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
Introduction
Cyclic AMP controls a wide variety of cellular functions [1]. As the only means of degrading this second messenger is through the action of cyclic nucleotide phosphodiesterases (PDEs), these enzymes provide a key regulatory system. Over 30 different PDEs, able to degrade cAMP in mammalian cells, have been identified [1], [2], [3], [4], [5]. These are expressed in a cell-type specific fashion, implying distinct functional roles.
Signalling through cAMP is compartmentalised [1], [6], [7], with gradients of cAMP identified in living cells and the pivotal role that PDEs play in determining these gradients demonstrated [8], [9], [10]. Undoubtedly the specific intracellular targeting that characterises many PDE isoforms plays a key role in establishing compartmentalised cAMP signalling [3].
There is currently considerable interest [11], [12], [13], [14], [15], [16], [17] in PDE4 cAMP specific phosphodiesterases as PDE4 selective inhibitors have potent anti-inflammatory action and are being developed as therapeutic agents for respiratory diseases. However, side effects, such as emesis, which are associated with certain PDE4 inhibitors such as the archetypal PDE4 selective inhibitor rolipram, have hindered their therapeutic deployment [11], [12]. PDE4 enzymes are encoded by four genes (A, B, C, D) [2], [3], [17]. Each PDE4 gene, as a consequence of alternative mRNA splicing, generates multiple PDE4 isoforms, which are each characterised by a unique N-terminal region. These various isoforms are then grouped into so-called ‘long’, ‘short’ and ‘super-short’ variants, dependent upon either the presence or absence of regulatory UCR1 and UCR2 modules. Various PDE4 isoforms have been shown to interact with other proteins and lipids, allowing specific isoforms to be targeted to distinct intracellular sites and signalling complexes within cells [3], [17]. Of key importance in achieving such recruitment are the isoform-specific unique N-terminal regions, which was first shown for the PDE4A1 isoform [18]. Thus, for example, the RACK1 [19] and AKAP signalling scaffold proteins [20], [21] can recruit PDE4D5 and PDE4D3, respectively, and the TAPAS-1 domain of PDE4A1 can insert into phospholipid bilayers [22].
Chimeras generated with Green Fluorescent Protein (GFP) have been used extensively to monitor the intracellular targeting and dynamics of a wide variety of proteins in living cells [23]. Here we exploit this technology to identify a novel action associated with the PDE4 selective inhibitor, rolipram, namely its ability to cause the intracellular redistribution of the PDE4A4 long isoform in living cells. We evaluate this action to show that it is specific for PDE4A4 and that not all PDE4 inhibitors can trigger such a response. This leads us to propose that rolipram and certain other PDE4 selective inhibitors may exert actions consequent upon binding to PDE4A4 that are not mediated by cAMP.
Section snippets
Materials and methods
[3H]-cyclic AMP and ECL reagent were from Amersham International (Amersham, UK). Dithiothreitol, Triton X-100 and N-{1-(2,3-dioleoyloxy)propyl}-N,N,N-trimethylammonium methylsulfate (DOTAP) and protease inhibitor tablets were obtained from Boehringer Mannheim (Mannheim, Germany). Bradford reagent was from Bio-Rad (Herts, UK). All other biochemicals were from Sigma (Poole, UK) with (R)-(−)- and (S)-(+)-enantiomers of rolipram from BioMol (Pennsylvania, USA).
Rolipram causes the redistribution of PDE4A4–GFP in living cells
PDE4A4 [30], having both UCR1 and UCR2, is a so-called PDE4 long isoform [2], [3], [17]. It has been found in brain and also in various cell types associated with immune responses [2], [3], [31], [32], [33]. It is characterised by its unique N-terminal region of 107 amino acids [30], which can functionally interact with the SH3 domains of various SRC family tyrosyl kinases [25], [26].
Here we show a fluorescence confocal microscopy analysis of living CHO (Fig. 1a) and HEK (Fig. 1c) cells that
Discussion
Here, for the first time, we show that the PDE4 selective inhibitor, rolipram causes the profound redistribution of the PDE4A4 isoform in a dose-dependent and reversible fashion. To facilitate the analysis of this effect and to follow it in living cells, we have exploited a PDE4A4 chimera formed with GFP. Indeed, GFP has proven itself as a powerful tool to shed insight into the workings of many biological systems as it allows an effective means of identifying dynamic events occurring in living
Acknowledgements
M.D.H. is funded by the MRC (UK) G8604010 and the European Union (QLG2-CT-2001-02278; QLK3-CT-2002-02149). BT thanks the EC for support in grant QLG2-CT-2001-02278. Our special thanks go to Dr. Sara P. Bjoern and her team in BioImage for expert advice on, and preparation of, PDE4A4B chimeras and mutants used in this study, and also to Grith Hagel for developing the extraction procedure used to measure immobile PDE4A4–GFP in cells.
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