PDE4B and PDE4D provide >90% of PDE4 cAMP phosphodiesterase activity in human embryonic kidney (HEK293B2) cells. Their selective small interference RNA (siRNA)-mediated knockdown potentiates isoprenaline-stimulated protein kinase A (PKA) activation. Whereas endogenous PDE4D co-immunoprecipitates with ␤arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with ␤arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the ␤ 2 -adrenergic receptor (␤ 2 -AR) by PKA and activation of extracellular signal-regulated kinase (ERK) through G i . Isoform-selective knockdown identifies PDE4D5 as the functionally important species regulating isoprenaline stimulation of both these processes. Ht31-mediated disruption of the tethering of PKA to AKAP scaffold proteins attenuates isoprenaline activation of ERK, even upon PDE4D knockdown. Selective siRNA-mediated knockdown identifies AKAP79, which is constitutively associated with the ␤ 2 -AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon ␤arrestin knockdown. A mutation that compromises interactions with ␤arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. ␤arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the ␤ 2 -AR and the consequential switching of its signaling to ERK. The ability to observe a cellular phenotype upon PDE4D5 knockdown demonstrates that other PDE4 isoforms, expressed at endogenous levels, are unable to afford rescue in HEK293B2 cells.It is now well appreciated that cAMP signaling is compartmentalized in cells (1-4). This notion arose originally from elegant studies done on cardiac myocytes (2) and gained considerable credence because the discovery of anchor proteins (AKAPs) for protein kinase A (PKA) 4 allowed gradients of cAMP to be sensed and acted upon accordingly (1, 4, 5). More recently, a number of cAMP sensors have been developed that have allowed gradients of cAMP to be identified and even visualized in cells (6 -9). Paramount to the generation and shaping of intracellular gradients is the action of cAMP phosphodiesterases, which provide the sole means of degrading cAMP in cells (2, 10 -14). Of these, the PDE4 family of enzymes has gained attention in view of the fact that chemical knock-out with selective inhibitors indicates that they perform an important role in regulating key processes such as inflammation and cognition (15)(16)(17)(18)(19)(20). Four genes (4A, 4B, 4C, and 4D) encode a large family of PDE4 isoforms (15,17). Little is known, however, about the functional significance of each PDE4 sub-family. Nevertheless, important insights into physiological function have come from gene knockout studies on the PDE4B and PDE4D sub-families, which have implied distinct roles for these sub-f...

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.

The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and beta-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and beta-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK1 and beta-arrestin interact at overlapping sites within the unique N-terminal region of PDE4D5 and at distinct sites within the conserved PDE4 catalytic domain. Screening scanning alanine substitution peptide arrays, coupled with mutagenesis and truncation studies, allowed definition of RACK1 and beta-arrestin interaction sites. Modelled on the PDE4D catalytic domain, these form distinct well-defined surface-exposed patches on helices-15-16, for RACK1, and helix-17 for beta-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased beta-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the beta2AR (beta2-adrenergic receptor) upon isoproterenol challenge approx. 4-fold and severely attenuated (approx. 4-5 fold) both isoproterenol-stimulated PKA (protein kinase A) phosphorylation of the beta2AR and activation of ERK (extracellular-signal-regulated kinase). The ability of a catalytically inactive form of PDE4D5 to exert a dominant negative effect in amplifying isoproterenol-stimulated ERK activation was ablated by a mutation that blocked the interaction of PDE4D5 with beta-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and beta-arrestin compete to sequester distinct 'pools' of PDE4D5. In this fashion, alterations in the level of RACK1 expression may act to modulate signal transduction mediated by the beta2AR.

Aeromonas hydrophila is an opportunistic Gram-negative pathogen that readily attaches to stainless steel to produce a thin biofilm with a complex 3D structure covering 40-50% of the available surface and producing large microcolonies. As A. hydrophila possesses an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system based on the ahyRI locus, the presence of the AhyI protein and C4-HSL within the biofilm phase was first established by Western blot and AHL biosensor analysis respectively. The ability of the A. hydrophila AH-1 N strain to form biofilms in a continuous-flow chamber was compared with isogenic ahyI and ahyR mutants. The ahyI mutant, which cannot produce C4-HSL, failed to form a mature biofilm. In addition, the viable count of biofilm, but not planktonic phase ahyI mutants, was significantly lower that the parent or ahyR mutant. This defect in the differentiation of the ahyI mutant biofilm could be partially restored by the addition of exogenous C4-HSL. A mutation in ahyR increased coverage of the available surface to around 80% with no obvious effect upon biofilm microcolony formation. These data support a role for AHL-dependent quorum sensing in A. hydrophila biofilm development. Exposure of the A. hydrophila AH-1N biofilm to N-(3-oxodecanoyl)homoserine lactone, which inhibits exoprotease production in planktonic cells, however, had no effect on biofilm formation or architecture within the continuous-flow chamber.