Post-translational Modification and Sequestration of cAMP-specific Phosphodiesterase 4 in signalling complexes

Li, Xiang (2006) Post-translational Modification and Sequestration of cAMP-specific Phosphodiesterase 4 in signalling complexes. PhD thesis, University of Glasgow.

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cAMP is a ubiquitous second messenger that is pivotal in controlling a wide array of cellular functions. The sole means to inactivate cAMP is to degrade it into 5'- AMP through the action of cyclic nucleotide phosphodiesterases (PDEs). It is now well appreciated that cAMP hydrolysis by PDEs is as important as its synthesis by adenylyl cyclases to achieve cAMP homeostasis in cells. The cAMP-specific phosphodiesterase-4 (PDE4) is encoded by four different genes (PDE4A, PDE4B, PDE4C and PDE4D), which generate over 16 different isoforms by alternative 5' mRNA splicing. This process gives rise to PDE4 isoforms with unique N-terminal region, complete or truncated UCR1/UCR2 modules, a central catalytic domain and an extreme C-terminal region. This plethora of PDE4 isoforms, complete with distinct N-terminal targeting domains and particular regulatory properties, confers compartmentalized action on cAMP signaling in various cell types. Understanding the distinct molecular mechanisms and functional outcomes of modification and sequestration of specific PDE4 isoforms can be expected to aid the development of more selective PDE4 inhibitor-based therapeutics. I have assessed the involvement of PDE4 isoenzymes in the β2-adrenergic receptor (β2AR) desensitization process in Chapter 3. It has been previously shown that agonist-activated P2ARS can recruit PDE4 isoforms in complex with β-arrestins and, in particular, PDE4D5 whereupon they undergo a dual desensitization. Using the PDE4-selective inhibitor rolipram or siRNA-mediated knockdown of PDE4B and PDE4D, I determined a new facet of PDE4-mediated process in the early stage of β2AR desensitization. In HEKB2 cells (stably overexpressing β2ARS) that have been pretreated with either rolipram or with siRNA to PDE4B and PDE4D, PKA phosphorylated GRK2 was accelerated in response to treatment with the β-agonist isoprenaline, as was isoprenaline-induced membrane translocation of GRK2, phosphorylation of the β2AR by GRK2, membrane translocation of β-arrestins/PDE4D complex and internalization of β2ARS, in comparison with the cells that were only challenged with isoprenaline. In the absence of isoprenaline, rolipram- induced inhibition of PDE4 activity in HEKB2 cells also stimulated the phosphorylation of GRK2 by PKA, with consequential effects on GRK2 membrane recruitment and GRK2 phosphorylation of β2AR. These results collectively suggest that PDE4 plays a fundamental role in influencing β2AR functioning by gating for the ability of PKA to phosphorylate GRK2 in resting cells and thereby regulating the degree of feedback through this system in stimulated cells. This provides a finely tuned adaptive response for preventing inappropriate activation of GRK through fluctuations in the basal levels of cAMP in resting/unstimulated cells. In Chapter 4 and 5 of this thesis, I analyzed the potential post-translational modification on one PDE4 isoform, namely PDE4D5. I have used site-directed mutagenesis to show that the ubiquitin-interacting motif (UIM) can confer the ability of PDE4D5 to undergo ubiquitination in response to extracellular stimuli. Furthermore, this ubiquitination was shown to be mediated by the E3 ligase Mdm2 that is brought in close proximity to PDE4D by the associated β-arrestin. My data shows that only a small subset of PDE4D5, namely that to which β-arrestins bind, is susceptible to ubiquitination.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Houslay, Prof. Miles
Date of Award: 2006
Depositing User: Mrs Monika Milewska-Fiertek
Unique ID: glathesis:2006-39014
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 18 Dec 2018 14:46
Last Modified: 18 Dec 2018 14:46
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