Purification and physical characterization of cloned human cAMP phosphodiesterases PDE-4D and-4C

Saldou, Natalie; Baecker, Preston A.; Li, Bin; Yuan, Zhengqiu; Obernolte, Rena; Ratzliff, James; Osen, Eric; Jarnagin, Kurt; Shelton, Earl R.

doi:10.1007/bf02737811

Cited by 5 publications

(2 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous studies, including our own, showed by gel filtration and density gradient centrifugation that the catalytic domains of PDE4A and PDE4B behave as oligomers (40,(42)(43)(44). As the catalytic domains of the PDE4 subtypes are highly conserved, it is unlikely that this discrepancy is due to sequence differences between PDE4A/B and the PDE4D used in our present study.…”

Section: Table I Physicochemical Properties Of the Pde4d Constructscontrasting

confidence: 39%

Dimerization of the Type 4 cAMP-specific Phosphodiesterases Is Mediated by the Upstream Conserved Regions (UCRs)

Richter

Conti

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

The cAMP-specific PDE4 family consists of four genes, each expressed as several splice variants. These variants are termed long and short forms depending on the presence or absence of two unique N-terminal domains called upstream conserved regions 1 and 2 (UCR1 and 2). UCR1 and UCR2 have been shown to form a module necessary for the activation of PDE4 upon phosphorylation by the cAMP-dependent kinase (PKA). Here we have uncovered PDE4 oligomerization as a novel function for the UCR1/UCR2 module. Using several different approaches including gel filtration, sucrose density gradient centrifugation, pull-down of differentially tagged PDE constructs, and yeast two-hybrid assay, we show that the long PDE4 splice variant PDE4D3 behaves as a dimer, whereas the short splice variant PDE4D2 is a monomer. Internal deletions of either the C-terminal portion of UCR1 or the N-terminal portion of UCR2 abolishes dimerization of PDE4D3 indicating that both domains are involved in this intermolecular interaction. The dimerization, however, is structurally distinguishable from a previously described intramolecular interaction involving the same domains. PKA phosphorylation and site-directed mutagenesis shown to ablate the latter do not interfere with dimerization. Therefore, dimerization of the long PDE4 forms may be an additional function of the UCR domains that further explains differences in the regulatory properties between the long and short PDE4 splice variants.The second messengers cAMP and cGMP are key molecules for transducing the action of extracellular signals such as hormones, neurotransmitters, and light into the most diverse cell functions, thus playing an important role in a wide array of physiological processes that include vision, cell growth and division, memory, and immune response (1-5). Intracellular cyclic nucleotide levels are determined by the rates of their synthesis by adenylyl cyclases and of their degradation through phosphodiesterases, enzymes that hydrolyze the phosphodiester bond and generate the corresponding 5Ј-nucleoside monophosphates.Cyclic nucleotide phosphodiesterases (PDEs) 1 compose a superfamily of isoenzymes that are divided into 11 PDE families on the basis of their sequence homology and enzymatic properties (2). They all share a highly conserved catalytic domain of about 270 amino acids fused to additional N-and/or C-terminal sequences that contain distinct domains unique to the members of a PDE family. These terminal domains determine several properties of the PDEs such as the regulation of enzyme activity by post-translational modifications (e.g. phosphorylation by PKA, PKB, ERK2, CaMK, and PKG, Refs. 6 -10) and binding of other messenger molecules (e.g. cGMP, Ca 2ϩ -calmodulin, and phosphatidic acid, Refs. 11-13), or by specifying the subcellular localization of the enzymes by protein-proteininteractions and membrane insertion (14 -16). The terminal domains of the PDEs, therefore, provide diverse modules for coordinating PDE activity with the overall signaling network specific to a cel...

show abstract

Section: Table I Physicochemical Properties Of the Pde4d Constructscontrasting

confidence: 39%

Dimerization of the Type 4 cAMP-specific Phosphodiesterases Is Mediated by the Upstream Conserved Regions (UCRs)

Richter

Conti

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…advantage of speed, economy, and efficacy compared to alternative expression systems such as the baculovirus system (17,18,23). All PDE4A constructs were found to be expressed as inclusion bodies and no free enzymatic activity could be detected.…”

Section: Figmentioning

confidence: 96%

Refolding, Purification, and Characterization of Human Recombinant PDE4A Constructs Expressed in Escherichia coli

Richter

Hermsdorf

Lilie

et al. 2000

Protein Expression and Purification

View full text Add to dashboard Cite

Phosphodiesterase Inhibitors: Factors That Influence Potency, Selectivity, and Action

Francis

Houslay

Conti

2011

Handbook of Experimental Pharmacology

View full text Add to dashboard Cite

Cyclic nucleotide phosphodiesterases (PDEs) are promising targets for pharmacological intervention. The presence of multiple PDE genes, diversity of the isoforms produced from each gene, selective tissue and cellular expression of the isoforms, compartmentation within cells, and an array of conformations of PDE proteins are some of the properties that challenge the development of drugs that target these enzymes. Nevertheless, many of the characteristics of PDEs are also viewed as unique opportunities to increase specificity and selectivity when designing novel compounds for certain therapeutic indications. This chapter provides a summary of the major concepts related to the design and use of PDE inhibitors. The overall structure and properties of the catalytic domain and conformations of PDEs are summarized in light of the most recent X-ray crystal structures. The distinctive properties of catalytic domains of different families as well as the technical challenges associated with probing PDE properties and their interactions with small molecules are discussed. The effect of posttranslational modifications and protein-protein interactions are additional factors to be considered when designing PDE inhibitors. PDE inhibitor interaction with other proteins needs to be taken into account and is also discussed.

show abstract

Purification and physical characterization of cloned human cAMP phosphodiesterases PDE-4D and-4C

Cited by 5 publications

References 42 publications

Dimerization of the Type 4 cAMP-specific Phosphodiesterases Is Mediated by the Upstream Conserved Regions (UCRs)

Dimerization of the Type 4 cAMP-specific Phosphodiesterases Is Mediated by the Upstream Conserved Regions (UCRs)

Refolding, Purification, and Characterization of Human Recombinant PDE4A Constructs Expressed in Escherichia coli

Phosphodiesterase Inhibitors: Factors That Influence Potency, Selectivity, and Action

Contact Info

Product

Resources

About