There is a growing appreciation that the cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA) signaling pathway is organized to form transduction units that function to deliver specific messages. Such organization results in the local activation of PKA subsets through the generation of confined intracellular gradients of cAMP, but the mechanisms responsible for limiting the diffusion of cAMP largely remain to be clarified. In this study, by performing real-time imaging of cAMP, we show that prostaglandin 1 stimulation generates multiple contiguous, intracellular domains with different cAMP concentration in human embryonic kidney 293 cells. By using pharmacological and genetic manipulation of phosphodiesterases (PDEs), we demonstrate that compartmentalized PDE4B and PDE4D are responsible for selectively modulating the concentration of cAMP in individual subcellular compartments. We propose a model whereby compartmentalized PDEs, rather than representing an enzymatic barrier to cAMP diffusion, act as a sink to drain the second messenger from discrete locations, resulting in multiple and simultaneous domains with different cAMP concentrations irrespective of their distance from the site of cAMP synthesis.

Isoproterenol challenge of Hek-B2 cells causes a transient recruitment of the endogenous PDE4D isoforms found in these cells, namely PDE4D3 and PDE4D5, to the membrane fraction. PDE4D5 provides around 80% of the total PDE4D protein so recruited, although it only comprises about 40% of the total PDE4D protein in Hek-B2 cells. PDE4D5 provides about 80% of the total PDE4D protein found associated with ␤-arrestins immunopurified from Hek-B2, COS1, and A549 cells as well as cardiac myocytes, whereas its overall level in these cells is between 15 and 50% of the total PDE4D protein. Truncation analyses indicate that two sites in PDE4D5 are involved in mediating its interaction with ␤-arrestins, one associated with the common PDE4 catalytic region and the other located within its unique amino-terminal region. Truncation analyses indicate that two sites in ␤-arrestin 2 are involved in mediating its interaction with PDE4D5, one associated with its extreme amino-terminal region and the other located within the carboxylterminal domain of the protein. We suggest that the unique amino-terminal region of PDE4D5 allows it to preferentially interact with ␤-arrestins. This specificity appears likely to account for the preferential recruitment of PDE4D5, compared with PDE4D3, to membranes of Hek-B2 cells and cardiac myocytes upon challenge with isoproterenol.The heptahelical ␤-adrenergic receptors (␤ 2 ARs) 1 act by coupling through the G-protein G s to stimulate adenylyl cyclase and thereby increase intracellular cAMP concentrations (1-4). It is now well established that the rapid uncoupling of this response is achieved by the action of the G-protein receptorcoupled kinase 2 (2). This phosphorylates the carboxyl-terminal tail of the plasma membrane-associated ␤ 2 AR, allowing the recruitment of ␤-arrestins from the cytosol (5-7). It is this recruitment of ␤-arrestin, rather than phosphorylation per se, that elicits uncoupling, presumably by sterically blocking coupling of the ␤ 2 AR to G s . The importance of ␤-arrestin interaction to G-protein-coupled receptors (GPCRs) in vivo has been clearly established in knockout mouse models (8 -10).The attenuation of cAMP signaling is also achieved through the action of phosphodiesterases (PDEs) that are able to hydrolyze cAMP to 5Ј-AMP (11-17). Since they provide the sole route for degradation of cAMP in cells, they are poised to play a key role in controlling cAMP signaling. Multiple genes encode a large superfamily of PDEs, which differ in their regulatory and kinetic properties. Of these, the PDE4 cAMP-specific phosphodiesterase family (13-16) has recently attracted much interest, since PDE4-selective inhibitors are currently being developed as potential therapeutic agents for various inflammatory diseases of the respiratory system, such as asthma and chronic obstructive pulmonary disease (18 -21). The PDE4 enzyme family is encoded by four genes (PDE4A, -B, -C, and -D), which generate over 16 different isoforms through the use of distinct promoters and alternative mRNA splicing (12,13,...

PDE4A11 is a novel cAMP-specific phosphodiesterase that is conserved in humans, mouse, rat, pig, and bat. Exon-1 4A11 encodes its unique, 81 amino acid N-terminal region. Reverse-transcriptase polymerase chain reaction performed across the splice junction, plus identification of expressed sequence tags, identifies PDE4A11 as a long isoform possessing UCR1 and UCR2 regulatory domains. Transcript analysis shows that PDE4A11 is widely expressed compared with PDE4A10 and PDE4A4B long isoforms. Truncation analysis identifies a putative promoter in a 250-base pair region located immediately upstream of the start site in Exon-1 4A11. Recombinant PDE4A11, expressed in COS-7 cells, is a 126-kDa protein localized predominantly around the nucleus and in membrane ruffles. PDE4A11 exhibits a K m for cAMP hydrolysis of 4 M, with relative V max similar to that of PDE4A10 and PDE4A4B. PDE4A11 is dose-dependently inhibited by rolipram, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone (Ro 20-1724), cilomilast, roflumilast, and denbufylline, with IC 50 values of 0.7, 0.9, 0.03, 0.004, and 0.3 M, respectively. Soluble and particulate PDE4A11 exhibit distinct rates of thermal inactivation (55°C; T (0.5) ϭ 2.5 and 4.4 min, respectively). Elevating cAMP levels in COS-7 cells activates PDE4A11 concomitant with its phosphorylation at Ser119 by protein kinase A (PKA). PDE4A11 differs from PDE4A4 in sensitivity to cleavage by caspase-3, interaction with LYN SH3 domain, redistribution upon long-term rolipram challenge, and sensitivity to certain PDE4 inhibitors. PDE4A11, PDE4A10, and PDE4A4 all can interact with ␤arrestin.

Memory formation requires cAMP signaling; thus, this cascade has been of great interest in the search for cognitive enhancers. Given that medications are administered long-term, we determined the effects of chronically increasing cAMP synthesis in the brain by expressing a constitutively active isoform of the G-protein subunit G␣s (G␣s*) in postnatal forebrain neurons of mice. Previously, we showed that G␣s* mice exhibit increased adenylyl cyclase activity but decreased cAMP levels in cortex and hippocampus due to a PKA-dependent increase in total cAMP phosphodiesterase (PDE) activity. Here, we extend previous findings by determining if G␣s* mice show increased activity of specific PDE families that are regulated by PKA, if G␣s* mice show PKA-dependent deficits in fear memory, and if these memory deficits are associated with PKA-dependent alterations in neuronal activity as mapped by Arc mRNA expression. Consistent with previous findings, we show here that G␣s* mice exhibit a significant compensatory increase in cAMP PDE1 activity and a trend toward increased cAMP PDE4 activity. Further, inhibiting the presumably elevated PKA activity in G␣s* mice fully rescues short-and long-term memory deficits in a fear-conditioning task, while extending the training session from one to four CS-US pairings partially rescues these deficits. Mapping of Arc mRNA levels suggests these PKA-dependent memory deficits may be related to decreased neuronal activity specifically within the cortex. G␣s* mice show decreased Arc mRNA expression in CA1, orbital cortex, and cortical regions surrounding the hippocampus; however, only the deficits in cortical regions surrounding the hippocampus are PKA dependent. Our results imply that chronically stimulating targets upstream of cAMP may detrimentally affect cognition.Regulated signaling through the cAMP cascade is key to the neurobiology of learning and memory (Livingstone et al. 1984;Connolly et al. 1996;Wong et al. 1999;Pineda et al. 2004). Studies in flies and mice show that both decreases (Livingstone et al. 1984;Wong et al. 1999) and increases (Connolly et al. 1996;) in adenylyl cyclase (AC) activity can result in memory deficits. Similarly, decreasing cAMP-dependent protein kinase A (PKA) activity impairs hippocampus-dependent memory (Abel et al. 1997), whereas increasing PKA activity impairs prefrontal cortex-dependent memory in mice (Ramos et al. 2003). There does, however, appear to be a window within which the cAMP cascade can be positively modulated to enhance memory. For example, overexpression of AC type 1 in mice enhances recognition memory . Further, pharmacological inhibition of cAMP phosphodiesterases (PDE), the only enzymes that degrade cAMP (cf. Beavo and Brunton 2002; Houslay and Adams 2003), improves baseline memory in mice (Barad et al. 1998;Monti et al. 2006) and rescues genetically (Bourtchouladze et al. 2003) and pharmacologically induced memory deficits (Zhang et al. 2000. As a result, the cAMP cascade has been of great interest in the search for therapeutics that wi...

The cAMP-specific phosphodiesterase-4 (PDE4) gene family is the target of several potential selective therapeutic inhibitors. The four PDE4 genes generate several distinct protein-coding isoforms through the use of alternative promoters and 5Ј-coding exons. Using mouse transcripts, we identified a novel, super-short isoform of human PDE4B encoding a novel 5Ј terminus, which we label PDE4B5. The protein-coding region of the novel 5Ј exon is conserved across vertebrates, chicken, zebrafish, and fugu. Reverse-transcription-polymerase chain reaction (PCR) and quantitative (PCR) measurements show that this isoform is brain-specific. The novel protein is 58 Ϯ 2 kDa; it has cAMP hydrolyzing enzymatic activity and is inhibited by PDE4-selective inhibitors rolipram and cilomilast (Ariflo). Confocal and subcellular fractionation analyses show that it is distributed predominantly and unevenly within the cytosol. The 16 novel N-terminal residues of PDE4B5 are identical to the 16 N-terminal residues of the super-short isoform of PDE4D (PDE4D6), which is also brain-specific. PDE4B5 is able to bind the scaffold protein DISC1, whose gene has been linked to schizophrenia. Microarray expression profiling of the PDE4 gene family shows that specific PDE4 genes are enriched in muscle and blood fractions; however, only by monitoring the individual isoforms is the brain specificity of the super-short PDE4D and PDE4B isoforms revealed. Understanding the distinct tissue specificity of PDE4 isoforms will be important for understanding phosphodiesterase biology and opportunities for therapeutic intervention.