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(Circulation Research. 2007;100:309.)
© 2007 American Heart Association, Inc.

Reviews

Overview of PDEs and Their Regulation

Kenji Omori, Jun Kotera

From the Discovery Research Laboratories, Tanabe Seiyaku Co Ltd, Saitama, Japan.

Correspondence to Kenji Omori, Discovery Research Laboratories, Tanabe Seiyaku Co Ltd, 2-50 Kawagishi 2-chome, Toda, Saitama 335-8505, Japan. E-mail k-omori{at}tanabe.co.jp

This Review is part of a thematic series on Phosphodiesterases, which includes the following articles:

Compartmentation of Cyclic Nucleotide Signaling in the Heart: The Role of Cyclic

Nucleotide Phosphodiesterases

Overview of PDEs and Their Regulation

Regulation of Phosphodiesterase 3 (PDE3) and Inducible cAMP Early Repressor (ICER) in the Heart

cAMP and cGMP Signaling Cross-Talk: Role of Phosphodiesterases and Implications for Cardiac Pathophysiology

cAMP Specific Phosphodiesterase-4 Enzymes in the Cardiovascular System: A

Molecular Toolbox for Generating Compartmentalized cAMP Signalling PDE5
David A. Kass Editor

	Abstract

Top Abstract Introduction PDE Family General Characteristics PDE Variants and Their... Gene Regulation in PDEs Enzymatic Regulation of PDEs... Subcellular Localization of... Phenotypes of Genetically... Conclusions References

 
Contraction and relaxation of vascular smooth muscle and cardiac myocytes are key physiological events in the cardiovascular system. These events are regulated by second messengers, cAMP and cGMP, in response to extracellular stimulants. The strength of signal transduction is controlled by intracellular cyclic nucleotide concentrations, which are determined by a balance in production and degradation of cAMP and cGMP. Degradation of cyclic nucleotides is catalyzed by 3',5'-cyclic nucleotide phosphodiesterases (PDEs), and therefore regulation of PDEs hydrolytic activity is important for modulation of cellular functions. Mammalian PDEs are composed of 21 genes and are categorized into 11 families based on sequence homology, enzymatic properties, and sensitivity to inhibitors. PDE families contain many splice variants that mostly are unique in tissue-expression patterns, gene regulation, enzymatic regulation by phosphorylation and regulatory proteins, subcellular localization, and interaction with association proteins. Each unique variant is closely related to the regulation of a specific cellular signaling. Thus, multiple PDEs function as a particular modulator of each cardiovascular function and regulate physiological homeostasis.

Key Words: cAMP • cGMP • cyclic nucleotide • cell signaling • phosphodiesterase inhibitor

	Introduction

Top Abstract Introduction PDE Family General Characteristics PDE Variants and Their... Gene Regulation in PDEs Enzymatic Regulation of PDEs... Subcellular Localization of... Phenotypes of Genetically... Conclusions References

 
Numerous cellular functions are regulated by second messengers, cAMP and cGMP (Figure 1). In the cardiovascular system, blood pressure is regulated by contraction and relaxation of vascular smooth muscle in association with vascular endothelial functions. Beating of cardiac myocytes is accurately controlled to pump blood out of the heart to other parts of the body according to environmental conditions. These events in hemodynamics are ingeniously regulated by extracellular stimulation through alteration of intracellular cyclic nucleotide levels, which are determined by a balance between their production and degradation by 3',5'-cyclic nucleotide phosphodiesterases (PDEs).^1–5 Downstream effector proteins of cAMP and cGMP are cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), cyclic nucleotide-gated ion channels, and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs), which are also called exchange proteins directly activated by cAMP (Epacs). PDEs are also downstream effectors of cAMP and cGMP. Recent studies on cyclic nucleotide–mediated signaling have revealed that the signal for each physiological event is independently regulated by compartmentation of certain signaling molecules.⁶ PDEs are closely related to the regulation of each specific transduction signal, and therefore multiple PDEs play important roles in modulating each cellular function. Our goal here is to give an overview of the expanding molecular characteristics of PDE families principally in humans.

View larger version (56K): [in this window] [in a new window]   Figure 1. Cyclic nucleotide signaling and regulation. Localization of fundamental molecules involved in cAMP and cGMP signaling is illustrated. Effector molecules of cAMP and cGMP are indicated by arrows from each cyclic nucleotide. Phosphorylation of PDEs by PKA and PKG is demonstrated by dotted arrows. Modulation of PDE activity by cGMP is shown by a thick arrow. Cellular and physiological outputs of cyclic nucleotide signaling are shown in gray-colored boxes. NPs indicate natriuretic peptides; NO, nitric oxide; NPRs, natriuretic peptide receptors; sGCs, soluble guanylyl cyclase; AC, adenylyl cyclase; Gs, GTP-binding protein subunit; GPCRs, G protein–coupled receptors; Epac, exchange protein directly activated by cAMP; CNG-channel, cyclic nucleotide-gated channel.

	PDE Family

Top Abstract Introduction PDE Family General Characteristics PDE Variants and Their... Gene Regulation in PDEs Enzymatic Regulation of PDEs... Subcellular Localization of... Phenotypes of Genetically... Conclusions References

 
PDEs are classified into classes I, II, and III. Mammalian PDEs, which belong to class I PDEs, have an HD domain (Pfam accession no. PF01966) in the C-terminal half and show high affinity for cAMP and/or cGMP. Protein domains involved in regulation of PDE enzymatic activity and subcellular localization are mainly present in the N-terminal half. Some PDEs have phosphorylation sites targeted by protein kinases and lipid modification sites (Figure 2).

View larger version (21K): [in this window] [in a new window]   Figure 2. Schematic representation of the eleven human PDE families. Representative members that constitute the 11 human PDE families are shown here. Each PDE protein is indicated by a thick line. Protein regions are represented by rectangles with patterns. N-terminal variation of PDE1A variants carrying N1, N2, and N3 sequences is shown in a box. Splice variants of PDE5, PDE10, and PDE11 families are also shown in boxes. The 3 isoforms in the PDE4 family are illustrated and boxed. PDE3 enzymes produced by alternative translation initiation are boxed. Reported phosphorylation sites are indicated with arrows. ERK phosphorylation site in PDE4A variants is absent.

Twenty-one class I PDE genes have been identified in human, rat, and mouse (Figure 3). They are categorized into 11 different families based on structural similarity such as sequence homology, protein domains, and enzymatic properties, including substrate specificity, kinetic properties, and sensitivity to endogenous regulators and inhibitors (Table 1 and the online supplemental Table, available at http://circres.ahajournals.org). Approximately 270 aa in the C-terminal catalytic domain are conserved, with a sequence identity of 35% to 50% among different PDE families. Some PDE families are composed of 2 to 4 subfamily genes showing sequence identity of more than 70% and having identical protein domains organization. Multiple transcriptional products, which are generated from most PDE genes by alternative splicing or transcription from distinct promoters, have actually been identified or predicted in human DNA databases. Thus, the mammalian PDE superfamily is composed of 21 genes and their multiple transcriptional splice variants.

View larger version (28K): [in this window] [in a new window]   Figure 3. Phylogenetic tree of human PDE families. The tree is created using NJ algorithm of PHILIP based on multiple alignment of amino acid sequences of PDEase I domain (Pfam accession no. PF00233) in human PDEs analyzed with CLUSTAL W.7 Each PDE family is indicated together with total aa number of reference sequence shown in parentheses. GAF-PDE subfamily is boxed.

View this table: [in this window] [in a new window]   Table 1. Biochemical Characteristics of Human PDE Families

According to the nomenclature used for each PDE isozyme (eg, HsPDE1A1), the first 2 letters indicate the animal species and the first Arabic number after PDE designates the PDE gene family. This number is followed by a single capital letter indicating a distinct subfamily gene. The last Arabic number indicates a specific splice variant or a specific transcript generated from a unique transcription initiation site (http://depts.washington.edu/pde/pde.html).

The unique characteristics of each PDE gene family are defined by protein domains located in the N terminal to the catalytic unit. As shown in Figure 2, approximately half of PDE gene families (PDE2, PDE5, PDE6, PDE10, and PDE11) have a protein domain termed GAF [Pfam accession no. PF01590] in tandem and are therefore designated GAF-PDE subfamily. The known functions of GAF domains are cGMP binding-mediated allosteric regulation and dimerization of GAF-PDEs. Some GAF domains have also been reported to bind cAMP. Other PDEs (PDE1, PDE3, PDE4, and PDE7–9) have no GAF domain and belong to the non–GAF-PDE subfamily. PDE1 contains a Ca²⁺/calmodulin (CaM)-binding site, PDE3 has a transmembrane domain, PDE4 has upstream conserved regions (UCRs), and PDE8 has a response regulator receiver (REC) domain [Response_reg; Pfam accession no. PF00072] and a per–arnt–sim (PAS) domain [Pfam accession no. PF00989]. PDE7 and PDE9 have no specific protein domain in addition to the PDE catalytic domain.

	General Characteristics

Top Abstract Introduction PDE Family General Characteristics PDE Variants and Their... Gene Regulation in PDEs Enzymatic Regulation of PDEs... Subcellular Localization of... Phenotypes of Genetically... Conclusions References

 
Fundamental information about the 11 human PDE gene families and PDE inhibitors are summarized in Table 1 and the online supplemental Table.

Phosphodiesterase 1
Three subfamily genes, PDE1A to -C, encode Ca²⁺/CaM-dependent cAMP- and cGMP-hydrolyzing PDEs. In humans, PDE1A shows high affinity for cGMP.⁸ PDE1B hydrolyzes cGMP with a K_m value lower and a V_max value higher than those for cAMP.⁹ High affinity for both cAMP and cGMP is observed with PDE1C.⁸

Phosphodiesterase 2
PDE2A hydrolyzes both cGMP and cAMP with similar maximal rates and relatively high K_m values. PDE2A is allosterically stimulated by cGMP binding to its GAF domain,¹⁰ which enables mutual regulation of both cAMP and cGMP signaling.

Phosphodiesterase 3
PDE3A and PDE3B are the subfamily genes of PDE3, which shows high affinity for both cAMP and cGMP. A low V_max value for cGMP compared with that for cAMP lets cGMP function as a competitive inhibitor for cAMP hydrolysis.² Therefore, PDE3s are termed cGMP-inhibited cAMP PDEs. The presence of a 44-aa insert in the catalytic domain is a unique characteristic of the PDE3 family. Another special feature is the presence of N-terminal hydrophobic membrane association domains (NHRs).¹¹

Phosphodiesterase 4
Four highly similar subfamily genes, PDE4A to -D, encode cAMP-specific rolipram-sensitive PDEs. The PDE4 family includes a number of splice variants categorized into 3 N-terminal variant groups ("long form," "short form," and "super-short form"¹²) based on the presence or absence of N-terminal UCR domains (Figure 2).¹³ The long form variants contain UCR1, linker region (LR) 1, UCR2, LR2, and the catalytic domain. The short form and the super-short form variants have LR1-UCR2-LR2 and UCR2 (truncated)-LR2 in the N-terminal region, respectively. UCR1, which includes 1 PKA phosphorylation site, is connected to UCR2 by LR1. UCR2 has a hydrophilic N-terminal region, which intramolecularly interacts with the hydrophobic C-terminal portion of UCR1.¹⁴ UCR1 and UCR2 are involved in PDE4 enzymatic regulation^15–17 through UCR2 interaction with the catalytic domain¹⁸ and have also been reported to participate in PDE4 dimerization.¹⁹ Although PDE4 catalytic site binds to the competitive inhibitor rolipram, the affinity of this site for rolipram can change markedly depending on the conformation of the enzyme. This can result in the so-called high-affinity rolipram-binding site, HARBS.

Phosphodiesterase 5
PDE5A, which has 2 GAF domains (GAF A and GAF B) in the N-terminal half, specifically hydrolyzes cGMP. PDE5A GAF A domain has been reported to be responsible for this enzyme allosteric binding to cGMP,²⁰ and therefore PDE5A is termed cGMP-binding cGMP-specific PDE. One PKG- and PKA-dependent phosphorylation site in the N-terminal region is related to activation of PDE5A enzyme.²¹ cGMP binding to PDE5A GAF A domain promotes phosphorylation, which not only activates the catalytic function but also increases cGMP-binding affinity.^21–23

Phosphodiesterase 6
In retinal rod and cone cells, the level of cGMP, a second messenger in visual signal transduction, is tightly controlled through regulated cGMP hydrolysis by 3 PDE6 subfamily genes (photoreceptor PDEs).⁵ Light-activated transducin stimulates PDE6 activity by removing the inhibitory subunit . Membrane hyperpolarization is caused by cGMP elimination, leading to electrical cellular response. PDE6 and PDE6ß subunits encoded by the PDE6A and PDE6B genes, respectively, form a holoenzyme, rod PDE, with 2 copies of the smaller inhibitory subunit (PDE6) encoded by PDE6G. GAF A domain is related to PDE6ß heterodimerization.²⁴ In cone cells, a homodimer of 2 ' subunits (PDE6') encoded by PDE6C comprises cone PDE with cone-specific inhibitory subunits encoded by PDE6H. Thus, regulation of PDE6 activity by small inhibitory subunits is a unique aspect of this PDE family. Sildenafil, vardenafil, and udenafil, but not tadalafil, inhibit PDE6 with substantially lower affinities than those for PDE5A.

Phosphodiesterase 7
Two subfamily genes, PDE7A and PDE7B, encode rolipram-insensitive high-affinity cAMP-specific PDEs (K_m value, approximately 0.2 µmol/L²⁵). A PKA pseudosubstrate site is present in the N terminus of PDE7A subfamily.²⁶ Dipyridamole nonselectively inhibits PDE7 activity.

Phosphodiesterase 8
PDE8A and PDE8B are the subfamily genes of PDE8. PDE8s are high-affinity cAMP-specific PDEs insensitive to rolipram and 3-isobutyl-1-methylxanthine (IBMX),²⁷ and contain REC and PAS domains in the N-terminal portion. REC domain functions as a receiver of signals from the sensor component in 2-component signal transduction system in lower organisms. PAS domain is involved in the binding of small ligands and protein–protein interaction. However, regulation of PDE8s via REC or PAS domain is unknown, and obvious endogenous PDE8 activity has not yet been demonstrated in either tissue or cell extracts. Actually, many studies on IBMX-insensitive cAMP PDE activity have been reported. It is true that this activity has, for the most part, not been definitively ascribed to PDE8, but it is very likely attributable to PDE8. Dipyridamole inhibits PDE8 activity but not selectively.

Phosphodiesterase 9
PDE9A specifically hydrolyzes cGMP with high affinity.^28,29 However, there is no report on the regulation of PDE9A activity or the presence of endogenous PDE9A activity in either tissue or cell extracts. IBMX-insensitive cGMP PDE activity, which has been shown so far, is likely attributable to PDE9.

Phosphodiesterase 10
PDE10A contains 2 N-terminal GAF domains and hydrolyzes both cAMP and cGMP.³⁰ High affinity for cAMP inhibits cGMP hydrolysis, making this enzyme a cAMP-inhibited dual-substrate PDE. Among newly discovered PDEs, the enzymatic activity of PDE10A is clearly demonstrated in tissue extracts.³¹ The enzymatic activity of a chimeric construct of PDE10A GAF domain and cyanobacterial adenylyl cyclase is stimulated by cAMP, suggesting a possible allosteric modulation of PDE10A activity by cAMP.³² Papaverine is known as the most potent inhibitor of PDE10A.³³

Phosphodiesterase 11
A full-length form, PDE11A4, contains 2 GAF domains and a catalytic domain. PDE11A hydrolyzes both cAMP and cGMP with similar K_m values.^34–36 Although a cyanobacterial adenylyl cyclase fused with PDE11A4 GAF domains is activated by cGMP,³² there is no report on the allosteric regulation of PDE11A enzyme. PDE11A activity has not yet been clearly demonstrated in tissue or cell extracts. Tadalafil has been shown to potently inhibit PDE11A activity still much less potently than it inhibits PDE5A.

	PDE Variants and Their Tissue-Expression Patterns

Top Abstract Introduction PDE Family General Characteristics PDE Variants and Their... Gene Regulation in PDEs Enzymatic Regulation of PDEs... Subcellular Localization of... Phenotypes of Genetically... Conclusions References

 
Phosphodiesterase 1
N-terminal and C-terminal variants of PDE1A are divided into 3 groups based on their N-terminal sequences (Figure 2). In humans, PDE1A5 (PDE1A2), PDE1A6 (PDE1A3), and PDE1A9 encode the N-terminal sequence N1, whereas PDE1A1, PDE1A4, and PDE1A8 have the N-terminal sequence N2. The N-terminal sequence N3 is encoded by PDE1A10 to -1A12.³⁷ These N-terminal sequences differ in Ca²⁺/CaM-binding domain, and therefore activation by Ca²⁺/CaM is N-terminal sequence dependent. PDE1As carrying the N2 sequence are activated by Ca²⁺/CaM, whereas activation of PDE1As with the N1 sequence is not statistically significant.³⁸ There is no report on the activation of PDE1As with the N3 sequence. In humans, PDE1A5 and PDE1A6 expression is brain specific, whereas PDE1A1 and PDE1A4 expression is ubiquitous but high in the kidney, liver, pancreas, and thyroid gland.^37,39 PDE1A10 expression is testis specific.³⁷ In mice, expression of the 61-kDa PDE1 protein (PDE1A) has been reported in the brain.⁴⁰

There are 2 N-terminal variants in humans, PDE1B1 and PDE1B2. PDE1B1 expression is predominant in the caudate nucleus and putamen of the brain³⁹ and low in the heart and skeletal muscle.⁴¹ PDE1B2 transcripts are found mostly in the spinal cord.³⁹ Detailed expression of the 63-kDa PDE1 protein (PDE1B) has been reported in mouse brain.⁴⁰

PDE1C1 and PDE1C3 are human N-terminal variants. Rat PDE1C2 encodes distinct N- and C-terminal sequences. PDE1C4 and PDE1C5, both of which encode the same 654-aa protein, are C-terminal variants of PDE1C1 in mice.⁴² Human PDE1C expression is high in the heart and brain.⁸ Rat PDE1C transcripts are very few in the heart but are abundant in olfactory epithelium.⁴⁴ Mouse PDE1C1 and PDE1C5 transcripts are very few in the heart but are abundant in the cerebellum.⁴²

Phosphodiesterase 2
The PDE2A gene encodes 3 N-terminal splice variants containing 2 GAF domains. PDE2A3, which is a human variant, is membrane associated probably because of its unique N-terminal sequence.⁴⁵ PDE2A3 transcripts are rich in the brain and moderate in the heart. Immunoreactive PDE2A protein is rich in the neocortex and low in other tissues, and its signals are also localized in capillary and venous endothelial cells and in microvessel endothelial cells but not in arterial endothelial cells of intact tissues.⁴⁶ In contrast, cultured endothelial cells exhibit PDE2 activity.⁴⁷

Phosphodiesterase 3
Transcripts of PDE3A1 (8.2 kb) and PDE3A2 (6.9 kb) carrying distinct 5' regions are produced by alternative transcription within exon 1 in human cardiovascular system.^11,48 These 2 transcripts encode 3 N-terminal variant forms, PDE3A-136 (136 kDa), PDE3A-118 (118 kDa), and PDE3A-94 (94 kDa) (Figure 2). PDE3A1 encodes PDE3A-136, which includes 2 N-terminal hydrophobic membrane association domains (NHR1 and NHR2) and the catalytic domain. PDE3A2 encodes PDE3A-118 (NHR2 to the catalytic domain) and PDE3A-94 (the catalytic domain), which are translated from alternative translation initiation sites.¹¹ In human placenta, the 4.4-kb transcript PDE3A3, which is generated from exon 4, encodes PDE3A-94.⁴⁹ Only PDE3A-136 has the Akt/protein kinase B (PKB) phosphorylation site, which is critical for enzymatic activation. PKA phosphorylation site is present in PDE3A-136 and PDE3A-118, but not in PDE3A-94. PDE3A1 expression is low in cardiac myocytes, but PDE3A2 expression is high in both cardiac and vascular myocytes.¹¹ PDE3A expression is observed in heart, vascular and placental smooth muscle, corpus cavernosum smooth muscle, and platelets.² Rat PDE3A transcripts are abundant in the myocardium, smooth muscle, epithelium, megakaryocytes, and oocytes.⁵⁰

Conversely, only PDE3B1 has been identified in humans. PDE3B transcripts are predominant in human adipose tissue.⁵¹ In rats, PDE3B expression is evident in white and brown adipose tissues and is also found in hepatocytes, renal collecting duct epithelium, and developing spermatocytes. PDE3B transcripts are also abundant in embryonic neuroepithelium including neural retina but not in mature nervous system.⁵⁰

Phosphodiesterase 4
PDE4A1 encodes a short form protein. Long forms of PDE4A proteins are encoded by PDE4A4B, PDE4A8, PDE4A10, and PDE4A11. In humans, PDE4A expression is fundamentally ubiquitous^52,53 and relatively high in the brain¹³ with variant-specific tissue distribution pattern. PDE4A4B transcripts are found in T cells,⁵² and PDE4A10 expression is high in the heart and small intestine.⁵⁴ PDE4A11 transcripts are widely observed in various tissues⁵⁵ with high expression in fetal brain but not in adult brain.⁵⁴ In rats, PDE4A transcripts are rich in brain neurons and the olfactory system.⁵⁶

The long forms PDE4B1 and PDE4B3 and the short form PDE4B2 are human PDE4B variants. PDE4B4, which encodes a long form protein, is an additional variant in rats.⁵⁷ PDE4B are widely distributed in various tissues with variant-specific tissue distribution pattern. In immune cells, PDE4B and PDE4D isoforms are predominant compared with PDE4A and PDE4C isoforms. Human PDE4B2 transcripts are abundant in leukocytes, especially neutrophils.⁵⁸ Transcripts of PDE4B2, which is the major PDE4B isoform in normal B cells, are abundant in naive and memory B cells and low in centroblasts and centrocytes.⁵⁹ In rats, PDE4B3 transcripts are present in the brain, heart, lung, and liver, whereas PDE4B4 expression is specific to the liver and brain.⁵⁷

The long forms PDE4C1, PDE4C2, and PDE4C3, which are generated from distinct promoters, have been identified in humans. PDE4C-54 and PDE4C-109 transcription is thought to be driven from a separated common promoter. PDE4C-791 is the 5' alternate of PDE4C1 but is predicted to encode the same protein. PDE4C expression is ubiquitous but has been reported to be low in the lung and absent in blood.⁵³ PDE4C-54 expression is testis specific. In general, expression of PDE4C variants is not fully understood.

In humans, PDE4D3 to -4D5 and PDE4D7 to -4D9 encode long forms, and PDE4D1 encodes a short form. Super-short forms are encoded by PDE4D2 and PDE4D6. In general, like PDE4B, PDE4D expression predominates in various tissues. However, PDE4D4 and PDE4D6 expression is brain specific, and PDE4D7 transcripts are ubiquitously distributed but are abundant in the lung and kidney. PDE4D8 transcripts are rich in the heart and skeletal muscle, suggesting muscle-specific expression.^61–63 PDE4D5 enzyme is the most dominant in well-differentiated human bronchial epithelium (WD-HBE) cells.⁶⁴ In rats, PDE4D1 to -4D3, PDE4D5, and PDE4D9 transcripts are broadly distributed in most tissues, whereas PDE4D4 and PDE4D6 to -4D8 show variant-specific tissue expression patterns.⁶⁵

Phosphodiesterase 5
N-terminal variants PDE5A1 to -5A3, which show similar K_m values (eg, 6 µmol/L) have been identified in humans (Figure 2).^66–68 One N-terminal phosphorylation site for PKA and PKG is conserved among all 3 variants. In humans, PDE5A transcripts are rich in various tissues, especially in smooth muscle tissues, and are also detected in platelets.^66,67 PDE5A1 and PDE5A2 transcripts are widely distributed.^67,68 In contrast, specific expression of PDE5A3 in smooth and/or cardiac muscle has been suggested.⁶⁸

Phosphodiesterase 6
Splice variants of PDE6A to -6C have not been reported. PDE6A and PDE6B transcripts are present in rod cells, whereas PDE6C transcripts are in cone cells.⁵

Phosphodiesterase 7
Three variant forms have been reported in human PDE7A subfamily. PDE7A1 and PDE7A2 are N-terminal variants,²⁶ and PDE7A3 is a C-terminal variant of PDE7A1.⁶⁹ PDE7A1 expression is ubiquitous, whereas PDE7A2 transcripts are confined to the heart, skeletal muscle, and kidney. PDE7A3 expression has been demonstrated in the heart, skeletal muscle, spleen, thymus, testis, and peripheral blood leukocytes.⁶⁹ In mice, PDE7A expression is highest in the skeletal muscle, followed by the spleen, uterus, heart, brain, and kidney but is insignificant in the testis.⁷⁰

Only PDE7B1 has been identified in humans and mice. In rats, there are 3 N-terminal splice variants, PDE7B1 to -7B3. Human PDE7B transcripts are observed in the caudate nucleus and putamen of the brain, heart, and several other tissues.⁷¹ In rats, PDE7B expression is particularly high in the testis and neuronal cells of several brain regions^72,73 and is also detected in the heart, lung, skeletal muscle, and kidney.⁷² In mice, PDE7B transcripts are rich in the pancreas and can also be found in the brain, heart, thyroid, and skeletal muscle.⁷⁴

Phosphodiesterase 8
In humans, 5 PDE8A splice variants, PDE8A1 to -8A5, have been identified. The longest form, PDE8A1, contains REC and PAS domains. PDE8A transcripts are expressed in various tissues and are abundant in the testis, ovary, small intestine, and colon.⁷⁵ In general, PDE8A1 expression is dominant as compared with that of other PDE8A variants.⁷⁶ In rats, PDE8A expression is high in the liver and testis.⁷⁷

Five splice variants of PDE8B have also been reported in humans. PDE8B1, the longest form, carries REC and PAS domains.²⁷ In humans, PDE8B transcripts are predominant in the thyroid gland and are low in most brain areas except the cerebellum.⁷⁸ Rat PDE8B transcripts are not confined to the thyroid gland. They are abundant in the brain and are detectable in neuronal cells of several brain regions other than the cerebellum.⁷⁷

Phosphodiesterase 9A
Although 21 splice variants of PDE9A have been identified in humans,⁷⁹ differences in functional characteristics and subcellular localization among these variants have not yet been reported in detail. PDE9A1 is the longest form of PDE9A variants. Expression of PDE9A transcripts is high in the spleen, small intestine, brain, colon, prostate, kidney, and placenta.^28,29 Transcripts of PDE9A1 and PDE9A6 (described as PDE9A5 in the literature) are predominant in some immune tissues.⁸⁰

Phosphodiesterase 10
Two major N-terminal variants, PDE10A1 and PDE10A2, and several minor variants have been identified in humans.^30,81 A PKA phosphorylation site in PDE10A2 is the most striking difference between PDE10A2 and PDE10A1.⁸² In most human tissues, PDE10A2 expression is higher than that of PDE10A1.⁸² PDE10A transcripts are particularly rich in the putamen, caudate nucleus, and testis.³⁰ PDE10A transcripts are present in neurons of the striatum, caudate nucleus, nucleus accumbens, and olfactory tubercles in rat brain.^31,83 In mice, PDE10A expression is highest in the testis and brain.⁸⁴ Histologically, PDE10A expression is high in the striatal medium spiny neurons (MSNs). However, signal intensity among brain areas in mice, rats, dogs, cynomolgus macaques, and humans is different.⁸⁵

Phosphodiesterase 11
Four N-terminal variants are encoded by PDE11A1 to -11A4 (Figure 2). PDE11A4 encodes the longest protein including 2 GAF domains and 2 N-terminal phosphorylation sites for PKA and PKG.³⁴ PDE11A variants are unique in showing various GAF domain organization.^34–36

In humans, PDE11A expression is strong in the prostate and moderate in the testis and several other tissues.^34,35 Transcripts of PDE11A1, PDE11A3, and PDE11A4 are mostly confined to the skeletal muscle, testis, and prostate, respectively.⁸⁶ Among PDE11A variant proteins, only PDE11A4 protein has been clearly detected in human tissues by immunochemical analysis.⁸⁷ Rat PDE11A4 transcripts are not prostate specific.⁸⁸

	Gene Regulation in PDEs

Top Abstract Introduction PDE Family General Characteristics PDE Variants and Their... Gene Regulation in PDEs Enzymatic Regulation of PDEs... Subcellular Localization of... Phenotypes of Genetically... Conclusions References

 
Expression of PDE transcripts has been reported to increase or decrease in various tissues; however, a molecular mechanism of PDE gene regulation has, in many cases, not been clarified in detail. In this section, regulation of PDEs in the cardiovascular and immune systems is highlighted.

Phosphodiesterase 1
Long-term nitrate treatment increases the expression of PDE1A1 transcripts and protein in rat aortas, which leads to nitrate tolerance.⁸⁹ PDE1B is known as an early response gene.⁹⁰ Granulocyte macrophage colony–stimulating factor upregulates PDE1B2 expression in differentiating monocytes.⁹¹ PDE1C expression is observed in proliferative phenotypes of human arterial smooth muscle cells (SMCs) but not in quiescent SMCs.⁹² Treatment with a stable prostacyclin derivative, 7-oxo-prostacyclin, upregulates PDE1C expression in rat heart.⁹³

Phosphodiesterase 2
PDE2A expression is induced by macrophage colony–stimulating factor in human monocytes differentiating into peritoneal macrophage.⁹¹ Epinephrine infusion has been reported to stimulate skeletal muscle PDE2A expression in young men.⁹⁴ Tumor necrosis factor (TNF) increases the levels of PDE2A3 transcripts in human umbilical vein endothelial cells (HUVECs) via p38 mitogen-activated protein kinase activation, which might lead to destabilization of endothelial barrier function.⁹⁵

Phosphodiesterase 3
Reduction in PDE3A expression has been reported in human failing hearts.⁹⁶ Angiotensin II and isoproterenol induce sustained downregulation of PDE3A expression and upregulation of inducible cAMP early repressor (ICER) expression in cardiac myocytes.⁹⁶ ICER induction represses PDE3A expression, leading to activation of cAMP/PKA signaling, which contributes to ICER expression. Sustained ICER induction leads to cardiomyocyte apoptosis. This is a unique autoregulatory positive feedback mechanism denoted as the PDE3A-ICER feedback loop.⁹⁷

PDE3B expression requires peroxisome proliferator–activated receptor (PPAR)⁹⁸ and is upregulated during differentiation to adipocytes in 3T3-L1 cells.⁹⁹ In contrast, treatment of fully differentiated 3T3-L1 adipocytes with TNF reduces PDE3B expression.¹⁰⁰ In addition, C₂-ceramide (a short-chain ceramide analog), which is involved in TNF signaling, decreases the level of PDE3B protein.¹⁰¹ Troglitazone, an antidiabetic drug, antagonizes ceramide action on PDE3B expression.

Phosphodiesterase 4
Expression of certain PDE4 isoforms is altered by rapid or chronic treatment with cAMP-stimulating agents.¹⁰² In human monocytic U937 cells, steady-state mRNA levels of PDE4A and PDE4B are upregulated by cAMP-stimulating agent ß agonist, or rolipram.¹⁰³ PDE4B expression is potently stimulated in monocytes by lipopolysaccharide via distinct transduction pathways.¹⁰⁴ Major PDE4 components, PDE4D3 and PDE4D5, are markedly downregulated during differentiation of monocytic U937 cells to macrophages, whereas PDE4B2 expression is induced.¹⁰⁵ Smoking elevates PDE4A4 and PDE4B2 transcription in peripheral blood monocytes, and significant PDE4A4 upregulation is observed in lung macrophages from smokers with chronic obstructive pulmonary disease compared with control smokers.¹⁰⁶ In rat heart, treatment with 7-oxo-prostacyclin, a stable prostacyclin derivative, causes upregulation of PDE4B3 transcription and downregulation of PDE4D1/2 and PDE4D3 expressions.⁹³

Rat PDE4D1/2 transcription is driven by a cAMP-responsive promoter, which is TATA-less and contains a number of GC-rich regions, Sp1, AP1, and AP2 sites and GC-rich regions.¹⁰⁷ Human PDE4D5 transcription starts from a putative promoter containing a number of CCAAT enhancer-binding protein binding sites (C/EBP) and 2 sites of cAMP response element (CRE).¹⁰⁸ Putative promoters for PDE4D6 to -4D8 also contain multiple CRE sites.⁶² The promoters for human PDE4A10 and PDE4A11 are TATA-less but include GC-rich islands and Sp1 site^54,55

Phosphodiesterase 5
Both human PDE5A1 and PDE5A2 promoters are TATA-less and contain putative Sp1 sites in the proximal region.^66,67 Lin et al¹⁰⁹ report PDE5A1 transcription from a further upstream region (318-bp upstream from the initiation ATG codon) and a core sequence for basal promoter activity. The PDE5A1 promoter is stimulated by cAMP or cGMP through Sp1 sites. The PDE5A2 promoter region, which is situated in an intron between the first exons for PDE5A1 and PDE5A2, is highly GC rich and includes potential Sp1 sites,^66,67,110 which are important for basal activity and responsiveness to cAMP and cGMP.¹¹⁰ Lin et al have analyzed PDE5A promoters with COS cells. However, whether these cells are suitable for this type of study is still questionable.

cAMP analogs stimulate transcription of PDE5A, especially PDE5A2 in rat vascular SMCs, whereas cGMP does not.^67,111 PDE5A upregulation is unlikely to occur during long-term tadalafil treatment accompanying cGMP stimulation in human corpora cavernosa SMCs.¹¹² No elevation of PDE5A protein, but a slight increase of PDE5A transcripts, has been observed on cGMP stimulation in Tunica albuginea fibroblasts.

Phosphodiesterase 6
The human PDE6A proximal promoter contains 2 indispensable cis elements, a Crx-binding element and an Nrl-response element (NRE).¹¹³ The human PDE6B minimal promoter includes ßAp1/NRE and the GC-rich sequence ß/GC necessary for in vitro interaction with Nrl and Sp1/Sp4 (especially Sp4) DNA-binding proteins, respectively.¹¹⁴ The molecular mechanism of PDE6C expression is currently unknown.

Phosphodiesterase 7
The proximal promoter region for human PDE7A1 and likely for PDE7A3 carries no typical TATA motif but contains a CpG island including 3 potential CRE sites.¹¹⁵ In Jurkat T cells, overexpression of CRE binding protein (CREB) increases the promoter activity. In human CD4⁺ T cells, T-cell activation quickly induces PDE7A1 transcription, whereas PDE7A3 induction is slow.⁶⁹ PDE7A1 expression is increased in response to intracellular cAMP levels in B lymphocytes.¹¹⁶ Phorbol myristate acetate stimulates the promoter activity, but the molecular mechanism of this stimulation is still unknown.

Expression of rat PDE7B is stimulated by cAMP stimulants in cultured striatal neurons via cAMP-dependent binding of CREB to CRE site in the putative PDE7B1 promoter region.¹¹⁷

Phosphodiesterase 8
Transcription of PDE8A1 is upregulated and reaches a maximum 8 hours after activation of human CD4⁺ T cells.⁶⁹

Phosphodiesterase 9 to 11
Regulation of the PDE9A, PDE10A, and PDE11A genes is unknown. Transcription of 2 major PDE10A variants, PDE10A1 and PDE10A2, start from the common promoter, which is highly GC rich and has neither a TATA motif nor a CAAT box.⁸¹ The putative promoters for PDE11A1 and PDE11A3 contain a TATA motif. PDE11A4 promoter lacks the TATA motif but includes a GC-rich region, with a CCAAT box and Sp1 site.⁸⁶

	Enzymatic Regulation of PDEs by Phosphorylation and Association Proteins

Top Abstract Introduction PDE Family General Characteristics PDE Variants and Their... Gene Regulation in PDEs Enzymatic Regulation of PDEs... Subcellular Localization of... Phenotypes of Genetically... Conclusions References

 
Regulators of PDEs such as kinases and association proteins are listed in Table 2 and illustrated in Figure 4.

View this table: [in this window] [in a new window]   Table 2. Regulatory Kinases and Association Proteins for PDEs

View larger version (32K): [in this window] [in a new window]   Figure 4. Regulation of enzymatic activity in PDE families. Expressive examples of enzymatic regulation in PDE1 to -5 are schematically illustrated. PDE proteins are shown as in Figure 2. Changes in the shape of each PDE enzyme show alteration of enzymatic activity. Activation and reduction of catalytic activity are indicated by open upward and downward arrows, respectively. ERK phosphorylation site is absent in PDE4A variants.

Ca²⁺/CaM binding to the N-terminal binding site of PDE1s causes marked stimulation of PDE activity. The binding affinities of PDE1A and PDE1B are reduced by PKA- and CaM kinase II–dependent phosphorylation, respectively.^118,119 PKA activation has been reported to reduce PDE1C activity in AtT20 cells.¹²⁰

PDE2A activation by Ca²⁺/phospholipid-dependent protein kinase has been reported in the liver Golgi–endosomal fraction; however, there is no direct evidence for PDE2A phosphorylation.¹²¹ Phosphorylation of rat PDE2A2 by its associated protein kinase inhibits PDE activity.¹²² However, the molecular basis and enzymatic alteration by PDE2 phosphorylation have not yet been investigated in detail.

Activation of PDE3s by PKA- and PKB-mediated phosphorylation has already been discussed (Figure 2).² PKA-mediated PDE3A activation functions as negative-feedback regulation in cAMP signaling, and PKB-mediated PDE3B activation in adipocytes greatly contributes to insulin action. However, reported phosphorylation sites of PDE3s are puzzling. In humans, PDE3A is phosphorylated at SHRRTS312 by PKA.¹²³ Human PDE3A contains a site, KXRXXS292, that is similar but not identical to the typical PKB phosphorylation motif RXRXXS. Although PKB phosphorylation of this site is intriguing, detailed analysis of human PDE3A phosphorylation has not been reported. Little is known about PKA- and PKB-mediated phosphorylation of rat PDE3A, which has potential phosphorylation sites.

With regard to PDE3B, human PDE3B contains typical PKB (Ser295) and PKA phosphorylation motifs. However, there is no report on the phosphorylation of these sites. In rat adipocytes, phosphorylation of PDE3B at MFRRPS302 by PKA and/or PKB¹²⁴ and at QLRRSS427 (Ser442 in humans) by PKA¹²⁵ has been reported. Mouse PDE3B is phosphorylated by insulin-stimulated PKB at RPRRRS273 (Ser295 in humans, Ser279 in rats), MFRRPS296 (Ser318 in humans, Ser302 in rats), and QLRRSS421 (Ser442 in humans).^126,127 Ser273 in mouse PDE3B is situated in a typical PKB phosphorylation site, but the other 2 sites, "RRXS," are fundamentally consensus PKA phosphorylation sites. In addition to PKA and PKB phosphorylation, human PDE3A enzyme is phosphorylated by PKC.¹²³

PDE4 activity is regulated by phosphorylation in UCR1 by PKA and in the C-terminal region by extracellular signal-regulated kinase (ERK), but the regulation is isoform dependent.^3,15,16 PKA phosphorylation of the long forms PDE4A4 and PDE4D3 causes their activation probably through disruption of UCR1-UCR2 interaction and UCR2 association with the catalytic region.^{18,19,128,129} ERK phosphorylates a consensus motif, RXSP, in cooperation with a kinase interaction motif (KIM) situated within the catalytic domains of PDE4B, PDE4C, and PDE4D.¹⁶ ERK phosphorylation of the long form PDE4D3 reduces cAMP-hydrolytic activity (ca. 50% inhibition), which in turn leads to PKA activation. Consequently, PKA phosphorylates PDE4 long forms, which escapes ERK-mediated inhibition. Conversely, ERK phosphorylation stimulates activity of the short forms PDE4D1 and PDE4B2 but does not affect that of the super-short form, suggesting the involvement of UCRs in this regulation.^16,130 Regulation of PDE4 activity by PKA and ERK phosphorylation depends on PDE4 isoforms and ERK regulatory components in the cells.

The long forms of PDE4 enzymes are activated by phosphatidic acid (acidic) and phosphatidylserine (acidic) but not by phosphatidylcholine (neutral). Alterations in the enzymatic properties of PDE4 isoforms by phosphatidic acid and PKA are similar and exclusive.¹³¹ Phosphatidic acid is believed to bind to a specific region of UCR1.¹⁷

Immunophilin XAP2 specifically binds to PDE4A5, resulting in inhibition of PDE4 activity, elevation of rolipram sensitivity, and reduction of PKA phosphorylation.¹³² The motif EELD in UCR2 of PDE4A5 and the C-terminal tetratricopeptide repeat (TPR) domain of XAP2 are involved in this interaction. DISC1 (Disrupted In SChizophrenia 1 gene), a key factor for psychiatric illnesses, interacts with UCR2 of PDE4B and inhibits PDE4B activity. Increased cAMP dissociates DISC1 from PDE4B via PKA activation and reverses PDE4B activity.¹³³ Rat PDE4A5 binds more efficiently to SH3 domains of several tyrosyl kinases, especially Lyn and Fyn, through an SH3-binding site within LR2.¹³⁴ This association with SH3 domains significantly reduces PDE activity and increases sensitivity to rolipram.¹³⁵

There are several reports on the regulation of PDE5 by phosphorylation. PDE5 activity is increased by PKA/PKG phosphorylation at Ser102 of human PDE5A1 in vitro.²¹ Although copurification of PDE6 (P) with PDE5¹³⁶ has been reported, the physiological significance of this association is unclear, owing to no inhibitory effect of PDE6 on PDE5 activity in vitro.¹³⁷ In contrast, PDE6ß is inhibited by PDE6 (see section General Characteristics, above). Association of PDE8A1 PAS domain with IB stimulates PDE8A activity but does not affect nuclear factor B activation.¹³⁸

Enzymatic regulation of other PDEs is not well understood. The N-terminal sequences of PDE7A, PDE7B, PDE10A2, and PDE11A4 are phosphorylated by PKA in vitro and/or in vivo. However, there is no evidence of enzymatic regulation of these PDEs by phosphorylation. Phosphorylation of PDE8s carrying putative PKA phosphorylation sites has not yet been investigated. There is no report on PDE9A phosphorylation.

	Subcellular Localization of PDEs, Interaction With Association Proteins, and Compartmentation of Cyclic Nucleotide Signaling

Top Abstract Introduction PDE Family General Characteristics PDE Variants and Their... Gene Regulation in PDEs Enzymatic Regulation of PDEs... Subcellular Localization of... Phenotypes of Genetically... Conclusions References

 
The PKA regulatory subunit binds to the PKA catalytic subunit, and the resultant complex is anchored to particulate fractions. cAMP binding to the regulatory subunit of PKA releases the catalytic subunit for enzymatic activation. A-kinase (PKA) anchoring proteins (AKAPs) play a role in PKA anchoring and function as scaffold molecules for many association proteins.⁶ Localization of each AKAP is among the important determinants of specific cAMP signaling in cells. With regard to PKG, association of G-kinase (PKG) anchoring proteins (GKAPs) with PKGs has been reported. Membrane anchoring of PKG II is mediated by N-terminal myristoylation. Cyclic nucleotides are generated in limited cellular space where their producer enzymes and/or receptors are located; however, target proteins for cyclic nucleotides are widely distributed. Some PDEs are reported to be localized in microdomains and/or to interact with association proteins including scaffold proteins. Regulation of intracellular PDE localization is recognized as among the key mechanisms in compartmentation of cyclic nucleotide signaling. Association proteins for PDEs are listed in Table 2 and illustrated in Figure 4.

Phosphodiesterase 1
PDE1A protein is predominant in the cytosol of contractile vascular SMCs but is localized in the nucleus of neointimal synthetic vascular SMCs.¹³⁸ Nuclear PDE1A is involved in regulation of cell proliferation and apoptosis of actively growing synthetic vascular SMCs.¹³⁹ However, the molecular mechanism of this altered localization is currently unclear. Diffuse cytoplasmic distribution of PDE1B protein has been shown in chick dorsal root ganglion neurons.

Phosphodiesterase 2
Rat PDE2A protein anchors to the membrane with the hydrophobic N-terminal portion¹⁴⁰ and is localized at lipid rafts in neurons.¹⁴¹ In cultured neonatal rat ventriculocytes, PDE2A protein is confined to membrane compartment with the sarcomeric Z line. Cyclic nucleotide signaling molecules such as ß-adrenoceptor, adenylyl cyclase, and nitric oxide synthase, are also localized with PDE2A in the lipid rafts, suggesting coupling of these molecules in cardiac signal transduction.¹⁴²

Phosphodiesterase 3
NHR modules greatly contribute to intracellular localization of PDE3 proteins. PDE3A-136 containing 2 NHRs is exclusively membrane bound. PDE3A-118 carrying NHR2 and PDE3A-94 containing no NHR module are found in both membrane-bound and cytosolic fractions of cardiac and vascular myocytes.¹¹ PDE3A and PDE3B proteins containing 2 NHRs are localized in the endoplasmic reticulum.¹⁴³ PDE3B protein is associated with caveolae and lipid raft in plasma membrane fractions of adipocytes, suggesting functional importance of this association in insulin signaling.¹⁴⁴ A scaffold molecule, 14-3-3ß protein, interacts with rat PDE3B protein phosphorylated via phosphatidylinositol 3-kinase/PKB pathway in adipocytes.¹²⁷ 14-3-3 protein also binds to phosphorylated human PDE3A at Ser428 by PKC.¹²³

Phosphodiesterase 4
PDE4 isoforms show unique intracellular distribution patterns characterized by interaction with several anchoring or targeting proteins such as immunophilin XAP2, DISC1, SH3 domain–containing proteins, ß-arrestin, receptor for activated C-kinase (RACK1), and AKAPs.

ß-Arrestin downregulates ß₂-adrenoceptor signaling by inhibiting further interaction with guanine nucleotide binding proteins (G proteins) (Figure 5). Adrenergic stimulation causes time-dependent recruitment of ß-arrestins and PDE4D isoforms to ß₂-adrenoceptor at the cell membrane level.¹⁴⁵ ß-Arrestin 1 and 2 bind to PDE4 isoforms of all 4 subfamilies through interaction with the C-terminal catalytic domain. PDE4D5 has a distinct ß-arrestin–binding site in the N-terminal region.¹⁴⁶ PKA colocalized with AKAP79¹⁴⁷ is first activated by cAMP and then phosphorylates the receptor, which causes G_s-to-G_i switch and ERK1/2 activation.¹⁴⁸ G protein–coupled receptor kinase recruited to the membrane phosphorylates ß₂-adrenoceptor, and then ß-arrestin/PDE4 complex is recruited to the receptor. This PDE4 recruitment contributes to local cAMP degradation, reduction of PKA activity, and modulation of ß₂-adrenoceptor function, thus constituting a negative-feedback regulation. Moreover, the recruitment may also regulate ERK signaling.^148,149 Regulation of PDE4 by this mechanism is largely dependent on the expression profile of PDE4 isoforms and coexisting PDE4-scaffolding proteins in the cell.

View larger version (60K): [in this window] [in a new window]   Figure 5. Compartmentation of PDE4 by ß-agonist stimulation. Proteins involved in the regulation of cAMP signaling derived from stimulated ß2-adrenoceptor are illustrated. cAMP produced by adenylyl cyclase stimulated by Gs activates PKA. PKA-mediated phosphorylation of ß2-adrenoceptor causes Gs-to-G switch and activation of ERK signaling. Phosphorylation of ß2-adrenoceptor by G protein–coupled receptor kinase (GRK) recruits ß-arrestin associated with PDE4 (PDE4D5). Association of PDE4D5 with RACK1 is involved in cytosolic localization of PDE4D5.

Ligation of T-cell Receptor (TCR) with CD28 costimulation causes T-cell activation by cAMP reduction through recruitment of ß-arrestin/PDE4 complex to lipid raft in microdomain. Regulation of compartmentalized cAMP signaling by ß-arrestin/PDE4 complex is considered to be crucial to T-cell regulation.¹⁵⁰

RACK1 specifically and directly binds to the N-terminal ß-arrestin–binding site of PDE4D5.¹⁴⁷ RACK1-PDE4D5 complex is present in cytosolic fractions. Because the binding of RACK1 and ß-arrestin to PDE4D5 is mutually exclusive, RACK1 might direct cytosolic localization of PDE4D5 by competing with ß-arrestin. RACK1 interaction does not alter PDE4D activity but increases its sensitivity to rolipram.

AKAPs are the most important molecules in compartmentation of cAMP/PDE4 signaling.⁶ AKAP149 (mitochondria), AKAP95 (perinucleus), and muscle AKAP (mAKAP) (perinucleus¹⁵¹) bind to PDE4A, PDE4A, and PDE4D3, respectively (Table 2). Nuclear membrane anchoring of mAKAP directs associating PDE4D3 to localize around the nucleus. PKA phosphorylation of PDE4D3 at Ser13 enhances the affinity of PDE4D3 for mAKAP, which functions as perinuclear negative-feedback regulation of cAMP signaling in addition to PKA-mediated activation.¹⁵² Furthermore, PDE4D3 acts as an adaptor protein of Epac1. Thus, mAKAP constitutes a complex with PKA, PDE4D3, and Epac1 for regulation of cAMP signaling.¹⁵¹ AKAP450, myomegalin, and myeloid translocation gene (MTG), which anchor to the Golgi, the Golgi, and the centrosome, interact with PDE4A, PDE4D3, and PDE4D3, respectively. PDE4 association with AKAPs has also been shown to prevent AKAP-anchored PKA from activation by cAMP at basal levels.¹⁵³

TAPAS-1 (tryptophan anchoring phosphatidic acid selective-binding domain 1) domain participates to PDE4A1 membrane anchoring through interaction with phosphatidic acid in phospholipid vesicles.¹⁵⁴ Membrane-anchored PDE4A1 is subject to Ca²⁺-stimulated intracellular redistribution depending on phospholipase D–mediated phosphatidic acid generation, which allows crosstalk among cAMP, phospholipase D, and Ca²⁺ signaling pathways.¹⁵⁵

Phosphodiesterase 5
Cellular localization of PDE5 is cytosolic. PDE5A is localized in Z band in cardiac myocytes, suggesting an association of PDE5 with certain proteins.¹⁵⁶ However, there is no evidence of direct binding. Sequestration of cGMP by localized cGMP-binding proteins such as PDE5 and PKG might be involved in the spatial regulation of cGMP signaling.¹⁵⁷

Phosphodiesterase 6
C termini of rod PDE (PDE6 and PDE6ß) contain a carboxyl-terminal motif for prenylation and carboxyl methylation. PDE6 and PDE6ß, which are farnesylated and geranylgeranylated, respectively, are membrane anchored. The small subunit PDE6, which is a prenyl-binding protein,¹⁵⁸ directly interacts with rod PDE and dissociates the complex from the rod outer segment disc membrane without changing PDE activity.¹⁵⁹

Phosphodiesterase 7
PDE7A2 with an N-terminal hydrophobic region is localized to particulate fractions.²⁶ PDE7A1 colocalizes with the RII PKA regulatory subunit in the Golgi–centrosome region of ß-TC3 insulinoma cells.¹⁶⁰ N-terminal PKA pseudosubstrate sites in human PDE7A1 bind to PKA catalytic subunits and inhibit kinase activity.^26,160 Myeloid translocation gene (MTG) protein also binds to and colocalizes with PDE7A in the Golgi of HuT78 cells.¹⁶¹ PDE7B localization is unknown. Recombinant human PDE7B1 protein with an N-terminal FLAG-tag expressed in COS-7 cells is cytosolic.⁷¹

Phosphodiesterase 8
A consensus sequence for myristoylation/palmitoylation motif, MGCAP, is present in human PDE8A1 and PDE8B1. However, subcellular localization of PDE8 proteins is unknown. N-terminal Xpress-tagged human PDE8A1 and PDE8B1 proteins are found in both cytosolic and particulate fractions of COS-7 cells.²⁷

Phosphodiesterase 9
PDE9A1, PDE9A16, and PDE9A17 have a pat7 nuclear localization signal, PLRDRRV. Nuclear localization of recombinant PDE9A1 is determined by the pat7 motif.⁸⁰

Phosphodiesterase 10
Recombinant proteins of PDE10A1 and PDE10A3 are cytosolic.¹⁶² By contrast, endogenous PDE10A2 protein from rat striatum as well as recombinant PDE10A2, which is found in the Golgi apparatus of transfected PC12H cells, is dominant in membrane fractions. By PKA phosphorylation, PDE10A2 alters subcellular localization from Golgi to cytosol.¹⁶² cAMP signaling in Golgi and cytosol of neurons is hypothesized to be controlled through alteration of PDE10A subcellular localization by PKA.

Phosphodiesterase 11
Subcellular localization of PDE11A is unknown. N-terminal Xpress-tagged recombinant PDE11A proteins are cytosolic in COS-7 cells.^34,88

	Phenotypes of Genetically Engineered Animals

Top Abstract Introduction PDE Family General Characteristics PDE Variants and Their... Gene Regulation in PDEs Enzymatic Regulation of PDEs... Subcellular Localization of... Phenotypes of Genetically... Conclusions References

 
Phenotypes of genetically engineered animals are summarized in Table 2. In this regard, there is no report on PDE1A, PDE1C, PDE2A, PDE4C, PDE5A, PDE6A-C, PDE7B, PDE8A-B, and PDE9A.

Phosphodiesterase 1
PDE1B-deficient mice¹⁶³ show increased locomotor activity and deficits in spatial learning.

Phosphodiesterase 3
Female PDE3A-deficient mice¹⁶⁴ are infertile because of immature oocytes, demonstrating the involvement of PDE3A in oocyte maturation and fertilization. However, alterations in contraction and relaxation of cardiac and vascular myocytes and platelet aggregation in PDE3A^–/– mice have not yet been reported.

PDE3B transgenic mice (ß-cell specific) demonstrate impaired acute insulin response to intravenous glucose loads and reduced insulin secretion in islets in vitro.¹⁶⁵ PDE3B plays a crucial role in regulation of cAMP signaling in ß-cell insulin secretion.

Phosphodiesterase 4
Knockout mice for PDE4A (PDE4A^–/–), PDE4B (PDE4B^–/–), and PDE4D (PDE4D^–/–) have been reported.^166,167 Although there are only a few reports on PDE4A^–/– mice, it is believed that no obvious phenotype exists in cardiac or immune system.

Immune response is altered in PDE4B^–/– mice.^167,168 PDE4B^–/– mice have impaired lipopolysaccharide-stimulated TNF production and are resistant to lipopolysaccharide-induced shock. PDE4B has critical function in lipopolysaccharide signaling. In an endotoxin inhalation–induced lung injury model, recruitment of neutrophils is markedly decreased in PDE4D^–/– and PDE4B^–/– mice. CD18 expression and chemotaxis response are decreased in PDE4D^–/– and PDE4B^–/– neutrophils.¹⁶⁹ Neutrophil function is regulated by PDE4B and PDE4D.

PDE4D^–/– but not PDE4B^–/– mice exhibit impaired airway contraction induced by cholinergic stimulation and abolished airway hyperreactivity caused by exposure to allergen, indicating the implication of PDE4D gene in cholinergic airway responsiveness and in development of hyperreactivity.^170,171

Progressive cardiomyopathy, accelerated heart failure after myocardial infarction, and cardiac arrhythmias are observed in PDE4D^–/– mice.¹⁷² PDE4D3 is associated with RyR2/calcium-release-channel complex, which is required for heart muscle excitation/contraction. Depletion of PDE4D3 in RyR2 complex enhances PKA phosphorylation of the complex and affects controlled intracellular Ca²⁺ release, resulting in cardiac dysfunction and arrhythmia.

In the central nervous system, PDE4D has been reported to be linked to cAMP signaling of ₂-adrenoceptor in noradrenergic neurons, which may explain the emetic side effect of PDE4 inhibitors.¹⁷³

Phosphodiesterase 6
Mutations in genes generating defective PDE6 enzymes (mainly rod PDE6ß) cause high-level cGMP accumulation in photoreceptor cells leading to cell death.¹⁷⁴

Phosphodiesterase 7
T-lymphocyte activation has been proposed to be linked to PDE7A activity.¹⁷⁵ However, PDE7A knockout mice (PDE7A^–/–) have been reported to show normal in vitro and in vivo T-cell functions, indicating that T-cell activation does not require PDE7A activity.¹⁷⁶

Phosphodiesterase 10
PDE10A knockout (PDE10A^–/–) mice¹⁷⁷ show decreased exploratory activity and delayed acquisition of conditioned avoidance behavior. A blunted locomotor response is observed in PDE10A^–/– mice following administration of antagonists for the ionotropic N-methyl-D-aspartate receptor that induce locomotor hyperactivity. PDE10A has a particular role in the responsiveness of MSNs to glutamatergic stimulation.

Phosphodiesterase 11
PDE11A knockout mice (PDE11^–/–)¹⁷⁸ demonstrate impaired sperm function and spermatogenesis, suggesting involvement of PDE11A activity in spermatogenesis. In humans, a protein corresponding to PDE11A3, which is a testis-specific variant, is undetectable in testicular tissue,⁸⁷ whereas PDE11A4 protein is present in the prostate, where prostatic fluid involved in sperm activity is produced. The effects of PDE11A disruption on prostatic function are unknown, and therefore the physiological role of PDE11A should not be linked simply to testicular functions.

	Conclusions

Top Abstract Introduction PDE Family General Characteristics PDE Variants and Their... Gene Regulation in PDEs Enzymatic Regulation of PDEs... Subcellular Localization of... Phenotypes of Genetically... Conclusions References

 
A number of splice variants have been identified in each PDE family, as described here. In regard to certain variants of classical PDEs (PDE1 to -6), molecular characteristics, tissue expression patterns, and expression regulation, all of which are indispensable information to figure out PDE functional roles in cells, have to some extent been investigated. By contrast, less information on newly discovered higher-numbered PDE families (PDE7 to -11) is currently available. Lack of clear evidence for the presence of apparent activity in most of the newly discovered PDEs in tissues and cells is the major obstacle to picture a precise and detailed network of intracellular signaling, where these PDE enzymes are involved in regulation of cellular functions.

Functional roles of PDEs have been studied using PDE family–specific inhibitors to demonstrate pharmacological effects of inhibition of each PDE family. However, PDE inhibitors cannot distinguish isoform-specific and variant-specific functions of PDE. Therefore, isoform-specific, highly selective PDE inhibitors, RNA interference, and genetically engineered animals, such as variant-specific knockout animals, seem to be potent and plausible tools to solve the specificity issue. Moreover, comparative studies looking at the whole picture of PDEs would be very important to evaluate the involvement of each PDE in specific cellular function and to understand regulation of cyclic nucleotide signaling. Establishment of a standardized platform for PDE research is necessary to interpret further intriguing observations.

	Acknowledgments

 
Sources of Funding

The preparat