Molecular Cloning and Expression of a Human Phosphodiesterase 4C

Owens, Raymond J.; Lumb, Simon; Rees-Milton, Karen; Russell, Annette; Baldock, Darren; Lang, Volker; Crabbe, Thomas; Ballesteros, Mercedes; Perry, M.J.

doi:10.1016/s0898-6568(97)00072-7

Cited by 30 publications

(16 citation statements)

References 32 publications

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“…Plasmids encoding FLAG-DISC1, mycHIS-fl-DISC1, mycHIS-(⌬1-187)-DISC1, PKA inhibitory peptide PKI, and PDE4 constructs PDE4A5, PDE4B1, PDEB2, PDEB3, PDE4C2, PDE4D3, PDE4D5, PDE4D3S54A, PDE4D3S54E, PDE4D3S13AS54A, and PDE4D-CAT have been described previously (Huston et al, 1996(Huston et al, , 1997Bolger et al, 1997;Owens et al, 1997;Hoffmann et al, 1998;Beard et al, 2000;Brandon et al, 2004;Fleming et al, 2004;Millar et al, 2005). DISC1 regions comprising residues 220 -283 and 359 -854 were subcloned into pEBG-2T vector to generate GST fusion proteins for expression in mammalian cells.…”

Section: Methodsmentioning

confidence: 99%

Isoform-Selective Susceptibility of DISC1/Phosphodiesterase-4 Complexes to Dissociation by Elevated Intracellular cAMP Levels

Murdoch¹,

Mackie²,

Collins³

et al. 2007

J. Neurosci.

146

164

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Disrupted-in-schizophrenia 1 (DISC1) is a genetic susceptibility factor for schizophrenia and related severe psychiatric conditions. DISC1 is a multifunctional scaffold protein that is able to interact with several proteins, including the independently identified schizophrenia risk factor phosphodiesterase-4B (PDE4B). Here we report that the 100 kDa full-length DISC1 isoform (fl-DISC1) can bind members of each of the four gene, cAMP-specific PDE4 family. Elevation of intracellular cAMP levels, so as to activate protein kinase A, caused the release of PDE4D3 and PDE4C2 isoforms from fl-DISC1 while not affecting binding of PDE4B1 and PDE4A5 isoforms. Using a peptide array strategy, we show that PDE4D3 binds fl-DISC1 through two regions found in common with PDE4B isoforms, the interaction of which is supplemented because of the presence of additional PDE4B-specific binding sites. We propose that the additional binding sites found in PDE4B1 underpin its resistance to release during cAMP elevation. We identify, for the first time, a functional distinction between the 100 kDa long DISC1 isoform and the short 71 kDa isoform. Thus, changes in the expression pattern of DISC1 and PDE4 isoforms offers a means to reprogram their interaction and to determine whether the PDE4 sequestered by DISC1 is released after cAMP elevation. The PDE4B-specific binding sites encompass point mutations in mouse Disc1 that confer phenotypes related to schizophrenia and depression and that affect binding to PDE4B. Thus, genetic variation in DISC1 and PDE4 that influence either isoform expression or docking site functioning may directly affect psychopathology.

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Section: Methodsmentioning

confidence: 99%

Isoform-Selective Susceptibility of DISC1/Phosphodiesterase-4 Complexes to Dissociation by Elevated Intracellular cAMP Levels

Murdoch¹,

Mackie²,

Collins³

et al. 2007

J. Neurosci.

146

164

View full text Add to dashboard Cite

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“…In still additional experiments, we determined that the human PDE4A4/5 isoform (3, 31), the human PDE4B isoforms PDE4B1, PDE4B2, and PDE4B3 (39), and also the human PDE4C2 isoform (41), when expressed in COS7 cells, could not be pulled down with GST-RACK1. These data imply that, of the known PDE4 isoforms, RACK1 binds selectively to PDE4D5.…”

Section: Figmentioning

confidence: 99%

The RACK1 Signaling Scaffold Protein Selectively Interacts with the cAMP-specific Phosphodiesterase PDE4D5 Isoform

Yarwood¹,

Steele²,

Scotland³

et al. 1999

Journal of Biological Chemistry

279

282

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The WD-repeat protein receptor for activated C-kinase (RACK1) was identified by its interaction with the cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4D5 in a yeast two-hybrid screen. The interaction was confirmed by co-immunoprecipitation of native RACK1 and PDE4D5 from COS7, HEK293, 3T3-F442A, and SK-N-SH cell lines. The interaction was unaffected by stimulation of the cells with the phorbol ester phorbol 2-myristate 3-acetate. PDE4D5 did not interact with two other WD-repeat proteins, ␤'-coatomer protein and G s ␤, in two-hybrid tests. RACK1 did not interact with other PDE4D isoforms or with known PDE4A, PDE4B, and PDE4C isoforms. PDE4D5 and RACK1 interacted with high affinity (K a approximately 7 pM) when they were expressed and purified from Escherichia coli, demonstrating that the interaction does not require intermediate proteins. The binding of the E. coli-expressed proteins did not alter the kinetics of cAMP hydrolysis by PDE4D5 but caused a 3-4-fold change in its sensitivity to inhibition by the PDE4 selective inhibitor rolipram. The subcellular distributions of RACK1 and PDE4D5 were extremely similar, with the major amount of both proteins (70%) in the high speed supernatant (S2) fraction. Analysis of constructs with specific deletions or single amino acid mutations in PDE4D5 demonstrated that a small cluster of amino acids in the unique amino-terminal region of PDE4D5 was necessary for its interaction with RACK1. We suggest that RACK1 may act as a scaffold protein to recruit PDE4D5 and other proteins into a signaling complex.Modulation of the levels of the second messenger cAMP in cells is important in the regulation of numerous physiological processes, including those in the immune/inflammatory systems, vascular smooth muscle, and the brain. Cyclic nucleotide phosphodiesterases (PDEs) 1 are a diverse family of enzymes that hydrolyze cAMP and cGMP and thus play an important role in modulating cAMP levels (1). The cAMP-specific phosphodiesterases (PDE4s) can be differentiated from other PDEs by sequence homology of the catalytic region of the enzymes (2) and by their ability to be specifically inhibited by the drug rolipram. Rolipram and other specific PDE4 inhibitors have been shown to have anti-depressant, anti-inflammatory, and smooth muscle relaxant activity in humans (2). The PDE4 enzymes are also characterized by the presence of unique regions of amino acid sequence outside the catalytic region of the proteins, which are called upstream conserved regions 1 and 2 (UCR1 and UCR2) and are located in the amino-terminal half of the proteins (3). The PDE4s are comprised of a large family of isoforms, encoded by four different genes (PDE4A, PDE4B, PDE4C, and PDE4D) in humans, with additional diversity being generated by alternative mRNA splicing (2). We and other groups (3-5) have characterized five different isoforms encoded by the human PDE4D gene, all of which appear to be conserved among mammals (6, 7). The five isoforms differ by the substitution of unique blocks of amino acids at the...

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“…[67][68][69][70][71][72][73][74][75] Rolipram, prototipik PDE4 inhibitörüdür, düşük afiniteli rolipram bağlanma sahası (LARBS) ve yüksek afiniteli bağlanma sitesi (HARBS) olarak adlandırı-lan iki siteye bağlanabilir.…”

Section: Genel Yapiunclassified

Phosphodiesterase Enzymes and Inhibitors: Review

Tuğrak¹,

Küçükoğlu²

2016

Turkiye Klinikleri J Pharm Sci

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Ö ÖZ ZE ET T Siklik nükleotid fosfodiesteraz enzimleri, vücutta her dokuda ve hücrede bulunabilen enzimlerdir. cAMP ve cGMP'yi hidroliz ettikleri için hücre sinyallerinde önemli rol oynarlar. PDE süper ailesi PDE1-PDE11 arasında olmak üzere 11 tanedir. Memeli hücrelerinde her bir aile 20'den fazla gen vermek için birdört farklı gen taşımaktadır. Bu genler 50'den fazla farklı PDE proteinini kodlarlar. PDE enzimleri içerisinde özellikle PDE1 ve PDE6 iyi karakterize edilmiş izozimlerdir. PDE'ler hücre ve dokularda spesifik dağılım gösteren enzimlerdir. Vücudun farklı doku ve organlarında bulunan PDE enzimleri farklılık gösterir. Bu sayede çeşitli hücre fonksiyonlarında farklı fizyolojik olaylara aracılık edebilirler. PDE izoenzimlerinin hücre ve doku fonksiyonlarına özel katkıları ve patofizyolojik düzenlenmeleri bugün önemli bir araştırma alanı oluşturmaktadır. Bu durum, etkileri henüz tam olarak belirlenmediği için özellikle PDE7-PDE11 gibi yeni PDE ailelerini daha çok ilgilendirmektedir. İnflamasyon, nörodejenerasyon ve kanser gibi birçok patolojik durumda meydana gelen değişiklikler, hücre içi sinyallerle ilgili PDE'lerin serbestleşmesini, bu patolojilerin önlenmesini, ayrıca bu tür patolojik durumların tedavisinde gözlemle-nen zorlukları açıklayabilir. Bu konularda yapılacak araştırmalar sonucu elde edilecek bulgular henüz kesin tedavisi bulunmayan hastalıkların tedavisinde yeni gelişmelere yol açabilir. Bu çalışmada, PDE enzimlerinin özellikleri, vücutta bulundukları doku ve organlar ile patofizyolojik olaylardaki rolleri açık-lanmış, ilaveten PDE inhibitörü ilaçlar anlatılmıştır. Ayrıca yeni geliştirilen ve ruhsatlandırma çalışmaları devam eden PDE inhibitörü bileşikler hakkında da bilgi verilmiştir.A An na ah ht ta ar r K Ke el li im me el le er r: : Fosfodiesteraz inhibitörleri; 2',3'-siklik AMP; 2',3'-siklik GMP A AB BS ST TR RA AC CT T Cyclic nucleotide phosphodiesterase enzymes can be found in every tissue and cell in the body. They play an important role in cell signaling because they hydrolyze cAMP and cGMP. PDE superfamily is 11 units, including PDE1-PDE11. Each family in mammalian cells encompasses 1 to 4 distinct genes, to give more than 20 genes. These genes encode more than 50 different PDE proteins. PDE enzymes in particular PDE1 and PDE6 are isozymes have been well characterized. PDEs are enzymes exhibiting specific distribution in cells and tissues. PDE enzymes located in different tissues and organs of the body vary. In this way, they can mediate different physiological events in various cell functions. Today, private contributions and pathophysiological regulations of PDE isoenzymes in cell and tissue functions constitute an important research field. This situation is more relevant for especially new PDE families such as PDE7-PDE11, because their effects have not been fully determined. Changes that occurs in many pathological situations such as inflammation, neurodegeneration and cancer, may explain the liberalization of PDEs related to the intracellular signaling as well as prevention of th...

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Molecular Cloning and Expression of a Human Phosphodiesterase 4C

Cited by 30 publications

References 32 publications

Isoform-Selective Susceptibility of DISC1/Phosphodiesterase-4 Complexes to Dissociation by Elevated Intracellular cAMP Levels

Isoform-Selective Susceptibility of DISC1/Phosphodiesterase-4 Complexes to Dissociation by Elevated Intracellular cAMP Levels

The RACK1 Signaling Scaffold Protein Selectively Interacts with the cAMP-specific Phosphodiesterase PDE4D5 Isoform

Phosphodiesterase Enzymes and Inhibitors: Review

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