Elsevier

Cellular Signalling

Volume 15, Issue 10, October 2003, Pages 955-971
Cellular Signalling

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

https://doi.org/10.1016/S0898-6568(03)00092-5Get rights and content

Abstract

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 Δb-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.

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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