Structure of PDE3A-SLFN12 complex and structure-based design for a potent apoptosis inducer of tumor cells.

Molecular glues are a class of small molecular drugs that mediate protein-protein interactions, that induce either the degradation or stabilization of target protein. A structurally diverse group of chemicals, including 17-ß-estradiol (E2), anagrelide, nauclefine, and DNMDP, induces apoptosis by forming complexes with phosphodiesterase 3A (PDE3A) and Schlafen 12 protein (SLFN12). They do so by binding to the PDE3A enzymatic pocket that allows the compound-bound PDE3A to recruit and stabilize SLFN12, which in turn blocks protein translation, leading to apoptosis. In this work, we report the high-resolution cryo-electron microscopy structure of PDE3A-SLFN12 complexes isolated from cultured HeLa cells pre-treated with either anagrelide, or nauclefine, or DNMDP. The PDE3A-SLFN12 complexes exhibit a butterfly-like shape, forming a heterotetramer with these small molecules, which are packed in a shallow pocket in the catalytic domain of PDE3A. The resulting small molecule-modified interface binds to the short helix (E552-I558) of SLFN12 through hydrophobic interactions, thus "gluing" the two proteins together. Based on the complex structure, we designed and synthesized analogs of anagrelide, a known drug used for the treatment of thrombocytosis, to enhance their interactions with SLFN12, and achieved superior efficacy in inducing apoptosis in cultured cells as well as in tumor xenografts.

Structure of PDE3A-SLFN12 complex reveals requirements for activation of SLFN12 RNase.

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.

The synthesis and biological evaluation of nucleobases/tetrazole hybrid compounds: A new class of phosphodiesterase type 3 (PDE3) inhibitors.

Spired by the chemical structure of Cilostazol, a selective phosphodiesterase 3A (PDE3A) inhibitor, several novel hybrid compounds of nucleobases (uracil, 6-azauracil, 2-thiuracil, adenine, guanine, theophylline and theobromine) and tetrazole were designed and successfully synthesized and their inhibitory effects on PDE3A as well as their cytotoxicity on HeLa and MCF-7 cancerous cell lines were studied. Obtained results show the linear correlation between the inhibitory effect of synthesized compounds and their cytotoxicity. In some cases, the PDE3A inhibitory effects of synthesized compounds are higher than the Cilostazol. Besides, compared to a standard anticancer drug methotrexate, some of the synthesized compounds showed the higher cytotoxicity against the HeLa and MCF-7 cancerous cell lines.

Mechanistic insights into cancer cell killing through interaction of phosphodiesterase 3A and schlafen family member 12.

Cytotoxic molecules can kill cancer cells by disrupting critical cellular processes or by inducing novel activities. 6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) is a small molecule that kills cancer cells by generation of novel activity. DNMDP induces complex formation between phosphodiesterase 3A (PDE3A) and schlafen family member 12 (SLFN12) and specifically kills cancer cells expressing elevated levels of these two proteins. Here, we examined the characteristics and covariates of the cancer cell response to DNMDP. On average, the sensitivity of human cancer cell lines to DNMDP is correlated with PDE3A expression levels. However, DNMDP could also bind the related protein, PDE3B, and PDE3B supported DNMDP sensitivity in the absence of PDE3A expression. Although inhibition of PDE3A catalytic activity did not account for DNMDP sensitivity, we found that expression of the catalytic domain of PDE3A in cancer cells lacking PDE3A is sufficient to confer sensitivity to DNMDP, and substitutions in the PDE3A active site abolish compound binding. Moreover, a genome-wide CRISPR screen identified the aryl hydrocarbon receptor-interacting protein (AIP), a co-chaperone protein, as required for response to DNMDP. We determined that AIP is also required for PDE3A-SLFN12 complex formation. Our results provide mechanistic insights into how DNMDP induces PDE3A-SLFN12 complex formation, thereby killing cancer cells with high levels of PDE3A and SLFN12 expression.

Phosphodiesterase 3A Represents a Therapeutic Target that Drives Stem Cell-like Property and Metastasis in Breast Cancer.

Considerable evidence suggests that as breast cancer progresses, genetic and epigenetic mechanisms contribute to the emergence of self-renewing cells (CSC), which may also arise as a consequence of metastasis. Although the molecular pathways that trigger stemness and metastasis are known, key molecular and mechanistic gaps in our understanding of these processes remain unclear. Here, we first screened the inflammation-associated stemness gene phosphodiesterase 3A (PDE3A) using a medium-throughput siRNA library, which was overexpressed in breast tumors and significantly correlated with clinical progression. PDE3A induced the inflammatory nuclear factor NFκB signaling pathway by suppressing cAMP/PKA, which promotes the expression of the stem cell marker OCT4. In addition, PDE3A also promoted the translocation of CCDC88A from the cytoplasm to nuclei, thereby boosting the invasion-metastasis cascade in breast cancer. Most importantly, the PDE3A-selective inhibitor cilostazol dramatically suppressed breast tumor growth and reduced metastasis to the lungs in xenograft breast cancer models, with minimum toxicity. Taken together, we show that PDE3A could predispose patients with breast cancer to metastases by acting as a mediator of cancer stemness. PDE3A is a potential therapeutic target for advanced breast cancer.

Acute Enhancement of Cardiac Function by Phosphodiesterase Type 1 Inhibition.

BACKGROUND: Phosphodiesterase type-1 (PDE1) hydrolyzes cAMP and cGMP and is constitutively expressed in the heart, although cardiac effects from its acute inhibition in vivo are largely unknown. Existing data are limited to rodents expressing mostly the cGMP-favoring PDE1A isoform. Human heart predominantly expresses PDE1C with balanced selectivity for cAMP and cGMP. Here, we determined the acute effects of PDE1 inhibition in PDE1C-expressing mammals, dogs, and rabbits, in normal and failing hearts, and explored its regulatory pathways. METHODS: Conscious dogs chronically instrumented for pressure-volume relations were studied before and after tachypacing-induced heart failure (HF). A selective PDE1 inhibitor (ITI-214) was administered orally or intravenously±dobutamine. Pressure-volume analysis in anesthetized rabbits tested the role of ß-adrenergic and adenosine receptor signaling on ITI-214 effects. Sarcomere and calcium dynamics were studied in rabbit left ventricular myocytes. RESULTS: In normal and HF dogs, ITI-214 increased load-independent contractility, improved relaxation, and reduced systemic arterial resistance, raising cardiac output without altering systolic blood pressure. Heart rate increased, but less so in HF dogs. ITI-214 effects were additive to ß-adrenergic receptor agonism (dobutamine). Dobutamine but not ITI-214 increased plasma cAMP. ITI-214 induced similar cardiovascular effects in rabbits, whereas mice displayed only mild vasodilation and no contractility effects. In rabbits, ß-adrenergic receptor blockade (esmolol) prevented ITI-214-mediated chronotropy, but inotropy and vasodilation remained unchanged. By contrast, adenosine A2B-receptor blockade (MRS-1754) suppressed ITI-214 cardiovascular effects. Adding fixed-rate atrial pacing did not alter the findings. ITI-214 alone did not affect sarcomere or whole-cell calcium dynamics, whereas ß-adrenergic receptor agonism (isoproterenol) or PDE3 inhibition (cilostamide) increased both. Unlike cilostamide, which further enhanced shortening and peak calcium when combined with isoproterenol, ITI-214 had no impact on these responses. Both PDE1 and PDE3 inhibitors increased shortening and accelerated calcium decay when combined with forskolin, yet only cilostamide increased calcium transients. CONCLUSIONS: PDE1 inhibition by ITI-214 in vivo confers acute inotropic, lusitropic, and arterial vasodilatory effects in PDE1C-expressing mammals with and without HF. The effects appear related to cAMP signaling that is different from that provided via ß-adrenergic receptors or PDE3 modulation. ITI-214, which has completed phase I trials, may provide a novel therapy for HF.

Crystal Structure of the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3B Provides Insights into Determinants of Substrate Specificity.

The enzyme acid sphingomyelinase-like phosphodiesterase 3B (SMPDL3B) was shown to act as a negative regulator of innate immune signaling, affecting cellular lipid composition and membrane fluidity. Furthermore, several reports identified this enzyme as an off target of the therapeutic antibody rituximab, with implications in kidney disorders. However, structural information for this protein is lacking. Here we present the high resolution crystal structure of murine SMPDL3B, which reveals a substrate binding site strikingly different from its paralogs. The active site is located in a narrow boot-shaped cavity. We identify a unique loop near the active site that appears to impose size constraints on incoming substrates. A structure in complex with phosphocholine indicates that the protein recognizes this head group via an aromatic box, a typical choline-binding motif. Although a potential substrate for SMPDL3B is sphingomyelin, we identify other possible substrates such as CDP-choline, ATP, and ADP. Functional experiments employing structure-guided mutagenesis in macrophages highlight amino acid residues potentially involved in recognition of endogenous substrates. Our study is an important step toward elucidating the specific function of this poorly characterized enzyme.

Copper regulates cyclic-AMP-dependent lipolysis.

Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining body weight and energy stores. Using a mouse model of genetic copper misregulation, in combination with pharmacological alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we found that copper regulates lipolysis at the level of the second messenger, cyclic AMP (cAMP), by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochemical studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue in a PDE3-specific loop that is essential for the observed copper-dependent lipolytic phenotype.

Design and Development of Novel 4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-6-morpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine as Cardiotonic Agent via Inhibition of PDE3.

The classical phosphodiesterase 3A (PDE3A) inhibitors provide relaxation of the vasculature system via increasing the cellular level of cyclic adenosine monophosphate (cAMP) and proved to be useful in the management of congestive heart failure. Consequently, the present paper deals with the development of novel pyrazole derivatives tethered with substituted 1,3,5-triazine derivatives in search for novel PDE3 inhibitors. The synthesis of designed inhibitors was realized in a multi-step reaction and the structures were ascertained with the help of various spectroscopic techniques. Subsequently, these analogs were tested for their inhibitory activities against PDE3 enzymes, where they exhibited considerable inhibition, revealing 9g as the most promising inhibitor of the class. In a docking study, the morpholine fragment of compound 9g was efficiently engulfed in the small pocket of the active site lined by Gly940 and Pro941. The substituted aromatic ring of the core scaffold was found to be positioned deep in the cavity bordered by Tyr829, Asn830, Leu850, Glu851, and Thr893. Moreover, it considerably improved the contractility of cardiac muscles without altering the heart beat frequency in experimental subjects.

Phosphodiesterase types 3 and 4 regulate the phasic contraction of neonatal rat bladder smooth myocytes via distinct mechanisms.

Activation of the cyclic AMP (cAMP) pathway reduces bladder contractility. However, the role of phosphodiesterase (PDE) families in regulating this function is poorly understood. Here, we compared the contractile function of the cAMP hydrolyzing PDEs in neonatal rat bladder smooth myocytes. RT-PCR and Western blotting analysis revealed that several isoforms of PDE1-4 were expressed in neonatal rat bladder. While 8-methoxymethyl-3-isobutyl-1-methylxanthine (a PDE1 inhibitor) and BAY-60-7550 (a PDE2 inhibitor) had no effect on the carbachol-enhanced phasic contractions of bladder strips, cilostamide (Cil, a PDE3 inhibitor) and Ro-20-1724 (Ro, a PDE4 inhibitor) significantly reduced these contractions. This inhibitory effect of Ro was blunted by the PKA inhibitor H-89, while the inhibitory effect of Cil was strongly attenuated by the PKG inhibitor KT 5823. Application of Ro in single bladder smooth myocytes resulted in an increase in Ca(2+) spark frequency but a decrease both in Ca(2+) transients and in sarcoplasmic reticulum (SR) Ca(2+) content. In contrast, Cil had no effect on these events. Furthermore, Ro-induced inhibition of the phasic contractions was significantly blocked by ryanodine and iberiotoxin. Taken together, PDE3 and PDE4 are the main PDE isoforms in maintaining the phasic contractions of bladder smooth myocytes, with PDE4 being functionally more active than PDE3. However, their roles are mediated through different mechanisms.

A role for phosphodiesterase 3B in acquisition of brown fat characteristics by white adipose tissue in male mice.

Obesity is linked to various diseases, including insulin resistance, diabetes, and cardiovascular disorders. The idea of inducing white adipose tissue (WAT) to assume characteristics of brown adipose tissue (BAT), and thus gearing it to fat burning instead of storage, is receiving serious consideration as potential treatment for obesity and related disorders. Phosphodiesterase 3B (PDE3B) links insulin- and cAMP-signaling networks in tissues associated with energy metabolism, including WAT. We used C57BL/6 PDE3B knockout (KO) mice to elucidate mechanisms involved in the formation of BAT in epididymal WAT (EWAT) depots. Examination of gene expression profiles in PDE3B KO EWAT revealed increased expression of several genes that block white and promote brown adipogenesis, such as C-terminal binding protein, bone morphogenetic protein 7, and PR domain containing 16, but a clear BAT-like phenotype was not completely induced. However, acute treatment of PDE3B KO mice with the ß3-adrenergic agonist, CL316243, markedly increased the expression of cyclooxygenase-2, which catalyzes prostaglandin synthesis and is thought to be important in the formation of BAT in WAT and the elongation of very long-chain fatty acids 3, which is linked to BAT recruitment upon cold exposure, causing a clear shift toward fat burning and the induction of BAT in KO EWAT. These data provide insight into the mechanisms of BAT formation in mouse EWAT, suggesting that, in a C57BL/6 background, an increase in cAMP, caused by ablation of PDE3B and administration of CL316243, may promote differentiation of prostaglandin-responsive progenitor cells in the EWAT stromal vascular fraction into functional brown adipocytes.

Modulating the cyclic guanosine monophosphate substrate selectivity of the phosphodiesterase 3 inhibitors by pyridine, pyrido[2,3-d]pyrimidine derivatives and their effects upon the growth of HT-29 cancer cell line.

Analogues with the scaffolds of 3-cyano-4-alkoxyphenyl-6-bromoaryl-2-pyridone and 2-amino-3-cyano-4-alkoxyphenyl-6-bromoarylpyridine were synthesized. Cyclization of the 2-amino derivatives with formic acid and formamide gave the corresponding pyrido[2,3-d]pyrimidin-4(3H)-one and the pyrido[2,3-d]-pyrimidin-4-amine derivatives, respectively. Active phosphodiesterase 3 (PDE3) inhibitors were identified from each of the four aforementioned scaffolds. This is the first report that pyrido[2,3-d]pyrimidin-4(3H)-one and pyrido[2,3-d]pyrimidin-4-amine derivatives can inhibit PDE3. The analogues with the pyridone and pyrido[2,3-d]pyrimidin-4(3H)-one scaffolds inhibited both cAMP and cyclic guanosine monophosphate (cGMP) hydrolysis by PDE3, while the amine containing scaffolds were more selective for cGMP hydrolysis. This observation may set the base for substrate-selective pharmacological modulation of this important class of drug targets and with less side effects, particularly tachcardia. The dual inhibitors of PDE3 were more potent inhibitor towards the growth of HT-29 cancer cell lines.

Identification of novel mutations confirms PDE4D as a major gene causing acrodysostosis.

Acrodysostosis is characterized by nasal hypoplasia, peripheral dysostosis, variable short stature, and intellectual impairment. Recently, mutations in PRKAR1A were reported in patients with acrodysostosis and hormone resistance. Subsequently, mutations in a phosphodiesterase gene (PDE4D) were identified in seven sporadic cases. We sequenced PDE4D in seven acrodysostosis patients from five families. Missense mutations were identified in all cases. Families showed de novo inheritance except one family with three affected children whose father was subsequently found to have subtle features of acrodysostosis. There were no recurrent mutations. Short stature and endocrine resistance are rare in this series; however, cognitive involvement and obesity were frequent. This last finding is relevant given PDE4D is insulin responsive and potentially involved in lipolysis. PDE4D encodes a cyclic AMP regulator and places PDE4D-related acrodysostosis within the same family of diseases as pseudohypoparathyroidism, pseudopseudohypoparathyroidism, PRKAR1A-related acrodysostosis and brachydactyly-mental retardation syndrome; all characterized by cognitive impairment and short distal extremities.

Ablation of calcineurin Aß reveals hyperlipidemia and signaling cross-talks with phosphodiesterases.

Insulin resistance, hyperlipidemia, and cardiovascular complications are common dysregulations of metabolic syndrome. Transplant patients treated with immunosuppressant drugs such as cyclosporine A (CsA), an inhibitor of calcineurin phosphatase, frequently develop similar metabolic complications. Although calcineurin is known to mediate insulin sensitivity by regulating ß-cell growth and adipokine gene transcription, its role in lipid homeostasis is poorly understood. Here, we examined lipid homeostasis in mice lacking calcineurin Aß (CnAß(-/-)). We show that mice lacking calcineurin Aß are hyperlipidemic and develop age-dependent insulin resistance. Hyperlipidemia found in CnAß(-/-) mice is, in part, due to increased lipolysis in adipose tissues, a process mediated by ß-adrenergic G-protein-coupled receptor signaling pathways. CnAß(-/-) mice also exhibit additional pathophysiological phenotypes caused by the potentiated GPCR signaling pathways. A cell autonomous mechanism with sustained cAMP/PKA activation is found in CnAß(-/-) mice or upon CsA treatment to inhibit calcineurin. Increased PKA activation and cAMP accumulation in CnAß(-/-) mice, however, are sensitive to phosphodiesterase inhibitor. Indeed, we show that calcineurin regulates degradation of phosphodiesterase 3B, in addition to phosphodiesterase 4D. These results establish a role for calcineurin in lipid homeostasis. These data also indicate that potentiated cAMP signaling pathway may provide an alternative molecular pathogenesis for the metabolic complications elicited by CsA in transplant patients.

[Phosphodiesterase 3 mediates cross-talk between the protein kinase- and cGMP- dependent pathways and cyclic AMP metabolism]. / Fosfodiesterazy rodziny 3 sprzegaja szlaki sygnalowe zalezne od kinaz bialkowych i cyklicznego GMP z metabolizmem cyklicznego AMP.

PDE3 is a dual-substrate phosphodiesterase responsible for hydrolyzing both cAMP and cGMP whilst being simultaneously inhibited by cGMP. This feature is related to presence of the 44 amino acid insert in the catalytic domain, which determines the mechanism of introduction of the cyclic nucleotide into the catalytic pocket of the enzyme. Once bound in the catalytic site cGMP results in steric hindrance for cAMP to enter the site. The regulatory domain of PDE3 consists of two hydrophobic regions: NHR1 and NHR2. Their presence defines the enzyme's intracellular localization, thus determining its participation in particular signaling cascades. Due to the properties of PDE3 this enzyme has exceptional importance for the cross-talk between cAMP-dependent signaling and other cascades. There are two different mechanisms of action of PDE3 enzymes in cell signaling pathways. In many signaling cascades assembly of a signalosome is necessary for phosphorylation and activation of the PDE3 proteins. In response to certain hormones and growth factors, PDE3 merges the metabolism of cAMP with protein kinase-dependent signaling pathways. PDE3 also controls the level of cAMP with regard to the alternating concentration of cGMP. This effect occurs in signaling cascades activated by natriuretic peptide.

Regulation of murine cardiac function by phosphodiesterases type 3 and 4.

Cyclic nucleotide phosphodiesterases (PDEs) encompass a large group of enzymes that regulate intracellular levels of two-second messengers, cAMP and cGMP, by controlling the rates of their degradation. More than 60 isoforms, subdivided into 11 gene families (PDE1-11), exist in mammals with at least six families (PDE1-5 and PDE8) identified in mammalian hearts. The two predominant families implicated in regulating contraction strength of the heart are PDE3 and PDE4. Studies using transgenic models in combination with family-specific PDE inhibitors have demonstrated that PDE3A, PDE4B, and PDE4D isoforms regulate cardiac contractility by modulating cAMP levels in various subcellular compartments. These studies have further uncovered contributions of PDE4B and PDE4D in preventing ventricular arrhythmias.

Role of PDE3A in regulation of cell cycle progression in mouse vascular smooth muscle cells and oocytes: implications in cardiovascular diseases and infertility.

Phosphodiesterase-3 (PDE3) is a major cAMP-hydrolyzing PDE in vascular smooth muscle cells (VSMCs) and oocytes. The exact role and contribution of the two PDE3 isoforms, PDE3A and PDE3B, in VSMC growth regulation and oocyte maturation was examined using PDE3A (3A) and PDE3B (3B) knockout (KO) mouse models. PDE3A-deficient VSMCs exhibit marked reduction in mitogen-induced cell growth due to cell cycle arrest at G0-G1 phase, which resulted from dysregulation of cAMP/protein kinase A (PKA)-activated and mitogen-activated protein kinase (MAPK)-signaling pathways, as well as from alterations in key cell cycle regulatory proteins. Similarly, PDE3A-deficient oocytes exhibit cell cycle arrest at G2/M phase because increased cAMP/PKA signaling in KO oocytes most likely inhibits Cdc25B-catalyzed dephosphorylation/activation of Cdc2 (maturation promoting factor (MPF)), a key regulator of G2/M transition.

From PDE3B to the regulation of energy homeostasis.

The incidence of obesity in the developed world is increasing at an alarming rate. Concurrent with the increase in the incidence of obesity is an increase in the incidence of type 2 diabetes. Cyclic AMP (cAMP) and cGMP are key second messengers in all cells; for example, when it comes to processes of relevance for the regulation of energy metabolism, cAMP is a key mediator in the regulation of lipolysis, glycogenolysis, gluconeogenesis and pancreatic ß cell insulin secretion. PDE3B, one of several enzymes which hydrolyze cAMP and cGMP, is expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, hypothalamic cells and ß cells. It has been shown, using PDE3 inhibitors and gene targeting approaches in cells and animals, that altered levels of PDE3B result in a number of changes in the regulation of glucose and lipid metabolism and in overall energy homeostasis. This article highlights the complexity involved in the regulation of PDE3B by hormones, and in the regulation of downstream metabolic effects by PDE3B in several interacting tissues.

PDE3 inhibition in dilated cardiomyopathy.

In dilated cardiomyopathy, a condition characterized by chamber enlargement and reduced myocardial contractility, decreases in ß-adrenergic receptor density and increases in Gαi and ß-adrenergic receptor kinase activities attenuate the stimulation of adenylyl cyclase in response to catecholamines. PDE3 inhibitors have been used to 'overcome' the reduction in cAMP generation by blocking cAMP hydrolysis. These drugs increase contractility in the short-term, but long-term administration leads to an increase in mortality that correlates with an increase in sudden cardiac death. Whether separate mechanisms account for these beneficial and harmful effects, and, if so, whether PDE3 can be targeted so as to increase contractility without increasing mortality are questions that remain unanswered.

Numerous distinct PKA-, or EPAC-based, signalling complexes allow selective phosphodiesterase 3 and phosphodiesterase 4 coordination of cell adhesion.

By activating two distinct classes of effector enzymes, namely Protein Kinases A [PKA] or Exchange Proteins Activated by cAMP [EPAC], the ubiquitous second messenger cAMP selectively coordinates numerous events simultaneously in virtually all cells. Studies focused on dissecting the manner by which cAMP simultaneously regulates multiple cellular events have shown that cAMP activates its effectors non-uniformly in cells and that this localized cAMP-mediated signalling is made possible, at least in part, by anchoring of cAMP effectors to selected subcellular structures. In the work described here, we report that HEK293T cells ["293T"] contain several PKA- and EPAC1-based signalling complexes. Interestingly, our data do not identify signalling complexes in which both PKA and EPAC are each present but rather are consistent with the idea that these two effectors operate in distinct complexes in these cells. Similarly, we report that while individual PKA- or EPAC-containing complexes can contain either phosphodiesterase 3B, [PDE3B] or phosphodiesterase 4D [PDE4D], they do not contain both these phosphodiesterases. Indeed, although PDE4D enzymes were identified in both PKA- and EPAC-based complexes, PDE3B was largely identified in EPAC-based complexes. Using a combination of approaches, we identified that integration of PDE3B into EPAC-based complexes occurred through its amino terminal fragment [PDE3B(AT)]. Consistent with the idea that integration of PDE3B within EPAC-based complexes was dynamic and regulated PDE3 inhibitor-mediated effects on cellular functions, expression of PDE3B(AT) competed with endogenous PDE3B for integration into EPAC-based complexes and antagonized PDE3 inhibitor-based cell adhesion. Our data support the concept that cells can contain several non-overlapping PKA- and EPAC-based signalling complexes and that these complexes may also represent sites within cells were the effects of family-selective PDE inhibitors could be integrated to affect cell functions, including adhesion.