Specific cGMP binding by the cGMP binding domains of cGMP-binding cGMP specific phosphodiesterase

doi:10.1016/S0898-6568(01)00216-9

Cellular Signalling

Volume 14, Issue 1, January 2002, Pages 45-51

https://doi.org/10.1016/S0898-6568(01)00216-9 Get rights and content

Abstract

The structure of cyclic GMP (cGMP)-binding (cGB), cGMP specific phosphodiesterase (PDE5) comprises several domains. We have used RT-PCR methods to clone the noncatalytic cGB domains of PDE5 from human colon cancer cell RNA and constructed glutathione-S-transferase (GST) fusion proteins to express and study the domains. One fragment showed 94% identity to bovine PDE5 and coded for the high affinity cGB domain of PDE5 (Val¹⁵⁶–Asp³⁹⁴, cGB-I). Another cloned fragment showed 92% identity to bovine PDE5 and coded for the phosphorylation site plus both high and low affinity cGB domains of PDE5 (Val³⁶–Glu⁵²⁹, cGB-II). Both fragments expressed as GST–cGB fusion proteins bound cGMP specifically, as determined by competitive [³H]-cGMP ligand binding. We found that cGB-I showed high affinity cGMP binding with K_d=0.33 μM. cGB-II showed two cGMP binding sites with similar affinities and specificity to the native enzyme. cGB-II was phosphorylated by cGMP-dependent protein kinase (PKG) as reported for bovine PDE5. These data show that recombinant regulatory regions of PDE5 form cGB sites similar to native enzyme sites and confirm proposed domain functions. These results establish that recombinant fusion proteins of PDE5 domains may be used to further characterize the structure of PDE5.

Introduction

Cyclic nucleotide phosphodiesterases (PDEs, EC3.1.4.17) are classified in 11 gene families (PDE 1–11) according to substrate specificity, regulatory factors, and sensitivity to inhibitors [1], [2], [3], [4], [5]. PDE2, PDE5, PDE6, PDE10, and PDE11A4 have two homologous noncatalytic cyclic GMP (cGMP)-binding (cGB) sites (GAFa and GAFb) located in the N-terminal half of the enzyme. The cGB domains of PDE2, PDE5, and PDE6 share a conserved N(K/R)XnFX₃DE motif similarity[6], [7] while PDE10A and PDE11A4 contains two GAF domains in the N-terminal regions [4], [5]. Because these cGB sites are not homologous to other nucleotide binding sites, the structure and function of these sites are undefined.

cGMP binding and cGMP-specific PDE (PDE5) from bovine (PDE5A1) [6], [8], rat (PDE5A1 and PDE5A2) [9], human lung tissues (PDE5A1 and PDE5A2) [10], [11], and human penile cavernosum (PDE5A1, PDE5A2, and PDE5A3)[12] have been cloned. The deduced amino acid sequences of human PDE5 reveals 95% and 93% similarity to bovine and rat PDE5, respectively. The C-terminal catalytic domain of PDE5 has been cloned and when expressed as a recombinant protein, showed a strong preference for cGMP substrate (K_m=5.5 μM) similar to the native enzyme [13]. However, the function and regulation of the isolated noncatalytic binding sites of PDE5 have not been studied extensively as recombinant proteins. During the preparation of this manuscript, Ho et al. [14] published a functional isolated high affinity cGB domain of PDE5. However, neither the low affinity cGB domain of PDE5 nor the fungal GAF domain protein YKG9 bound cGMP. Our study shows that isolated recombinant N-terminal cGB domains can be made with similar high and low affinity cGMP binding properties and specific Ser92 phosphorylation features with fidelity to the native PDE5 enzyme.

Binding of cGMP to both allosteric sites of PDE5 has been suggested as a prerequisite for phosphorylation, but not for catalysis [15]. In vitro, PDE5 can be phosphorylated by cGMP-dependent protein kinase (PKG) and less efficiently by cAMP-dependent protein kinase A (PKA) at serine-92 [8], [16]. Phosphorylation is influenced by cGMP binding to allosteric sites [15], [16]. As with binding, the role of PDE5 phosphorylation in catalysis is not well studied. Phosphorylation of partially purified PDE5 by PKA resulted in an increased V_max for cGMP hydrolysis and decreased sensitivity to zaprinast inhibition [17]. Phosphorylation of recombinant PDE5 by PKG also increased its catalytic activities but with no change of sensitivity to either zaprinast or sildenafil [18]. Phosphorylation of PDE5 in intact vascular smooth muscle cells was correlated with increased catalytic activity [19]. These studies suggest that the binding of cGMP to both allosteric sites and/or phosphorylation of PDE5 may be involved in cellular regulation of PDE5.

PDE5 is important in regulating smooth muscle cell relaxation and signal transduction pathways [20]. We have recently found that PDE5 inhibition is also involved in colon tumor cell apoptosis induction [21]. To better understand the complex structure and phosphorylation regulations of PDE5, we have studied a domain containing an isolated cGMP high affinity site and a domain containing high and low affinity binding and phosphorylation sites using GST fusion recombinant proteins. Expressed cGB domains of PDE5 from HT29 colon cancer cells showed binding and phosphorylation with fidelity to the native protein. PDE5 inhibitors including exisulind and CP461 did not compete for cGMP binding. Site mutagenesis of serine 92 (S92A) confirmed the PKG phosphorylation site. Part of this data has been published in abstract form [22].

Section snippets

Materials

[8-³H]-cGMP was purchased from NEN Life Science Products (Boston, MA, USA). cGMP and cAMP were obtained from ICN Pharmaceuticals, (Costa Mesa, CA, USA). cIMP, cUMP, cCMP, 2′-O-monobutyl-cAMP and 2′-O-monobutyl-cGMP were from Biolog Life Science Institute (La Jolla, CA, USA). 8-Bromo-cGMP, 8-bromo-cAMP, and zaprinast were obtained from Calbiochem (San Diego, CA, USA). 8-Bromo-cGMP was further chromatography purified with sephadex G-25 (Amersham Pharmacia Biotechnology, Piscataway, NJ, USA) [23].

Cloning of cGB domain sequences from HT-29 cells

The 720-bp cDNA fragment obtained by RT-PCR coding for the high affinity cGB-I from HT-29 cells showed 94% identity to bovine PDE5A1 (561–1280 bp) and a deduced amino acid sequence relative to bovine PDE5 Val¹⁵⁶–Asp³⁹⁴ with 96% similarity. The 1484 bp fragment coding for the phosphorylation site (Ser⁹²) and both high and low affinity cGB sites (cGB-II) showed 92% identity to bovine PDE5 (204–1685 bp) and complete amino acid residue identity to human PDE5A1 (Val⁴⁶–Glu⁵³⁹) and PDE5A2 (Thr⁸–Glu⁴⁹⁷

Discussion

The results of these studies show the successful expression of cGMP binding domains of PDE5 in E. coli bacteria as GST fusion proteins. These two proteins are stable in solution, show high expression yields into correct sizes, and are readily purified by affinity chromatography. The recombinant GST fusion proteins retain the specific cGMP binding and PKG/PKA phosphorylation features of the native PDE5 protein. These studies compliment those using the catalytic domain of PDE5 [13].

The expression

Acknowledgements

We thank Dr. Sharron Francis from the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine for her help with preparation of this manuscript. We are also grateful to Linn Ayers and Mary David for their technical assistance in GST-recombinant protein preparation and Western blots.

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The phosphodiesterase 5 (PDE5) inhibitors, including sildenafil (Viagra™), vardenafil (Levitra™), and tadalafil (Cialis™) have been developed for treatment of erectile dysfunction. Moreover, sildenafil and tadalafil are used for the management of pulmonary arterial hypertension in patients. Since our first report showing the cardioprotective effect of sildenafil in 2002, there has been tremendous growth of preclinical and clinical studies on the use of PDE5 inhibitors for cardiovascular diseases and cancer. Numerous animal studies have demonstrated that PDE5 inhibitors have powerful protective effect against myocardial ischemia/reperfusion (I/R) injury, doxorubicin cardiotoxicity, ischemic and diabetic cardiomyopathy, cardiac hypertrophy, Duchenne muscular dystrophy and the improvement of stem cell efficacy for myocardial repair. Mechanistically, PDE5 inhibitors protect the heart against I/R injury through increased expression of nitric oxide synthases, activation of protein kinase G (PKG), PKG-dependent hydrogen sulfide generation, and phosphorylation of glycogen synthase kinase-3β — a master switch immediately proximal to mitochondrial permeability transition pore and the end effector of cardioprotection. In addition, PDE5 inhibitors enhance the sensitivity of certain types of cancer to standard chemotherapeutic drugs, including doxorubicin. Many clinical trials with PDE5 inhibitors have focused on the potential cardiovascular and anti-cancer benefits. Despite mixed results of these clinical trials, there is a continuing strong interest by basic scientists and clinical investigators in exploring their new clinical uses. It is our hope that future new mechanistic investigations and carefully designed clinical trials would help in reaping additional benefits of PDE5 inhibitors for cardiovascular disease and cancer in patients.
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On the other hand, the bands for fragments 89–518 and 98–518 became smeared in the presence of 2 mm cGMP and migrated much slower, implying that N-terminal residues 98–115 affect the conformation of the GAF domains upon cGMP binding. This phenomenon is consistent with previous reports that the N-terminal fragments or full-length holoenzyme migrated slower in the presence of cGMP on native gels (16, 35). To determine the impact of PDE5A N-terminal loop 98–147 on cGMP binding, we measured the cGMP dissociation constants of the various fragments.
The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98–147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.
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This chapter focuses on the atomic structures of cGMP- or cAMP-binding GAF domains from the PDEs and adenylyl cyclases. These include the X-ray crystal structures of the regulatory segments, consisting of tandem GAF domains, from the mammalian phosphodiesterase PDE2A, and from a cyanobacterial adenylyl cyclase. cAMP and cGMP are important second messengers in cells. Their levels are controlled by activities of adenylyl and guanylyl cyclases (AC or GC), the enzymes that catalyze their synthesis, and cyclic nucleotide phosphodiesterases (PDEs), which hydrolyze these 3′,5′ cyclic nucleotides to their inactive 5′-nucleotide monophosphates. Of the 11 PDE families identified in mammalian tissues, all contain an N-terminal regulatory segment and a C-terminal catalytic domain. The first crystal structures of cyclic nucleotide-binding GAF domains have answered several basic questions about the mode of ligand binding and dimerization, but have also raised many others. A putative GAF ligand was found when the cAMP-stimulated cyanobacterial adenylyl cyclase cyaB1 was shown to be negatively regulated through its GAF domains by Na⁺, with an IC50 of 14.3 mM. Ultimately, crystal or NMR structures will be needed for the various PDE domains with and without bound ligands to visualize the conformational changes that occur upon binding. Structures of the full-length holoenzymes may also be required to visualize the allosteric mechanism. Since these small molecule binding GAF domains are intimately involved in regulation of so many different enzymes, they are also likely to become good targets for drug development.
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This chapter gives an overview on cyclic GMP-specific cGMP-binding phosphodiesterase (PDE5), a class I mammalian PDE with a catalytic site that specifically hydrolyzes the phosphodiester bond of cGMP and an allosteric site that binds cGMP with high specificity. The single human PDE5 gene is located on chromosome 4q26, contains 23 exons, and encodes mRNA for three variants, PDE5A1, -A2, and -A3. The protein products, 100 kDa, 95 kDa, and 95 kDa, respectively, share similar properties and have different amino-termini, a result of alternative mRNA splicing. Phosphorylation of Ser-102 (human PDE5) by PKG or PKA is enhanced by cGMP binding to the regulatory region or ligand (cGMP or PDE5 inhibitors) occupation of the catalytic site. PDE5 is phosphorylated in intact cells in response to stimuli that elevate cGMP, but elevation of cAMP does not mimic this. Cloning of PDE5 cDNA led to identification of two conserved Zn²⁺ binding motifs in catalytic domains of all mammalian PDEs. Zinc that binds to PDE5 with high stoichiometry is effective at a far lower concentration than either Mn²⁺ or Mg²⁺ in supporting catalytic activity. An allosteric site in unphosphorylated PDE5 binds cGMP with a K_D ∼0.2-1.5 μM; affinity varies depending on conditions. Cyclic GMP in the anti conformation binds to GAF-A, which is composed of ∼110 residues. A recombinant protein containing only GAF-A binds cGMP with high affinity, and cGMP binding causes a large conformational change. GAF-A also provides high-affinity dimerization contacts between PDE5 monomers. GAF-B provides high-affinity dimerization contacts between PDE5 monomers and reduces affinity of the GAF-A subdomain for cGMP.

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^☆: This work was supported in part by NIH grant HL46494 (W.J.T.).

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Specific cGMP binding by the cGMP binding domains of cGMP-binding cGMP specific phosphodiesterase☆

Abstract

Introduction

Section snippets

Materials

Cloning of cGB domain sequences from HT-29 cells

Discussion

Acknowledgements

Pharmacol Ther

J Biol Chem

Am J Respir Crit Care Med

Proc Natl Acad Sci USA

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Eur J Biochem

Gene

Biochem Biophys Res Commun

Biochem Biophys Res Commun

J Biol Chem

EMBO J

Biochem J

J Biol Chem