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. 2003 Feb 4;100(3):940-5.
doi: 10.1073/pnas.262787199. Epub 2003 Jan 27.

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

George S Baillie  1 , Arvind Sood, Ian McPhee, Irene Gall, Stephen J Perry, Robert J Lefkowitz, Miles D Houslay
Affiliations

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

George S Baillie et al. Proc Natl Acad Sci U S A. .

Retraction in

Abstract

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.

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Figures

Figure 1
Figure 1
Recruitment of PDE4D to the β2AR in HEK 293 cells. HEK 293 cells were challenged with isoprenaline (iso, 10 μM) and harvested at the indicated times, and the β2AR, immunopurified on anti-FLAG antibody, was conjugated to beads. (a) HEK 293 cells transfected to express a FLAG-tagged β2AR. Blotting detects a doublet for the β2AR, PDE4D5 and PDE4D3 isoforms, and β-arrestin1/2 (βArr) isoforms. (a Lower) An identical FLAG immunopurification protocol was performed but on native HEK 293 cells, showing no PDE4D recruitment. (b) Total immunoreactive PDE4D recruited over time in the β2AR-transfected HEK 293 cells (mean ± SD for n = 3 experiments).
Figure 2
Figure 2
Isoprenaline-induced phosphorylation of the β2AR in HEK 293 cells. In all these experiments at the indicated times, HEK 293 cells overexpressing the β2AR were harvested, and the PKA phosphorylation status of the anti-FLAG-immunopurified β2AR was assessed by using the PKA substrate antibody. In all instances lanes were loaded equally with immunopurified receptor. (a) Cells were challenged with either isoprenaline (Iso, 10 μM) alone or together with rolipram (Iso + roli, 10 μM). Lanes were loaded equally with immunopurified receptor. (b) Quantification of the data in a for n = 3 experiments with means ± SD. (c) Cells were treated for 8 min with isoprenaline (10 μM) plus rolipram (10 μM) in either the absence or presence of the PKA inhibitor H89 (1 μM). (d) Cells were treated for 8 min with isoprenaline (10 μM) plus rolipram (10 μM) either without or with transfection of protein kinase inhibitor (PKI), the PKA inhibitor (21). (e) As indicated, cells were transfected to express D556A-PDE4D5 before being treated for 8 min with isoprenaline (10 μM) before analysis. (f) Quantification of the data shown in d for n = 3 experiments with means ± SD.
Figure 3
Figure 3
β2AR-mediated activation of ERK in HEK 293 cells. These analyses were all done on HEK 293 cells overexpressing the β2AR. Each experiment was done three times. (a) Cells were treated with either isoprenaline (Iso, 10 μM) alone or together with rolipram (Iso + roli, 10 μM) before harvesting as indicated and immunoblotting for either total ERK or active P-ERK. The two immunoreactive species identified reflect the 42- and 44-kDa forms of ERK. (b) Cells were treated with isoprenaline (10 μM) together with rolipram (10 μM) either in the absence or presence of H89 (1 μM), harvested at 4 min, and immunoblotted for ERK and active P-ERK. (c) Cells were pretreated with pertussis toxin (25 ng/ml for 16 h) before challenge with isoprenaline (10 μM) together with rolipram (10 μM) and immunoblotted for ERK and P-ERK. A control (ctr) experiment was done in which cells that had not been subjected to pretreatment with pertussis toxin were challenged with isoprenaline and rolipram for 4 min before harvesting. (d) Cells were transfected with D556A-PDE4D5 before challenge with isoprenaline (10 μM) alone and then immunoblotted for ERK and P-ERK. A control experiment was done by using cells that had not been transfected with D556A-PDE4D5 but were challenged with isoprenaline for 4 min. (e) Quantification of the time course for ERK activation in cells treated with either isoprenaline (10 μM) alone or together with rolipram (10 μM) as well as the effect of isoprenaline on cells that had been transfected to express the D556A-PDE4D5 construct. Shown are means ± SD.
Figure 4
Figure 4
β2AR-mediated activation of ERK in primary cardiac myocytes. (a) Myocytes were challenged as indicated with isoprenaline (iso, 10 μM) and harvested, and the immunopurified β2AR was immunoblotted for PDE4D. PDE4D5 (105 kDa, Upper) and PDE4D3 (95 kDa, Lower) isoforms were identified. (b) Quantification of the data shown in a (means ± SD). (c) Myocytes were challenged with the indicated ligands for 10 min before Western blotting for ERK and P-ERK. In some instances, as indicated, cells were pretreated for 16 h with 25 ng/ml pertussis toxin, 10 μM isoprenaline, 10 μM rolipram, or 1 μM H89. (d) Quantification of the data shown in c for three experiments with means ± SD. (e) Myocytes were challenged for the indicated times with isoprenaline (10 μM) before Western blotting for ERK and P-ERK. DN-4D5 indicates cells transfected to express D556A-PDE4D5 by adenovirus-mediated gene transfer as evaluated by immunoblotting. The level of recombinant inactive PDE4D5 was 50- to 60-fold greater than that of endogenous PDE4D3 and PDE4D5 species, which made up the greater fraction (≈60%) of the total PDE4 activity in these cells. Control experiments (ctrA) were performed with cells exposed to adenovirus allowing GFP expression alone. Here ctrA indicates cells exposed to control virus and treated for 10 min with isoprenaline (10 μM), and ctrB indicates such cells treated for 10 min with both isoprenaline (10 μM) and rolipram (10 μM). (f) Quantification of the time-course data shown in e for three experiments with means ± SD. Each experiment was performed three times.
Figure 5
Figure 5
A schematic representation of the role of arrestin-recruited PDE4 in regulating the “switching” of the β2AR from Gs to Gi stimulation. Agonist occupancy of the β2AR initially leads to coupling to Gs, which causes activation of adenylyl cyclase, elevated cAMP levels, and activation of PKA, which is able to phosphorylate the β2AR. Concomitantly, agonist occupancy also leads to GRK-mediated phosphorylation of the β2AR, which allows for the recruitment of β-arrestin together with bound PDE4. PKA phosphorylation of the β2AR confers switching from Gs to Gi, with consequent activation of ERK1/2. However, β-arrestin-recruited PDE4 provides a negative feedback loop, the role of which is to attenuate local cAMP levels and thus the ability of membrane PKA to phosphorylate the β2AR. This action of β-arrestin-recruited PDE4 is uncovered by dominant negative PDE4, which replaces the active endogenous recruited PDE4 to ablate the negative feedback loop and thus accentuate switching to Gi.
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