified TcrPDEB2 (formerly known as TcPDE1). These are two different PDE enzymes of the TcrPDEB family, named in accordance with the recent recommendations of the Nomenclature Committee for Kinetoplast PDEs [Kunz, Beavo, D'Angelo, Flawia, Francis, Johner, Laxman, Oberholzer, Rascon, Shakur et al. (2006) Mol. Biochem. Parasitol. 145, 133-135]. Both enzymes show resistance to inhibition by many mammalian PDE inhibitors, and those that do inhibit do so with appreciable differences in their inhibitor profiles for the two enzymes. Both enzymes contain two GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA) domains and a catalytic domain highly homologous with that of the T. brucei TbPDE2/TbrPDEB2 family. The N-terminus+GAF-A domains of both enzymes showed significant differences in their affinities for cyclic nucleotide binding. Using a calorimetric technique that allows accurate measurements of low-affinity binding sites, the TcrPDEB2 N-terminus+GAF-A domain was found to bind cAMP with an affinity of approximately 500 nM. The TcrPDEB1 N-terminus+GAF-A domain bound cAMP with a slightly lower affinity of approximately 1 muM. The N-terminus+GAF-A domain of TcrPDEB1 did not bind cGMP, whereas the N-terminus+GAF-A domain of TcrPDEB2 bound cGMP with a low affinity of approximately 3 muM. GAF domains homologous with those found in these proteins were also identified in related trypanosomatid parasites. Finally, a fluorescent cAMP analogue, MANT-cAMP [2'-O-(N-methylanthraniloyl)adenosine-3',5'-cyclic monophosphate], was found to be a substrate for the TcPDEB1 catalytic domain, opening the possibility of using this molecule as a substrate in non-radioactive, fluorescence-based PDE assays, including screening for trypanosome PDE inhibitors.

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The mRNA encoding a high-affinity cAMP phosphodiesterase is regulated by hormones and cAMP.

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The mRNA encoding a high-affinity cAMP phosphodiesterase is regulated by hormones and cAMP.

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