Biomedical subjects

George S Baillie

Publications and source records attributed to George S Baillie.

At least 19 recordsLinked to original sources

PubMed2006

Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells.

The cAMP/protein kinase A (PKA)-dependent insertion of water channel aquaporin-2 (AQP2)-bearing vesicles into the plasma membrane in renal collecting duct principal cells (AQP2 shuttle) constitutes the molecular basis of arginine vasopressin (AVP)-regulated water reabsorption. cAMP/PKA signaling systems are compartmentalized by A kinase anchoring proteins (AKAP) that tether PKA to subcellular sites and by phosphodiesterases (PDE) that terminate PKA signaling through hydrolysis of localized cAMP. In primary cultured principal cells, AVP causes focal activation of PKA. PKA and cAMP-specific phosphodiesterase-4D (PDE4D) are located on AQP2-bearing vesicles. The selective PDE4 inhibitor rolipram increases AKAP-tethered PKA activity on AQP2-bearing vesicles and enhances the AQP2 shuttle and thereby the osmotic water permeability. AKAP18delta, which is located on AQP2-bearing vesicles, directly interacts with PDE4D and PKA. In response to AVP, PDE4D and AQP2 translocate to the plasma membrane. Here PDE4D is activated through PKA phosphorylation and reduces the osmotic water permeability. Taken together, a novel, compartmentalized, and physiologically relevant cAMP-dependent signal transduction module on AQP2-bearing vesicles, comprising anchored PDE4D, AKAP18delta, and PKA, has been identified.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Scanning peptide array analyses identify overlapping binding sites for the signalling scaffold proteins, beta-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5.

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.

Amino Acid Sequence↗

PubMed2006

Reduced PDE4 expression and activity contributes to enhanced catecholamine-induced cAMP accumulation in adipocytes from FOXC2 transgenic mice.

Overexpression of forkhead transcription factor FOXC2 in white adipose tissue (WAT) leads to a lean phenotype resistant to diet-induced obesity. This is due, in part, to enhanced catecholamine-induced cAMP-PKA signaling in FOXC2 transgenic mice. Here we show that rolipram treatment of adipocytes from FOXC2 transgenic mice did not increase isoproterenol-induced cAMP accumulation to the same extent as in wild type cells. Accordingly, phosphodiesterase-4 (PDE4) activity was reduced by 75% and PDE4A5 protein expression reduced by 30-50% in FOXC2 transgenic WAT compared to wild type. Thus, reduced PDE4 activity in adipocytes from FOXC2 transgenic mice contributes to amplified beta-AR induced cAMP responses observed in these cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Phosphodiesterase-4 influences the PKA phosphorylation status and membrane translocation of G-protein receptor kinase 2 (GRK2) in HEK-293beta2 cells and cardiac myocytes.

Membrane-recruitment of GRK2 (G-protein receptor kinase 2) provides a fundamental step in the desensitization process controlling GPCRs (G-protein-coupled receptors), such as the beta2AR (beta2-adrenergic receptor). In the present paper, we show that challenge of HEK-293beta2 [human embryonic kidney cells stably overexpressing the FLAG-tagged beta2AR-GFP (green fluorescent protein)] cells with the beta-adrenoceptor agonist, isoprenaline, causes GRK2 to become phosphorylated by PKA (cAMP-dependent protein kinase). This action is facilitated when cAMP-specific PDE4 (phosphodiesterase-4) activity is selectively inactivated, either chemically with rolipram or by siRNA (small interfering RNA)-mediated knockdown of PDE4B and PDE4D. PDE4-selective inhibition by rolipram facilitates the isoprenaline-induced membrane translocation of GRK2, phosphorylation of the beta2AR by GRK2, membrane translocation of beta-arrestin and internalization of beta2ARs. PDE4-selective inhibition also enhances the ability of isoprenaline to trigger the PKA phosphorylation of GRK2 in cardiac myocytes. In the absence of isoprenaline, rolipram-induced inhibition of PDE4 activity in HEK-293beta2 cells acts to stimulate PKA phosphorylation of GRK2, with consequential effects on GRK2 membrane recruitment and GRK2-mediated phosphorylation of the beta2AR. We propose that a key role for PDE4 enzymes is: (i) to gate the action of PKA on GRK2, influencing the rate of GRK2 phosphorylation of the beta2AR and consequential recruitment of beta-arrestin subsequent to beta-adrenoceptor agonist challenge, and (ii) to protect GRK2 from inappropriate membrane recruitment in unstimulated cells through its phosphorylation by PKA in response to fluctuations in basal levels of cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Spatial organisation of AKAP18 and PDE4 isoforms in renal collecting duct principal cells.

A plethora of stimuli including hormones and neurotransmitters mediate a rise of the cellular level of cAMP and thereby activation of protein kinase A (PKA). PKA phosphorylates and thereby modulates the activity of a wide range of cellular targets. It is now appreciated that different stimuli induce the activation of PKA at specific sites where the kinase phosphorylates particular substrates in close proximity. The tethering of PKA to cellular compartments is facilitated by A kinase-anchoring proteins (AKAPs). The incorporation of phosphodiesterases (PDEs) into AKAP-based signalling complexes provides gradients of cAMP that regulate PKA activity locally. An example for a process depending on compartmentalised cAMP/PKA signalling is the arginine-vasopressin (AVP)-mediated water reabsorption in renal collecting duct principal cells. Upon activation through AVP, PKA phosphorylates the water channel aquaporin-2 (AQP-2) located on intracellular vesicles. The phosphorylation triggers the redistribution of AQP2 to the plasma membrane. AKAP-anchored PKA has been shown to be involved in AQP2 shuttling. Here, AKAP18 isoforms and members of the PDE4 family of PDEs are shown to be differentially localised in renal principal cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2005

DISC1 and PDE4B are interacting genetic factors in schizophrenia that regulate cAMP signaling.

The disrupted in schizophrenia 1 (DISC1) gene is a candidate susceptibility factor for schizophrenia, but its mechanistic role in the disorder is unknown. Here we report that the gene encoding phosphodiesterase 4B (PDE4B) is disrupted by a balanced translocation in a subject diagnosed with schizophrenia and a relative with chronic psychiatric illness. The PDEs inactivate adenosine 3',5'-monophosphate (cAMP), a second messenger implicated in learning, memory, and mood. We show that DISC1 interacts with the UCR2 domain of PDE4B and that elevation of cellular cAMP leads to dissociation of PDE4B from DISC1 and an increase in PDE4B activity. We propose a mechanistic model whereby DISC1 sequesters PDE4B in resting cells and releases it in an activated state in response to elevated cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2005

RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with beta arrestin to control the protein kinase A/AKAP79-mediated switching of the beta2-adrenergic receptor to activation of ERK in HEK293B2 cells.

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 beta arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with beta arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the beta2-adrenergic receptor (beta2-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 beta2-AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon beta arrestin knockdown. A mutation that compromises interactions with beta arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. Beta arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the beta2-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.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2005

Compartmentalisation of phosphodiesterases and protein kinase A: opposites attract.

Understanding the molecular organisation of intracellular signalling pathways is a topic of considerable research interest. Since many signalling enzymes are widely distributed and have several substrates, a critical component in signal transduction is the control of specificity. This is achieved, in part by the assembly of multiprotein complexes where clusters of signalling enzymes create focal points to disseminate the intracellular action of many hormones. This is particularly true for the cAMP dependent protein kinase (PKA) that is localised throughout the cell via its association with A-kinase anchoring proteins (AKAPs). Recent data suggest that some AKAPs also interact with phosphodiesterases (PDEs). Compartmentalisation of PDEs not only provides an elegant means to control PKA activation by monitoring the local cAMP flux, but also serves to concentrate and segregate the action of these important regulatory enzymes.

Animals↗

PubMed2005

Arrestin times for compartmentalised cAMP signalling and phosphodiesterase-4 enzymes.

Various methods reveal that cyclic AMP (cAMP) signalling in cells is compartmentalised. These methods use FRET probes based upon either protein kinase A (PKA) or EPAC, cAMP-gated ion channels, or the selective activation of AKAP-anchored PKA isoforms. The basis of compartmentalisation involves point sources of cAMP generation within sub-domains of the plasma membrane coupled to degradation by spatially segregated, anchored forms of cAMP phosphodiesterases. cAMP-specific phosphodiesterase-4 (PDE4) isoforms play a central role in determining compartmentalisation, as exemplified in cardiac myocytes and T cells. The PKA phosphorylation status of the beta2-adrenoreceptor, and hence its ability to switch its signalling from G(s) to G(i) and thus to activate ERK, is regulated dynamically by the agonist-stimulated recruitment of PDE4 to the receptor in complex with beta-arrestin. The co-receptor CD28 enhances signalling through the T-cell receptor by recruiting a PDE4/beta-arrestin complex, which then attenuates PKA phosphorylation of Csk.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2005

Investigation of extracellular signal-regulated kinase 2 mitogen-activated protein kinase phosphorylation and regulation of activity of PDE4 cyclic adenosine monophosphate-specific phosphodiesterases.

Recently, it has been shown that enzymes of the cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase (PDE) family 4 can be directly phosphorylated by extracellular signal-regulated kinase 2 (ERK2). Phosphorylation of PDE4s by ERK2 is dependent on two docking domains on either side of the target serine that allow specificity and high-fidelity binding of the kinase. The functional consequence of PDE4 phosphorylation by ERK is either an increase or a decrease in PDE activity, depending on whether the PDE4 contains only one of the upstream conserved regions (UCR1) that are typical of PDE4s or both (UCR1 and UCR2). We detail some of the methods that have been crucial in elucidating these important discoveries that represent a novel point of cross talk between the cAMP signaling system and the ERK mitogen-activated protein kinase cascade.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2004

Expression, intracellular distribution and basis for lack of catalytic activity of the PDE4A7 isoform encoded by the human PDE4A cAMP-specific phosphodiesterase gene.

PDE4A7 is an isoform encoded by the human PDE4A cAMP-specific phosphodiesterase gene that fails to hydrolyse cAMP and whose transcripts are widely expressed. Removal of either the N- or C-terminal unique portions of PDE4A7 did not reconstitute catalytic activity, showing that they did not exert a chronic inhibitory effect. A chimera (Hyb2), formed by swapping the unique N-terminal portion of PDE4A7 with that of the active PDE4A4C form, was not catalytically active. However, one formed (Hyb1) by swapping the unique C-terminal portion of PDE4A7 with that common to all active PDE4 isoforms was catalytically active. Compared with the active PDE4A4B isoform, Hyb1 exhibited a similar K(m) value for cAMP and IC50 value for rolipram inhibition, but was less sensitive to inhibition by Ro-20-1724 and denbufylline, and considerably more sensitive to thermal denaturation. The unique C-terminal region of PDE4A7 was unable to support an active catalytic unit, whereas its unique N-terminal region can. The N-terminal portion of the PDE4 catalytic unit is essential for catalytic activity and can be supplied by either highly conserved sequence found in active PDE4 isoforms from all four PDE4 subfamilies or the unique N-terminal portion of PDE4A7. A discrete portion of the conserved C-terminal region in active PDE4A isoforms underpins their aberrant migration on SDS/PAGE. Unlike active PDE4A isoforms, PDE4A7 is exclusively localized to the P1 particulate fraction in cells. A region located within the C-terminal portion of active PDE4 isoforms prevents such exclusive targeting. Three functional regions in PDE4A isoforms are identified, which influence catalytic activity, subcellular targeting and conformational status.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2004

Remodelling of the PDE4 cAMP phosphodiesterase isoform profile upon monocyte-macrophage differentiation of human U937 cells.

Monocytes and macrophages provide key targets for the action of novel anti-inflammatory therapeutics targeted at inhibition of PDE4 cAMP-specific phosphodiesterases. PDE4 enzymes provide the dominant cAMP phosphodiesterase activity in U937 human monocytic cells. Differentiation of U937 monocytic cells to a macrophage-like phenotype causes a marked reduction in total cellular PDE4 activity. Monocytic U937 cells express the long PDE4A4, PDE4D5 and PDE4D3 isoforms plus the short PDE4B2 isoform. Differentiation of U937 cells to a macrophage-like phenotype causes a marked downregulation of PDE4D3 and PDE4D5, elicits a marked upregulation of PDE4B2 and induces the novel PDE4A10 long isoform. Comparable patterns are found in human peripheral blood monocytes and macrophages differentiated from them. Immunopurification of PDE4 subfamilies identifies long PDE4D isoforms as providing the major PDE4 activity in U937 monocytic cells. In U937 macrophage-like cells, the activity of the short PDE4B2 isoform predominates. No indication of either the expression or induction of PDE4C was evident. Activation of ERK exerts an inhibitory effect on total PDE4 activity in monocytic U937 cells, where the activity of long PDE4 isoforms predominates. The effect of ERK activation is switched to one of overall stimulation of total PDE4 activity in macrophage U937 cells, where the activity of the short PDE4B2 isoform predominates.10 The profound differentiation-induced changes in PDE4 isoform profile identified here suggests that the development of inhibitors specific for particular PDE4 isoforms may allow for selective effects on monocytes and macrophages to be achieved.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2004

Differential expression of PDE4 cAMP phosphodiesterase isoforms in inflammatory cells of smokers with COPD, smokers without COPD, and nonsmokers.

The expression profile of a panel of 15 cAMP phosphodiesterase isoforms was determined for inflammatory cell types of relevance to chronic obstructive pulmonary disease (COPD). In particular, the expression profiles for bronchoalveolar macrophages, peripheral blood monocytes, T lymphocytes, and neutrophils from smokers with and without COPD were compared. The phosphodiesterase expression profile was also analyzed for peripheral blood monocytes, T lymphocytes, and neutrophils from nonsmokers and compared with smokers. Qualitative RT-PCR identified transcripts for PDE4A10, PDE4A7, PDE4B1, PDE4B2, PDE4D1, and PDE4D2 isoforms as well as transcripts for both PDE3B and PDE7A in T cells, monocytes, and macrophages in all subjects. Transcripts for PDE4B3 and PDE4D4 were not observed in any of the cell types investigated. PDE4C was detected in all cells analyzed except for T cells. The long PDE4A4, PDE4D3, and PDE4D5 isoforms exhibited cell type-specific expression patterns. Semiquantitative and real-time quantitative RT-PCR were used to analyze differential expression between disease states and between cell types. PDE4A4 was found significantly upregulated in lung macrophages from smokers with COPD when compared with control smokers. Furthermore, PDE4A4 as well as PDE4B2 transcripts were detected in higher amounts in peripheral blood monocytes of smokers when compared with nonsmokers. Finally, PDE4D5 and PDE4C were differentially regulated in lung macrophages when compared with monocytes of the same subjects, irrespective of the disease state. The data obtained suggest that PDE4A4 may be relevant as a macrophage-specific anti-inflammatory target for COPD.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2003

The unique amino-terminal region of the PDE4D5 cAMP phosphodiesterase isoform confers preferential interaction with beta-arrestins.

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 beta-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 beta-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 beta-arrestin 2 are involved in mediating its interaction with PDE4D5, one associated with its extreme amino-terminal region and the other located within the carboxyl-terminal domain of the protein. We suggest that the unique amino-terminal region of PDE4D5 allows it to preferentially interact with beta-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.

Amino Acid Sequence↗

PubMed2003

Attenuation of the activity of the cAMP-specific phosphodiesterase PDE4A5 by interaction with the immunophilin XAP2.

The cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4A5 interacted with the immunophilin XAP2 in a yeast two-hybrid assay. The interaction was confirmed in biochemical pull-down analyses. The interaction was specific, in that PDE4A5 did not interact with the closely related immunophilins AIPL1, FKBP51, or FKBP52. XAP2 also did not interact with other PDE4A isoforms or typical isoforms from the three other PDE4 subfamilies. Functionally, XAP2 reversibly inhibited the enzymatic activity of PDE4A5, increased the sensitivity of PDE4A5 to inhibition by the prototypical PDE4 inhibitor 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) and attenuated the ability of cAMP-dependent protein kinase to phosphorylate PDE4A5 in intact cells. XAP2 maximally inhibited PDE4A5 by approximately 60%, with an IC50 of 120 nm, and reduced the IC50 for rolipram from 390 nm to 70-90 nm. Co-expression of XAP2 and PDE4A5 in COS7 cells showed that they could be co-immunoprecipitated and also reduced both the enzymatic activity of PDE4A5 and its IC50 for rolipram. Native XAP2 and PDE4A5 could be co-immunoprecipitated from the brain. The isolated COOH-terminal half of XAP2 (amino acids 170-330), containing its tetratricopeptide repeat domain, but not the isolated NH2-terminal half (amino acids 1-169), containing the immunophilin homology region, similarly reduced PDE4A5 activity and its IC50 for rolipram. Mutation of Arg271 to alanine, in the XAP2 tetratricopeptide repeat region, attenuated its ability to both interact with PDE4A5 in two-hybrid assays and to inhibit PDE4A5 activity. Either the deletion of a specific portion of the unique amino-terminal region or specific mutations in the regulatory UCR2 domain of PDE4A5 attenuated its ability be inhibited by XAP2. We suggest that XAP2 functionally interacts with PDE4A5 in cells.

1-Methyl-3-isobutylxanthine↗

PubMed2003

Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform.

We have isolated cDNAs encoding PDE4B4, a new cAMP-specific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of 'long' PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85 kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within the cytosol, which was confirmed by confocal microscopic analysis of living COS7 cells transfected with a green fluorescent protein-PDE4B4 chimaera. PDE4B4 exhibited a K(m) for cAMP of 5.4 microM and a V(max), relative to that of the long PDE4B1 isoform, of 2.1. PDE4B4 was inhibited by the prototypical PDE4 inhibitor rolipram [4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone] with an IC(50) of 83 nM. Treatment of COS7 cells with forskolin, to elevate cAMP levels, produced activation of PDE4B4, which was associated with the phosphorylation of PDE4B4 on Ser-56 within UCR1. The unique tissue distribution and intracellular targeting of PDE4B4 suggests that this isoform may have a distinct functional role in regulating cAMP levels in specific cell types.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2003

beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from Gs to Gi.

Phosphorylation of the beta(2) adrenoreceptor (beta(2)AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (G(s)) to inhibitory guanine nucleotide regulatory protein (G(i)). beta-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the beta(2)AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the beta(2)AR. In human embryonic kidney 293 cells overexpressing a recombinant beta(2)AR, stimulation with isoprenaline recruits beta-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the beta(2)AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the beta(2)AR. Rolipram and PDE4D5-D556A also enhance beta(2)AR-mediated activation of extracellular signal-regulated kinases ERK12. This is consistent with a switch in coupling of the receptor from G(s) to G(i), because the ERK12 activation is sensitive to both inhibitors of PKA (H89) and G(i) (pertussis toxin). In cardiac myocytes, the beta(2)AR also switches from G(s) to G(i) coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK12. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK12 activation. Thus, receptor-stimulated beta-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the beta(2)AR in a physiological system, the cardiac myocyte.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2003

Occupancy of the catalytic site of the PDE4A4 cyclic AMP phosphodiesterase by rolipram triggers the dynamic redistribution of this specific isoform in living cells through a cyclic AMP independent process.

In cells transfected to express wild-type PDE4A4 cAMP phosphodiesterase (PDE), the PDE4 selective inhibitor rolipram caused PDE4A4 to relocalise so as to form accretion foci. This process was followed in detail in living cells using a PDE4A4 chimera formed with Green Fluorescent Protein (GFP). The same pattern of behaviour was also seen in chimeras of PDE4A4 formed with various proteins and peptides, including LimK, RhoC, FRB and the V5-6His tag. Maximal PDE4A4 foci formation, occurred over a period of about 10 h, was dose-dependent on rolipram and was reversible upon washout of rolipram. Inhibition of protein synthesis, using cycloheximide, but not PKA activity with H89, inhibited foci generation. Foci formation was elicited by Ro20-1724 and RS25344 but not by either Ariflo or RP73401, showing that not all PDE4 selective inhibitors had this effect. Ariflo and RP73401 dose-dependently antagonised rolipram-induced foci formation and dispersed rolipram pre-formed foci as did the adenylyl cyclase activator, forskolin. Foci formation showed specificity for PDE4A4 and its rodent homologue, PDE4A5, as it was not triggered in living cells expressing the PDE4B2, PDE4C2, PDE4D3 and PDE4D5 isoforms as GFP chimeras. Altered foci formation was seen in the Deltab-LR2-PDE4A4 construct, which deleted a region within LRZ, showing that appropriate linkage between the N-terminal portion of PDE4A4 and the catalytic unit of PDE4A4 was needed for foci formation. Certain single point mutations within the PDE4A4 catalytic site (His505Asn, His506Asn and Val475Asp) were shown to ablate foci formation but still allow rolipram inhibition of PDE4A4 catalytic activity. We suggest that the binding of certain, but not all, PDE4 selective inhibitors to PDE4A4 induces a conformational change in this isoform by 'inside-out' signalling that causes it to redistribute in the cell. Displacing foci-forming inhibitors with either cAMP or inhibitors that do not form foci can antagonise this effect. Specificity of this effect for PDE4A4 and its homologue PDE4A5 suggests that interplay between the catalytic site and the unique N-terminal region of these isoforms is required. Thus, certain PDE4 selective inhibitors may exert effects on PDE4A4 that extend beyond simple catalytic inhibition. These require protein synthesis and may lead to redistribution of PDE4A4 and any associated proteins. Foci formation of PDE4A4 may be of use in probing for conformational changes in this isoform and for sub-categorising PDE4 selective inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗