Delphine Mika

Cyclic AMP compartments and signaling specificity: role of cyclic nucleotide phosphodiesterases.

### Introduction The concept that cAMP has restricted access to different pools of intracellular effectors was articulated more than 30 years ago. Observations made by [Corbin et al. (1977)][1] demonstrated that the two isoenzymes of the major intracellular cAMP target, PKA I and II, are located in

A CaMKII/PDE4D negative feedback regulates cAMP signaling.

Significance Cardiovascular disease is the leading cause of death worldwide. A better understanding of the pathophysiological mechanisms governing such disease states as heart failure is essential to the identification of novel therapeutic targets. We have discovered a regulatory loop that coordinates the function of proteins and secondary messengers involved in cardiac contractility and the development of heart failure. This regulatory loop may be a novel target for pharmacologic intervention to restore cardiac homeostasis. cAMP production and protein kinase A (PKA) are the most widely studied steps in β-adrenergic receptor (βAR) signaling in the heart; however, the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is also activated in response to βAR stimulation and is involved in the regulation of cardiac excitation-contraction coupling. Its activity and expression are increased during cardiac hypertrophy, in heart failure, and under conditions that promote arrhythmias both in animal models and in the human heart, underscoring the clinical relevance of CaMKII in cardiac pathophysiology. Both CaMKII and PKA phosphorylate a number of protein targets critical for Ca2+ handling and contraction with similar, but not always identical, functional consequences. How these two pathways communicate with each other remains incompletely understood, however. To maintain homeostasis, cyclic nucleotide levels are regulated by phosphodiesterases (PDEs), with PDE4s predominantly responsible for cAMP degradation in the rodent heart. Here we have reassessed the interaction between cAMP/PKA and Ca2+/CaMKII signaling. We demonstrate that CaMKII activity constrains basal and βAR-activated cAMP levels. Moreover, we show that these effects are mediated, at least in part, by CaMKII regulation of PDE4D. This regulation establishes a negative feedback loop necessary to maintain cAMP/CaMKII homeostasis, revealing a previously unidentified function for PDE4D as a critical integrator of cAMP/PKA and Ca2+/CaMKII signaling.

Functional selectivity of GPCR-directed drug action through location bias

G protein-coupled receptors (GPCRs) are increasingly recognized to operate from intracellular membranes as well as the plasma membrane. The β2-adrenergic GPCR can activate Gs-linkedcyclic AMP (cAMP) signaling from endosomes. We show here that the homologous human β1-adrenergic receptor initiates an internal Gs-cAMP signal from the Golgi apparatus. By developing a chemical method to acutely squelch G protein coupling at defined membrane locations, we demonstrate that Golgi activation contributes significantly to the overall cellular cAMP response. Golgi signalling utilizes a pre-existing receptor pool rather than receptors delivered from the cell surface, requiring separate access of extracellular ligands. Epinephrine, a hydrophilic endogenous ligand, accesses the Golgi-localized receptor pool by facilitated transport requiring the organic cation transporter 3 (OCT3) whereas drugs can access the Golgi pool by passive diffusion according to hydrophobicity. We demonstrate marked differences among both agonist and antagonist drugs in Golgi-localized receptor access, and show that β-blocker drugs presently used in the clinic differ markedly in ability to antagonize the Golgi signal. We propose ’location bias’ as a new principle for achieving functional selectivity of GPCR-directed drug action.

PDE4B mediates local feedback regulation of β1-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes

ABSTRACT Multiple cAMP phosphodiesterase (PDE) isoforms play divergent roles in cardiac homeostasis but the molecular basis for their non-redundant function remains poorly understood. Here, we report a novel role for the PDE4B isoform in &bgr;-adrenergic (&bgr;AR) signaling in the heart. Genetic ablation of PDE4B disrupted &bgr;AR-induced cAMP transients, as measured by FRET sensors, at the sarcolemma but not in the bulk cytosol of cardiomyocytes. This effect was further restricted to a subsarcolemmal compartment because PDE4B regulates &bgr;1AR-, but not &bgr;2AR- or PGE2-induced responses. The spatially restricted function of PDE4B was confirmed by its selective effects on PKA-mediated phosphorylation patterns. PDE4B limited the PKA-mediated phosphorylation of key players in excitation–contraction coupling that reside in the sarcolemmal compartment, including L-type Ca2+ channels and ryanodine receptors, but not phosphorylation of distal cytosolic proteins. &bgr;1AR- but not &bgr;2AR-ligation induced PKA-dependent activation of PDE4B and interruption of this negative feedback with PKA inhibitors increased sarcolemmal cAMP. Thus, PDE4B mediates a crucial PKA-dependent feedback that controls &bgr;1AR-dependent cAMP signals in a restricted subsarcolemmal domain. Disruption of this feedback augments local cAMP/PKA signals, leading to an increased intracellular Ca2+ level and contraction rate.

PDE4D phosphorylation: A coincidence detector integrating multiple signaling pathways

In Eukaryotes, more than 100 different phosphodiesterase (PDE) proteins serve to fine-tune cyclic nucleotide (cAMP and cGMP) signals and contribute to specificity of signaling. In mammals, PDEs are divided into 11 families, of which PDE4 represents the largest family. Four genes (pde4a, pde4b, pde4c and pde4d) encode for this class of enzymes in mammals and give rise to more than 20 variants. Within this family of genes, PDE4D was discovered on the basis of its regulatory properties and its induction by hormones and cAMP. PDE4D has often been used as the prototype PDE4 and large body of work has been generated on the biochemical, pharmacological, and physiological properties of this enzyme. This review covers the regulation of PDE4D by phosphorylation, the impact of this regulation in the context of the structure of this protein, and the functional consequences of this complex pattern of posttranslational modifications.

β1‐adrenergic receptor antagonists signal via PDE4 translocation

It is generally assumed that antagonists of Gs‐coupled receptors do not activate cAMP signalling, because they do not stimulate cAMP production via Gs‐protein/adenylyl cyclase activation. Here, we report a new signalling pathway whereby antagonists of β1‐adrenergic receptors (β1ARs) increase cAMP levels locally without stimulating cAMP production directly. Binding of antagonists causes dissociation of a preformed complex between β1ARs and Type‐4 cyclic nucleotide phosphodiesterases (PDE4s). This reduces the local concentration of cAMP‐hydrolytic activity, thereby increasing submembrane cAMP and PKA activity. Our study identifies receptor/PDE4 complex dissociation as a novel mechanism of antagonist action that contributes to the pharmacological properties of β1AR antagonists and might be shared by other receptor subtypes.

The upstream conserved regions (UCRs) mediate homo- and hetero-oligomerization of type 4 cyclic nucleotide phosphodiesterases (PDE4s).

PDE4s (type 4 cyclic nucleotide phosphodiesterases) are divided into long and short forms by the presence or absence of conserved N-terminal domains termed UCRs (upstream conserved regions). We have shown previously that PDE4D2, a short variant, is a monomer, whereas PDE4D3, a long variant, is a dimer. In the present study, we have determined the apparent molecular masses of various long and short PDE4 variants by size-exclusion chromatography and sucrose density-gradient centrifugation. Our results indicate that dimerization is a conserved property of all long PDE4 forms, whereas short forms are monomers. Dimerization is mediated by the UCR domains. Given their high sequence conservation, the UCR domains mediate not only homo-oligomerization, but also hetero-oligomerization of distinct PDE4 long forms as detected by co-immunoprecipitation assays and FRET microscopy. Endogenous PDE4 hetero-oligomers are, however, low in abundance compared with homo-dimers, revealing the presence of mechanisms that predispose PDE4s towards homo-oligomerization. Oligomerization is a prerequisite for the regulatory properties of the PDE4 long forms, such as their PKA (protein kinase A)-dependent activation, but is not necessary for PDE4 protein-protein interactions. As a result, individual PDE4 protomers may independently mediate protein-protein interactions, providing a mechanism whereby PDE4s contribute to the assembly of macromolecular signalling complexes.

Anchored PDE4 regulates chloride conductance in wild-type and ΔF508-CFTR human airway epithelia

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that impair its expression and/or chloride channel function. Here, we provide evidence that type 4 cyclic nucleotide phosphodiesterases (PDE4s) are critical regulators of the cAMP/PKA‐dependent activation of CFTR in primary human bronchial epithelial cells. In non‐CF cells, PDE4 inhibition increased CFTR activity under basal conditions (ΔISC 7.1 μA/cm2) and after isoproterenol stimulation (increased ΔISC from 13.9 to 21.0 μA/cm2) and slowed the return of stimulated CFTR activity to basal levels by > 3‐fold. In cells homozygous for ΔF508‐CFTR, the most common mutation found in CF, PDE4 inhibition alone produced minimal channel activation. However, PDE4 inhibition strongly amplified the effects of CFTR correctors, drugs that increase expression and membrane localization of CFTR, and/or CFTR potentiators, drugs that increase channel gating, to reach ~25% of the chloride conductance observed in non‐CF cells. Biochemical studies indicate that PDE4s are anchored to CFTR and mediate a local regulation of channel function. Taken together, our results implicate PDE4 as an important determinant of CFTR activity in airway epithelia, and support the use of PDE4 inhibitors to potentiate the therapeutic benefits of CFTR correctors and potentiators.—Blanchard, E., Zlock, L., Lao, A., Mika, D., Namkung, W., Xie, M., Scheitrum, C., Gruenert, D.C., Verkman, A.S., Finkbeiner, W.E., Conti, M., Richter, W. Anchored PDE4 regulates chloride conductance in wild type and ΔF508‐CFTR human airway epithelia. FASEB J. 28, 791–801 (2014). www.fasebj.org