Abstract-Cardiac myocytes have provided a key paradigm for the concept of the compartmentalized cAMP generation sensed by AKAP-anchored PKA. Phosphodiesterases (PDEs) provide the sole route for degrading cAMP in cells and are thus poised to regulate intracellular cAMP gradients. PDE3 and PDE4 represent the major cAMP degrading activities in rat ventriculocytes. By performing real-time imaging of cAMP in situ, we establish the hierarchy of these PDEs in controlling cAMP levels in basal conditions and on stimulation with a ␤-adrenergic receptor agonist. PDE4, rather than PDE3, appears to be responsible for modulating the amplitude and duration of the cAMP response to beta-agonists. PDE3 and PDE4 localize to distinct compartments and this may underpin their different functional roles.

Challenge of COS1 cells with the adenylyl cyclase activator forskolin led to the activation of recombinant PDE4A8, PDE4B1, PDE4C2 and PDE4D5 cAMP‐specific phosphodiesterase long isoforms. Forskolin challenge did not activate mutant long PDE4 isoforms where the serine target residue (STR) within the protein kinase A (PKA) consensus phosphorylation site in Upstream Conserved Region 1 (UCR1) was mutated to alanine. The PKA inhibitor, H89, ablated forskolin activation of wild‐type long PDE4 isoforms. Activated PKA caused the in vitro phosphorylation of recombinant wild‐type long PDE4 isoforms, but not those where the STR was mutated to alanine. An antiserum specific for the phosphorylated form of the STR detected a single immunoreactive band for recombinant long PDE4 isoforms expressed in COS1 cells challenged with forskolin. This was not evident in forskolin‐challenged cells treated with H89. Neither was it evident in forskolin‐challenged cells expressing long isoforms where the STR had been mutated to alanine. In transfected COS cells challenged with forskolin, only the phosphorylated PDE4D3 long form showed a decrease in mobility in Western blotting analysis. This decreased mobility of PDE4D3 was ablated upon mutation of either of the two serine targets for PKA phosphorylation in this isoform, namely Ser54 in UCR1 and Ser13 in the isoform‐specific N‐terminal region. Activation by forskolin challenge did not markedly alter the sensitivity of PDE4A8, PDE4B1, PDE4C2 and PDE4D5 to inhibition by rolipram. Long PDE4 isoforms from all four sub‐families can be phosphorylated by protein kinase A (PKA). This leads to an increase in their activity and may thus contribute to cellular desensitization processes in cells where these isoforms are selectively expressed. British Journal of Pharmacology (2002) 136, 421–433; doi:

The b-adrenergic receptor/cyclic AMP/protein kinase A (PKA) signalling pathway regulates heart rate and contractility. Here, we identified a supramolecular complex consisting of the sarcoplasmic reticulum Ca 2 þ -ATPase (SERCA2), its negative regulator phospholamban (PLN), the A-kinase anchoring protein AKAP18d and PKA. We show that AKAP18d acts as a scaffold that coordinates PKA phosphorylation of PLN and the adrenergic effect on Ca 2 þ re-uptake. Inhibition of the compartmentalization of this cAMP signalling complex by specific molecular disruptors interferes with the phosphorylation of PLN. This prevents the subsequent release of PLN from SERCA2, thereby affecting the Ca 2 þ re-uptake into the sarcoplasmic reticulum induced by adrenergic stimuli.

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,...

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