Figure 1: Role of PDE3 in cAMP- and cGMP-mediated signal transduction. PK-A: Protein kinase A (cAMP dependent). PK-G: Protein kinase G (cGMP-dependent).
PDE3 is a phosphodiesterase. The PDEs belong to at least eleven related gene families, which are different in their primary structure, substrate affinity, responses to effectors, and regulation mechanism. Most of the PDE families are composed of more than one gene. PDE3 is clinically significant because of its role in regulating heart muscle, vascular smooth muscle and platelet aggregation. PDE3 inhibitors have been developed as pharmaceuticals, but their use is limited by arrhythmic effects and they can increase mortality in some applications.
Both PDE3A and PDE3B are expressed in vascular smooth muscle cells and are likely to modulate contraction. Their expression in vascular smooth muscle is altered under specific conditions such as elevated cAMP and hypoxia.[1]
The PDE3 family in mammals consists of two members, PDE3A and PDE3B. The PDE3 isoforms are structurally similar, containing an N-terminal domain important for the localization and a C-terminus end.[2] The 44-amino acid insertion in the catalytic domain differs in the PDE3 isoforms, and the N-terminal portions of the isoforms are quite divergent. PDE3A and PDE3B have strikingly similar pharmacological and kinetic properties, but the distinction is in expression profiles and affinity for cGMP.[3]
The PDE3 family is composed of two genes, PDE3A and PDE3B. In cells expressing both genes, PDE3A is usually dominant. Three different variants of PDE3A (PDE3A1-3) are products of alternate startcodon usage of the PDE3A gene. The PDE3B encodes a single isoform only.[1][4]
In their full-length both PDE3A and PDE3B contain two N-terminal hydrophobic membrane association regions, NHR1 and NHR2 (figure 2). The difference of the PDE3A1-3 variants lies in whether they include:
PDE3A is mainly implicated in cardiovascular function and fertility but PDE3B is mainly implicated in lipolysis.[3] Table 1 is an overview of localization of the PDE3 isoforms.
PDE3A can be either membrane-associated or cytosolic, depending on the variant and the cell type it is expressed in.[1]
Regulation
PDE3A and PDE3B activity is regulated by several phosphorylation pathways. Protein kinase A and protein kinase B both activate PDE3A and PDE3B via phosphorylation at two different phosphorylation sites (P1 and P2) between NHR1 and NHR2 (figure 2). Hydrolysis of cAMP by PDE3 isoforms is also directly inhibited by cGMP, although PDE3B is only ≈10% as sensitive to cGMP inhibition as PDE3A.[4] The PDE3B has been extensively studied for its importance in mediating the antilipolytic and antiglycogenlytic effect of insulin in adipose and livertissues. The activation of PDE3B in adipocytes is associated with phosphorylation of serineresidue by an insulin-stimulated protein serine kinase (PDE3IK). By blocking insulin activation of PDE3IK, and in turn phosphorylation/activation of PDE3B, the antilipolytic effect of insulin can be antagonized. Activation of PDE3B decreases concentrations of cAMP, which in turn reduces protein kinase A activity. Protein kinase A is responsible for activation of lipase, which induces lipolysis as well as other physiological pathways.[6][4]
Whether phosphorylation pathways, which regulate activity of PDE3A or PDE3B, could serve as potential drug targets rather than the catalytic domain of the PDE3 enzyme itself is unclear and beyond the scope of this text.
Structure
The mammalian PDEs share a common structural organization and contain three functional domains, which include the conserved catalytic core, a regulatory N-terminus, and the C-terminus. The conserved catalytic core is much more similar within PDE families, with about 80% amino acid identity, than between different families. It is believed that the core contains common structural elements that are important for the hydrolysis of cAMP and cGMP phosphodiester bonds. It is also believed that it contains family-specific determinants for differences in affinity for substrates and sensitivity for inhibitors.[6]
The catalytic domain of PDE3 is characterized by a 44-amino acid insert, but this insert is unique to the PDE3 family, and is a factor when determining a structure for a potent and selective PDE3 inhibitor.[6]
The crystal structure of the catalytic domains of several PDEs, including PDE3B, have shown that they contain three helical subdomains:
At the interface of these domains a deep hydrophobic pocket is formed by residues that are highly conserved among all PDEs. This pocket is the active site and is composed of four subsites:
The M site is at the bottom of the hydrophobic binding pocket and contains two divalent metal binding sites. The metal ions that can bind to these sites are either zinc or magnesium. The zinc binding site has two histidine and two aspartic acid residues that are absolutely conserved among those PDEs studied to date.[3][1]
The N-terminal portions of PDEs are widely divergent and contain determinants that are associated with regulatory properties specific to different gene families. For PDE3, those determinants are the hydrophobic membrane association domains and cAMP-dependent protein kinase phosphorylation sites.[6]
Substrate affinity
At first, the PDE3s were purified and described as enzymes that hydrolyse both cGMP and cAMP with Km values of 0.1–0.8 μM. However the Vmax for cAMPhydrolysis is 4–10 times higher than Vmax for cGMP hydrolysis.[6]
When different PDEs were first identified, two types of PDEs (PDE3 and PDE4) that exhibited high affinities for cAMP were isolated. PDE3 exhibited high affinity for both cGMP and cAMP, but PDE4 had high affinity for only cAMP. For that reason, the PDE3 was called the cGMP-inhibited PDE to distinguish it from PDE4.[6]
The 44-amino acid insertion in the catalytic domain of PDE3s is believed to be involved in PDE3's interaction with its substrate and inhibitors, but that remains to be established.[6]
The proposed molecular mechanism of cyclic nucleotide specificity of PDEs is the so-called glutamine switch mechanism.
In the PDEs that have had their structure solved, there seems to be an invariant glutamine residue that stabilizes the binding of the purine ring in the active site (binding pocket). The g-amino group of the glutamine residue can alternatively adopt two different orientations:
The hydrogen bond network supports guanine binding – cGMP selectivity
The hydrogen bond network supports adenine binding – cAMP selectivity.
In PDEs that can hydrolyse both cGMP and cAMP (PDE3s), the glutamine can rotate freely and therefore switch between orientations.[3][1]
Active site
From early studies an initial model of PDE, active site topography was derived. This early model can be summarized into the following steps concerning cAMP active site topography:
cAMP substrate with its adenine and ribose moieties in an "anti" relationship
The phosphate atom in cAMP binds to PDE active site, using an arginine residue and a water molecule, which was initially associated with Mg2+. A second arginine residue and the Mg2+ may also play roles during binding and/or play roles in the next step
SN2 attack of phosphorus by H2O with formation of a trigonal bipyramid transition state
5´-AMP is formed as an "inverted" product. Electronic charges conserve the net charge overall and across the transition state[7]
It has been demonstrated that PDE3A inhibition prevents oocyte maturation in vitro and in vivo.[1] For example, when mice are made completely deficient of PDE3A, they become infertile.[2]
Aggregation of platelets is highly regulated by cyclic nucleotides. PDE3A is a regulator of this process, and PDE3 inhibitors effectively prevent aggregation of platelets. Cilostazol is approved for treatment of intermittent claudication and is thought to involve inhibition of platelet aggregation and also inhibition of smooth muscle proliferation and vasodilation.
The most studied roles of PDE3B have been in the areas of insulin, IGF1, and leptin signaling.[1] When PDE3B is overexpressed in β-cells in mice, it causes impaired insulin secretion and glucose intolerance.[2]
Cancer
PDE3a expression has been described as a biomarker for sensitivity for PDE3-inhibitor Zardaverine in different types of cancer.[8]
Asthma
Targeting PDE3 with optimal doses and timing, enoximone prevents allergic inflammation in HDM-driven models of allergic airway inflammation.[9] PDE3 inhibitors enoximone and milrinone can be used as a rescue drug in life-threatening bronchial asthma/acute severe asthma.[10][11][12]
Cyclic adenosine monophosphate is a second messenger, or cellular signal occurring within cells, that is important in many biological processes. cAMP is a derivative of adenosine triphosphate (ATP) and used for intracellular signal transduction in many different organisms, conveying the cAMP-dependent pathway.
A protein kinase is a kinase which selectively modifies other proteins by covalently adding phosphates to them (phosphorylation) as opposed to kinases which modify lipids, carbohydrates, or other molecules. Phosphorylation usually results in a functional change of the target protein (substrate) by changing enzyme activity, cellular location, or association with other proteins. The human genome contains about 500 protein kinase genes and they constitute about 2% of all human genes. There are two main types of protein kinase. The great majority are serine/threonine kinases, which phosphorylate the hydroxyl groups of serines and threonines in their targets. Most of the others are tyrosine kinases, although additional types exist. Protein kinases are also found in bacteria and plants. Up to 30% of all human proteins may be modified by kinase activity, and kinases are known to regulate the majority of cellular pathways, especially those involved in signal transduction.
A cyclic nucleotide (cNMP) is a single-phosphate nucleotide with a cyclic bond arrangement between the sugar and phosphate groups. Like other nucleotides, cyclic nucleotides are composed of three functional groups: a sugar, a nitrogenous base, and a single phosphate group. As can be seen in the cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) images, the 'cyclic' portion consists of two bonds between the phosphate group and the 3' and 5' hydroxyl groups of the sugar, very often a ribose.
A phosphodiesterase (PDE) is an enzyme that breaks a phosphodiester bond. Usually, phosphodiesterase refers to cyclic nucleotide phosphodiesterases, which have great clinical significance and are described below. However, there are many other families of phosphodiesterases, including phospholipases C and D, autotaxin, sphingomyelin phosphodiesterase, DNases, RNases, and restriction endonucleases, as well as numerous less-well-characterized small-molecule phosphodiesterases.
In cell biology, protein kinase A (PKA) is a family of serine-threonine kinase whose activity is dependent on cellular levels of cyclic AMP (cAMP). PKA is also known as cAMP-dependent protein kinase. PKA has several functions in the cell, including regulation of glycogen, sugar, and lipid metabolism. It should not be confused with 5'-AMP-activated protein kinase.
Cyclic guanosine monophosphate (cGMP) is a cyclic nucleotide derived from guanosine triphosphate (GTP). cGMP acts as a second messenger much like cyclic AMP. Its most likely mechanism of action is activation of intracellular protein kinases in response to the binding of membrane-impermeable peptide hormones to the external cell surface. Through protein kinases activation, cGMP can relax smooth muscle. cGMP concentration in urine can be measured for kidney function and diabetes detection.
Transducin (Gt) is a protein naturally expressed in vertebrate retina rods and cones and it is very important in vertebrate phototransduction. It is a type of heterotrimeric G-protein with different α subunits in rod and cone photoreceptors.
Adenylate kinase is a phosphotransferase enzyme that catalyzes the interconversion of the various adenosine phosphates. By constantly monitoring phosphate nucleotide levels inside the cell, ADK plays an important role in cellular energy homeostasis.
Glycogen phosphorylase is one of the phosphorylase enzymes. Glycogen phosphorylase catalyzes the rate-limiting step in glycogenolysis in animals by releasing glucose-1-phosphate from the terminal alpha-1,4-glycosidic bond. Glycogen phosphorylase is also studied as a model protein regulated by both reversible phosphorylation and allosteric effects.
Cyclic guanosine monophosphate-specific phosphodiesterase type 5 is an enzyme from the phosphodiesterase class. It is found in various tissues, most prominently the corpus cavernosum and the retina. It has also been recently discovered to play a vital role in the cardiovascular system.
cGMP-dependent protein kinase or protein kinase G (PKG) is a serine/threonine-specific protein kinase that is activated by cGMP. It phosphorylates a number of biologically important targets and is implicated in the regulation of smooth muscle relaxation, platelet function, sperm metabolism, cell division, and nucleic acid synthesis.
Biological crosstalk refers to instances in which one or more components of one signal transduction pathway affects another. This can be achieved through a number of ways with the most common form being crosstalk between proteins of signaling cascades. In these signal transduction pathways, there are often shared components that can interact with either pathway. A more complex instance of crosstalk can be observed with transmembrane crosstalk between the extracellular matrix (ECM) and the cytoskeleton.
Phosphoinositide phospholipase C is a family of eukaryotic intracellular enzymes that play an important role in signal transduction processes. These enzymes belong to a larger superfamily of Phospholipase C. Other families of phospholipase C enzymes have been identified in bacteria and trypanosomes. Phospholipases C are phosphodiesterases.
3′,5′-cyclic-nucleotide phosphodiesterases (EC 3.1.4.17) are a family of phosphodiesterases. Generally, these enzymes hydrolyze a nucleoside 3′,5′-cyclic phosphate to a nucleoside 5′-phosphate:
Phosphodiesterase 1, PDE1, EC 3.1.4.1, systematic name oligonucleotide 5′-nucleotidohydrolase) is a phosphodiesterase enzyme also known as calcium- and calmodulin-dependent phosphodiesterase. It is one of the 11 families of phosphodiesterase (PDE1-PDE11). Phosphodiesterase 1 has three subtypes, PDE1A, PDE1B and PDE1C which divide further into various isoforms. The various isoforms exhibit different affinities for cAMP and cGMP.
The PDE2 enzyme is one of 21 different phosphodiesterases (PDE) found in mammals. These different PDEs can be subdivided to 11 families. The different PDEs of the same family are functionally related despite the fact that their amino acid sequences show considerable divergence. The PDEs have different substrate specificities. Some are cAMP selective hydrolases, others are cGMP selective hydrolases and the rest can hydrolyse both cAMP and cGMP.
cAMP-specific 3',5'-cyclic phosphodiesterase 4D is an enzyme that in humans is encoded by the PDE4D gene.
cAMP-specific 3',5'-cyclic phosphodiesterase 4A is an enzyme that in humans is encoded by the PDE4A gene.
In the field of molecular biology, the cAMP-dependent pathway, also known as the adenylyl cyclase pathway, is a G protein-coupled receptor-triggered signaling cascade used in cell communication.
Phosphodiesterases (PDEs) are a superfamily of enzymes. This superfamily is further classified into 11 families, PDE1 - PDE11, on the basis of regulatory properties, amino acid sequences, substrate specificities, pharmacological properties and tissue distribution. Their function is to degrade intracellular second messengers such as cyclic adenine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) which leads to several biological processes like effect on intracellular calcium level by the Ca2+ pathway.
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