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| Item Type: | Article |
|---|---|
| Title: | 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 |
| Creators Name: | Lynch, M.J. and Baillie, G.S. and Mohamed, A. and Li, X. and Maisonneuve, C. and Klussmann, E. and van Heeke, G. and Houslay, M.D. |
| Abstract: | 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. |
| Keywords: | 3',5'-Cyclic-AMP Phosphodiesterases, Arrestins, Western Blotting, Cell Line, Cyclic AMP-Dependent Protein Kinases, Type 3 Cyclic Nucleotide Phosphodiesterases, Enzyme Activation, Isoenzymes, Mitogen-Activated Protein Kinases, Biological Models, Phosphoric Diester Hydrolases, Precipitin Tests, RNA Interference, Adrenergic beta-2 Receptors |
| Source: | Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Volume: | 280 |
| Number: | 39 |
| Page Range: | 33178-33189 |
| Date: | 30 September 2005 |
| Official Publication: | https://doi.org/10.1074/jbc.M414316200 |
| PubMed: | View item in PubMed |
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