Occupancy of the catalytic site of the PDE4A4 cyclic AMP phosphodiesterase by rolipram triggers the...

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Cellular Signalling 15 (2003) 955–971

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

Robert Terrya,*, York-Fong Cheungb, Morten Praestegaarda, George S. Baillieb,Elaine Hustonb, Irene Gallb, David R. Adamsc, Miles D. Houslayb

aBioImage A/S, Moerkhoej Bygade 28, Soeborg DK-2860, DenmarkbMolecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Wolfson Building, Institute of Biomedical and Life Sciences,

University of Glasgow, Glasgow G12 8QQ, Scotland, UKcDepartment of Chemistry, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, Scotland, UK

Received 21 March 2003; accepted 30 May 2003

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 ArifloR or RP73401,

showing that not all PDE4 selective inhibitors had this effect. ArifloR 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 Db-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.

D 2003 Elsevier Inc. All rights reserved.

Keywords: PDE4A4; Rolipram; Cyclic AMP phosphodiesterase; Green flourescent protein; Protein kinase A

0898-6568/03/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/S0898-6568(03)00092-5

Abbreviations: PDE, cyclic nucleotide phosphodiesterase; PDE4,

cAMP specific family 4 PDE; UCR, Upstream Conserved Region;

PDE4A4, human PDE4 long form known specifically as HSPDE4A4B

(GenBank Accession number, L20965); IBMX, isobutylmethylxanthine;

rolipram, 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (racemic

mixture used unless specified otherwise); IC50, concentration of inhibitor

yielding 50% inhibition; EC50, concentration of compound yielding 50%

stimulatory effect; LR2, Linker Region 2.

* Corresponding author. Tel.: +45-4443-7506.

E-mail address: [email protected] (R. Terry).

1. 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 [1–5]. These are expressed in a

cell-type specific fashion, implying distinct functional roles.

R. Terry et al. / Cellular Signalling 15 (2003) 955–971956

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 [8–10]. 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 [11–17] in PDE4

cAMP specific phosphodiesterases as PDE4 selective inhib-

itors 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 mul-

tiple 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 iso-

forms 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, respec-

tively, 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 intracel-

lular 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 redis-

tribution 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.

2. 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-trimethylammo-

nium 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).

2.1. Molecular biology constructs

Analyses were done on the long PDE4A4B isoform

(GenBank accession number L20965) and a chimera formed

by making an in-frame fusion of PDE4A4B linked at its C-

terminus to Green Fluorescent Protein (GFP; GenBank

accession number U55672). Similar C-terminus chimeras

were made to PDE4A4B using, in place of GFP, human

LimK-1, RhoC, the FRB fragment of the FKBP12 rapamycin

associated protein, FRAP (FRB= FRAP[2025–2114]) and

the minimal V5-6His tag peptide (V5 epitope plus 6 histi-

dines). The latter, derived from the pEF6/V5-His C cloning

vector (Invitrogen) adds 47 amino acids to PDE4A4B. Site

directed mutagenesis was performed using a QuickChange

DNA mutagenesis kit (Stratagene, La Jolla, CA, USA)

according to the manufacturer’s instructions. All mutagenesis

and deletion constructs were confirmed by DNA sequencing.

The cDNA clones and expression vectors used for the various

PDE4 isoforms have been described previously [24,25].

2.2. Generation of stable cells lines

Chinese hamster ovary cells (CHO), human HEK293 and

rat RBL cells were transfected with the plasmids described

using the transfection agent FuGENEk 6 (Boehringer

Mannheim, USA) according to the method recommended

by the suppliers. Stable transfectants were selected using 1

mg/ml G418 sulphate (Geneticin; Invitrogen, Denmark) in

the growth medium most appropriate to each cell type. CHO

cells were cultured in HAM’s F12 nutrient mix with

Glutamax-1, 10% foetal bovine serum (FBS) plus 0.1%

penicillin/streptomycin (10000 units ml� 1). For the culture

of HEK293 and RBL cells, Dulbecco’s Modified Eagles

Medium (DMEM) containing 4500 mg/l glucose and Glu-

tamax was used in place of HAM’s F12. All cells were

cultured at 37 jC in 95% humidity and conditions of normal

atmospheric gases supplemented with 5% CO2. Clonal cell

lines were established from populations of stably transfected

cells by transferring single cells to individual culture com-

partments on Lab-Tek chambered coverglasses (Nunc, Den-

mark) containing fresh culture medium with 1 mg/ml G418

sulphate.

2.3. Analysis of GFP-PDE4 chimera in living cells

Cells were cultured to about 80% confluence in Lab-Tek

chambered coverglasses. Cells were also grown in plastic

96-well plates (Polyfiltronics Packard 96-View Plate or

Costar Special Optics Plate; both types tissue culture trea-

ted). Prior to experiments, the cells were cultured over night

in the appropriate medium, without G418 sulphate but with

100 Ag/ml penicillin–streptomycin mixture and 10% FBS.

Confocal images were collected using a Zeiss LSM 410

microscope (Carl Zeiss, Jena, Germany) equipped with

argon-ion and HeNe lasers giving excitation lines at 488

and 546 nm, respectively. GFP fluorescence was collected at

R. Terry et al. / Cellular Signalling 15 (2003) 955–971 957

488-nm excitation using a FT510 dichroic beam splitter and

a 510- to 525-nm band pass emission filter. Red fluoro-

phores (Texas Red, Alexa Fluor 546, BODIPY 558/568,

etc.) were collected at a 546-nm excitation using a FT560

dichroic beam splitter and an OG590 long-pass emission

filter. Images were typically collected with a Fluar 40X, NA:

1.3 oil immersion objective, the microscope’s confocal

aperture set to a value of 10 units (optimum for this lens).

Image sequences of live cells over time, used to monitor the

time course for the formation of accretion foci and their

behaviour during cell division, were collected using a Zeiss

Axiovert 135M fluorescence microscope fitted with a Fluar

40X, NA: 1.3 oil immersion objective and coupled to an

Orca ER charged coupled device (CCD) camera (Hamma-

matsu Photonics, Japan). Filters used for GFP were a 470F20-nm excitation filter, a 510-nm dichroic mirror and a

515F 15-nm emission filter for minimal image background.

The cells were maintained at 37 jC with a custom-built stage

heater, and the cell chambers were sealed with high-vacuum

grease to prevent significant pH shifts in the full culture

media used for these time lapse experiments. Image analysis

methods were used to measure the number of accretion foci

per cell. Cells were generally fixed and their nuclei stained

prior to imaging using: 4% formaldehyde in phosphate

buffered saline (PBS, pH7.4; InVitrogen, Denmark) for 15

min, followed by 10 AM Hoechst 33258 (Molecular Probes,

Eugene, OR, USA) in PBS for a further 10 min, then washed

three times in PBS.

Automated images were collected on a Nikon Diaphot

300 (Nikon, Japan) using a Nikon Plan Fluor 20X/0.5NA

objective lens. The microscope was fitted with a motorised

specimen stage and motorised focus control (Prior Scientific,

Cambridge UK), excitation filter wheel (Sutter Instruments,

Novato, CA, USA) and Orca ER camera. Automation of

stage positioning, focus, excitation filter selection and image

acquisition was controlled using macros written in-house,

running under IPLab for Windows (Scanalytics, Fairfax, VA,

USA). Images were collected in pairs, the first using a 340/

10-nm excitation filter, the second with a 475RDF40 exci-

tation filter (Chroma, Brattleboro, VT). Both images were

collected via the same dichroic and emission filters opti-

mised for EGFP applications (XF100 filter set, Omega

Optical, Brattleboro, VT). Image pairs were automatically

analysed using custom macros written in IPLab software,

involving for each image pair the identification and counting

of individual nuclei from the Hoechst image, then identifi-

cation and counting of accretion foci from the corresponding

GFP image. The ratio of foci number to nuclear count for

each pair represents an estimate of the average number of

accretion foci per cell in that image pair. The analysis also

returned the total area and intensity of pixels in identified

accretion foci, allowing these parameters to be appreciated

as average values per cell. The amount of GFP in cells was

also quantified using a fluorescence plate reader optimised

for reading fluorescence from adherent cells (Fluoroskan

Ascent CF, Labsystems, Finland) equipped with appropriate

filter sets (EGFP: excitation 485 nm, emission 527 nm;

Hoechst 33258: excitation 355 nm, emission 460 nm). For

such measurements, it was usual to increase the cell density

to approximately 1�105 cells/well (200 Al/well) to obtain

best signal to background ratio. Total GFP signal/well was

measured first from live cells in full growth medium, then

cells were treated with an extraction buffer to simultaneous-

ly fix the cells and extract the mobile GFP, leaving immobile

GFP for subsequent measurement. Extraction buffer con-

sisted of 0.4% formaldehyde buffer (pH 7.4) plus 1% Triton

X-100. Extraction buffer was applied to cells for 10 min at

room temperature. Full fixation and nuclear staining was

completed with 4% formaldehyde buffer + 10 AM Hoechst

33258 for a further 10 min, then cells were washed three

times in PBS to complete the procedure. Once the cells had

been extracted, fixed and their nuclei stained with Hoechst

dye, the immobile GFP signal was measured and corrected

per well for cell number using the Hoechst signal. All plate

reader data have been corrected for relevant background

offsets.

2.4. Antibodies

Antisera specific for the human PDE4A sub-family have

been described elsewhere by us [26]. Briefly, this was

generated against a fusion protein formed between GST

and a portion of the extreme C-terminal region of PDE4A

that is unique to this sub-family. Such a region is found in

common to all active PDE4 isoforms. Attempts to character-

ise components potentially associated with the PDE4A4

accretion foci included use of specific antisera raised against

the following proteins: pericentrin (Covance); h-tubulin and

g-tubulin (both from Sigma); Human transferrin receptor

(Zymed); Fyn (15) and Lyn (44) (both from Santa Cruz

Biotech); PKA-RI, PKA-RII, PKA-RII, AKAP121,

AKAP149, AKAP KL, AKAP82, AKAP220 and AKAP450

were all from BD Transduction Labs. Secondary antibodies

to detect these antisera were obtained from Molecular

Probes, and were generally conjugated to Alexa Fluor 546

or similar fluorophore with fluorescence characteristics

distinct from GFP. BODIPY 558/568 Phalloidin (Molecular

Probes) was used to identify F-actin structures in cells.

2.5. Transient expression in COS7 cells

As described previously by us [24], transfection was done

using the COS7 SV40-transformed monkey kidney cell line

maintained at 37 jC in an atmosphere of 5% CO2/95% air in

complete growth medium containing DMEM supplemented

with 0.1% penicillin/streptomycin (10000 units ml� 1), glu-

tamine (2 mM) and 10% FCS. Briefly, COS7 cells were

transfected using DEAE-dextran. The DNA to be transfected

(5 Ag) was mixed, and incubated for 15 min with 250 Al of 10mg ml� 1 DEAE-dextran in PBS to give a ‘DNA-dextran’

mix. When cells reached 70% confluency, in 100-mm dishes,

the medium was removed and the cells were given 10 ml of


fresh DMEM containing 0.1 mM chloroquine and the DNA-

dextran mix (250 Al). The cells were then incubated for 4 h at37 jC. After this period, the medium was removed and the

cells shocked with 10% DMSO in PBS. After PBS washing,

the cells were returned to normal growth medium and left for

a further 2 days before use. For determination of PDE

activity, the cells were homogenized in KHEM buffer (50

mM KCl, 10 mM EGTA, 1.92 mM MgCl2, 1 mM dithio-

threitol, 50 mM Hepes, final pH7.2,) containing ‘complete’

protease inhibitors (Boehringer Mannheim) of final concen-

trations 40 Ag/ml PMSF, 156 Ag/ml benzamine, 1 Ag/ml

aprotonin, 1 Ag/ml leupeptin, 1 Ag/ml pepstatin A and 1 Ag/ml antipain. As described previously, in such transfected

cells then >98% of the total PDE activity was due to the

recombinant PDE4 isoenzyme.

2.6. Assay of cAMP PDE activity

PDE activity was determined [25] by a modification of

the two-step procedure of Thompson and Appleman [27] in

10 mM MgCl2, 20 mM Tris–HCl buffer final pH7.4. All

assays were conducted at 30 jC with initial rates taken from

Fig. 1. Rolipram causes the recruitment of PDE4A4–GFP into accretion foci in

fluorescence images of PDE4A4 chimeras in living cells. CHO cells stably transf

after having been treated with rolipram (3 AM) for 7 h. HEK cells stably transfe

having been treated with rolipram (10 AM) for 16 h. RBL cells (e) stably transfec

AM) for 24 h. In image (f) is shown a confocal fluorescent image of CHO cells tran

for 18 h prior to fixation and subsequent visualisation of PDE4A4 using a PDE4A4

images from experiments repeated at least three times.

linear time courses. Activity was linear with added protein

concentration. Dose-effect inhibitor analyses were per-

formed with 1 AM cAMP as a substrate over a range of

rolipram concentrations. Rolipram was dissolved in 100%

DMSO as a 10-mM stock and diluted in 20 mM Tris–HCl

(pH 7.4), 10 mM MgCl2 buffer to provide a range of

concentrations for use in the assay. The residual levels of

DMSO were not found to affect PDE activity over the

ranges used in this study. Inhibitor studies were analysed

using the Kaleida-Graph software package (Synergy Soft-

ware, Reading, PA). The activities of the various PDE4A4

mutant forms are expressed relative (%) to that of wild-type

PDE4A4 by assaying, at a substrate concentration of 1 AMcAMP, equal amounts of immunoreactive PDE4A4 protein,

as determined with PDE4A-specific antibodies in both

ELISA and quantitative immunoblotting [25].

2.7. SDS/PAGE and Western blotting

Eight-percent acrylamide gels were used and the samples

boiled for 5 min after being resuspended in Laemmli buffer

[28]. Gels were run at 8 mA/gel overnight or 50 mA/gel for

living CHO and HEK cells. Images (a) through (e) show confocal GFP

ected to express PDE4A4–GFP in (a) the absence of rolipram and also (b)

cted to express PDE4A4–GFP in (c) the absence of rolipram and (d) after

ted to express PDE4A4–GFP shown here after treatment with rolipram (10

sfected to express untagged PDE4A4 and challenged with rolipram (10 AM)

-specific antibody. White arrows indicate examples of foci. These are typical


4–5 h with cooling. For detection of transfected PDE by

Western blotting, 2–50 Ag protein samples were separated

by SDS/PAGE and then transferred to nitrocellulose before

being immunoblotted using the indicated specific antisera.

Labelled bands were identified using peroxidase linked to

anti-rabbit IgG, and the Amersham ECLWestern blotting kit

was used as a visualisation protocol.

2.8. Protein analysis

Protein concentration was determined using BSA as

standard [29].

3. Results

3.1. 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 [2,3,31–33]. It is characterised by its unique N-

terminal region of 107 amino acids [30], which can func-

tionally interact with the SH3 domains of various SRC

family tyrosyl kinases [25,26].

Fig. 2. Rolipram causes the recruitment of PDE4A4 tagged with various different p

confocal fluorescence images of the various indicated PDE4A4 chimeras in fixed

transfected to express PDE4A4-LimK-1 (full length, 647 residues; a,b), PDE4A4-R

residues; e, f). Shown are untreated cells (a, c, e) and cells challenged with rolipram

HEK (h) cells that have been transiently transfected with PDE4A4 tagged at its C-te

rolipram (10 AM) for 18 h prior to fixation. White arrows indicate examples of fo

Here we show a fluorescence confocal microscopy

analysis of living CHO (Fig. 1a) and HEK (Fig. 1c) cells

that have been stably transfected so as to express a chimera

formed from PDE4A4 and Green Fluorescent Protein

(PDE4A4–GFP). While fluorescence is dispersed through

the cell interior, it is clearly excluded from nuclei and

certain other structures in the cytoplasm of these cells and

which, in terms of disposition and appearance, are most

consistent with mitochondrial and endoplasmic reticulum

compartments. Strikingly, however, the addition of the

PDE4 selective inhibitor, rolipram (10 AM), causes a pro-

found change in the distribution of PDE4A4–GFP in both

CHO (Fig. 1b) and HEK (Fig. 1d) cells which took the form

of the appearance of accretion foci. Such foci are also

generated by rolipram treatment of RBL monocytic cells

expressing PDE4A4–GFP (Fig. 1e). However, they are not

seen upon rolipram treatment of cells transfected to express

GFP by itself, where fluorescence remains spread uniformly

throughout the cell, including the nucleus (data not shown).

Rolipram-induced foci are also evident for untagged

PDE4A4 expressed in CHO cells treated with rolipram

(Fig. 1f), as well as with PDE4A4 C-terminally tagged with

the V5 epitope plus six histidine residues (V5-6His chimera)

expressed in both CHO and HEK cells (Fig. 2g,h). PDE4A4

fusions to a range of very different proteins also respond to

rolipram in the same way as PDE4A4–GFP. LimK-1 (full-

roteins into accretion foci in living CHO cells. Images (a) through (h) show

cells visualised using a PDE4A4-specific antibody. CHO cells were stably

hoC (full length, 193 residues; c, d) and PDE4A4-FRB (FRAP fragment, 89

(10 AM) 18 h prior to fixation. Also shown are examples of both CHO (g) and

rminus with the minimal V5-6His peptide and that had been challenged with

ci. These are typical images from experiments repeated at least three times.


length, 647 residues; Fig. 2a,b), RhoC (full-length, 193

residues; Fig. 2c,d) and FRB (FRAP fragment, 89 residues;

Fig. 2e,f) each fused to PDE4A4 in place of GFP success-

fully form accretion foci when challenged with rolipram.

Foci are clearly separate from centrioles, as detected by

immunostaining for pericentrin and from the microtubule

motor centre, as indicated by staining for gamma-tubulin

(data not shown). They are also separate from the cellular

actin network and from the anchored form of PKA, as

detected using antisera to the RIIa and RIIh subunits of

PKA (data not shown).

The other major PDE4 isoforms that are widely expressed

in cells, including those of the immune system, are the

PDE4B2 short isoform and the PDE4D3 and PDE4D5 long

isoforms. We show here, however, that GFP chimeras made

with these species and expressed in CHO cells do not

redistribute to form accretion foci in the presence of rolipram

(Fig. 3a–c). Neither does the PDE4C2 long isoform (data

not shown). However, rolipram-induced accretion foci were

seen in CHO cells stably expressing a GFP chimera formed

Fig. 3. Analysing the potential of rolipram to alter the intracellular distribution of

confocal, widefield fluorescence images of PDE4-GFP chimeras in living cells.

PDE4D5-GFP, (d) PDE4A5-GFP were all challenged with rolipram (10 AM) for

arrows in (d) indicate the only examples of foci formed by rolipram in these studie

with PDE4A5 (Fig. 3d), the rodent homologue of the human

PDE4A4 isoform.

3.2. Properties of rolipram-induced redistribution of

PDE4A4–GFP in living cells

We show here the time dependence of the ability of 10

AM rolipram to cause the dynamic redistribution of

PDE4A4–GFP in living cells over a period of 9 h (Fig.

4a). During this time, the total GFP fluorescence, expressed

per cell, increased by 52F 3%. The increase in total GFP

fluorescence is very likely to be due in part to cAMP-

mediated stimulation of the hCMV promoter used to drive

expression of the PDE4A4–GFP chimera, as shown previ-

ously by others [34,35]. However, strikingly, the immobile

fraction of fluorescence, reflecting PDE4A4–GFP in accre-

tion foci, increased by more than 10-fold during the 9-

h period of treatment with 10 AM rolipram (Fig. 4a). The

formation of accretion foci is completely reversible (Fig. 4b)

as removal of rolipram leads to complete dispersal of foci.

chimera of various other PDE4-GFP isoforms in living CHO cells. Shows

CHO cells transfected to express (a) PDE4B2-GFP, (b) PDE4D3-GFP, (c)

18 h prior to visualisation of GFP fluorescence in these living cells. White

s. Typical experiments of the ones done at least three times are shown here.

Fig. 4. The rolipram-induced formation accretion foci in PDE4A4–GFP

expressing CHO cells. (a) Shows the change over time in both the total

PDE4A4–GFP signal and also the signal due to the immobilised

PDE4A4–GFP localised in accretion foci in CHO cells treated with

rolipram (10 AM). Values are meanF S.D. (n= 8); (b) CHO cells expressing

PDE4A4–GFP were treated for 16 h with rolipram (10 AM) so as to allow

the formation and assessment of accretion foci (time = zero). They were

then washed and incubated in fresh medium without rolipram for 4.5 h

before re-assessment in order to define total PDE4A4–GFP fluorescence

and the immobile GFP fraction remaining after the removal of rolipram.

During this period of rolipram-free incubation, total GFP content of the

cells decreased by approximately 17%. Values are meanF S.D. (n= 32).


As an example, within 4.25 h after washout of rolipram, all

accretion foci disperse and the immobile PDE4A4–GFP

signal accordingly falls dramatically to approximately 10%

of the value measured in cells that have not had rolipram

removed; during the same time period, these washout cells

lose only 17F 3% of their total PDE4A4–GFP (Fig. 4b).

Interestingly, reappearance of foci in cells that had been

previously challenged with rolipram occurs at a consider-

ably quicker rate than in naı̈ve cells challenged with

rolipram for the first time (Fig. 5a,b). This indicates that

rolipram sensitises the cell in some fashion and that this

‘memory’ extends past the time taken for dispersal of foci

by washout of rolipram. In this regard, we see here that the

addition of cycloheximide (30 Ag/ml) to PDE4A4–GFP

expressing CHO cells challenged for 16 h with a range of

rolipram concentrations up to 100 AM completely ablated

foci formation (Fig. 6a). These data indicate that additional

protein synthesis is needed for rolipram-induced foci for-

mation to ensue despite the presence of high levels of

PDE4A4–GFP in these cells.

Rolipram elicits PDE4A4–GFP foci generation in a

dose-dependent fashion (Fig. 6a), with an EC50 value of

0.50F 0.08 AM (meanF S.D.; n = 4 experiments). This

value is similar to that which we observe for the dose-

dependent inhibition of PDE4A4–GFP activity by rolipram

(Table 1) and to that reported previously by us [25] for the

inhibition of wild-type PDE4A4 by rolipram.

Additionally, challenge with the adenylyl cyclase activa-

tor, forskolin (up to 100 AM), either alone or together with

the nonselective PDE inhibitor IBMX (100 AM), failed to

induce foci formation in PDE4A4–GFP expressing CHO

cells (data not shown). Thus, foci generation is unlikely to

be due to any elevation in intracellular cAMP levels. Indeed,

challenging cells with forskolin actually serves to inhibit

foci generation dose-dependently, and will disperse foci pre-

formed with rolipram (Fig. 6b; 5 AM) with an EC50 value of

0.44F 0.04 AM (meanF S.D., n = 3) in the continued

presence of rolipram (5 AM). These data suggest that

interaction of the reversible competitive inhibitor rolipram

with PDE4A4 engenders a conformational change in this

isoform that directly leads to generation of accretion foci,

and that elevated cAMP levels may reverse this effect by

competing with rolipram for interaction with the active site

of PDE4A4.

3.3. PDE4 selective inhibitors can be segregated into those

that elicit foci formation of PDE4A4–GFP in living cells

and those that do not

We set out to evaluate whether PDE4 selective inhibitors

(Fig. 7a), other than rolipram, might elicit the generation of

foci in PDE4A4–GFP expressing CHO cells. Like rolipram,

the PDE4 selective inhibitors Ro20–1724 and RS25344

[36–38] both cause the dose-dependent formation of foci in

PDE4A4–GFP expressing cells (Fig. 7b), with EC50 values

of 3.8F 0.2 AM and 17F 3 nM, respectively (meanF S.D.;

n = 3 experiments). These values are very similar to the IC50

values of 4.3F 0.5 AM and 22F 7 nM we note here for the

inhibition of PDE4A4–GFP by Ro20–1724 and RS25344,

respectively (meanF S.D.; n = 3 experiments). Intriguingly,

(S)-(+)-rolipram is much less effective than its (R)-(�)-

enantiomer (Fig. 7b). This is with regard to both the

magnitude of foci generation, with (S)-(+)-rolipram gener-

ating maximum levels of foci around 20% of that of (R)-

(�)-rolipram, and the dose-dependency of its action, with

Fig. 5. Rolipram causes the formation of accretion foci more rapidly in PDE4A4–GFP expressing CHO cells that have previously been subjected to challenge

with rolipram. In (a) are shown CHO cells expressing PDE4A4–GFP with images taken at the indicated times after the addition of rolipram (10 AM). In the

first panel of images (i, ii, iii), cells had not previously been exposed to rolipram. In the second panel of images (iv, v, vi), cells were pretreated with rolipram

(10 AM) for 16 h so as to allow the formation and assessment of accretion foci. They were then washed and incubated in fresh medium without rolipram for 4.5

h at 37 jC so that all rolipram-induced foci of PDE4A4–GFP had dispersed. Cells were then returned to incubator conditions between images; (b) open circles

show the time dependence of PDE4A4–GFP foci reappearance in cells that have been pretreated for 16 h with rolipram (10 AM), then washed to allow foci to

disperse and then, finally, rechallenged (zero time) with rolipram (10 AM). The second curve (filled circles) shows the time dependence of PDE4A4–GFP foci

formation in naive cells exposed to rolipram (10 AM) for the first time. Both curves report foci number per cell vs. time. Values for the reappearance curves

were derived from single fields of more than 240 cells followed over the period shown. All images used in this analysis were collected using the same

microscope settings and counts made using a fixed threshold for spot recognition. Values have been normalised to values from cells exposed to rolipram (10

AM) for 16 h, taken, for this purpose, as the maximum response of the system. The de novo foci formation curve uses values for the immobile GFP fluorescence

(cell corrected) derived from wells treated for various times with rolipram (10 AM). The values for this curve are given as a percentage of the immobile GFP

fluorescence from cells treated for 25 h with rolipram (10 AM), taken again as the maximal response of the system.


Fig. 6. Formation and dispersal properties of rolipram-induced PDE4A4–

GFP accretion foci in CHO cells. (a) Shows the dose dependence for the

formation of accretion foci (immobile GFP per cell) in response to rolipram

alone (filled circles) and in the presence of 30 Ag/ml cycloheximide (open

circles); in each case, PDE4A4–GFP expressing CHO cells were incubated

with the indicated concentrations of rolipramF 30 Ag/ml cycloheximide for

16 h. Each value is the meanF S.D. (n= 8). (b) Shows the dose dependence

for the forskolin-mediated dispersal of rolipram-induced formation of

accretion foci of PDE4A4–GFP in CHO cells. Cells treated with 5 AMrolipram for 18 h, then in continuous presence of 5 AM rolipram, exposed to

a range of forskolin concentrations + 100 AM IBMX (all treatments) for a

period of 2 h. Values are meanF S.D. (n= 8).

Table 1

Inhibition of PDE4A4 constructs by rolipram

PDE4A4–GFP

construct

IC50 (AM) for

inhibition by

rolipram

EC50 (AM) for

foci formation

by rolipram

EC50 (mM)

for activation

by Mg2 +

Relative

activity

(%)

Wild-type 4A4 0.83F 0.06 0.50F 0.08 1.5F 0.5 (100)

H506N-4A4 0.77F 0.13 Not formed 12F 2 48F 8

H505N-4A4 0.27F 0.08 Not formed 4F 1 48F 4

V475D-4A4 0.63F 0.15 Not formed 17F 3 23F 4

This table shows the concentration of rolipram (IC50; AM) giving 50%

inhibition of the indicated recombinant PDE4A4–GFP constructs assayed

in the presence of 1 AM cAMP as substrate (n= 4 separate determinations).

Shows the concentration of Mg2 + (EC50; mM) giving 50% activation of the

indicated recombinant PDE4A4–GFP constructs assayed in the presence of

1 AM cAMP as substrate (n = 3 separate determinations). PDE4A4

unconjugated had an EC50 value of 2F 1 mM Mg2 +. Also shown is the

relative activity (%) that the mutant forms have with respect to the wild-

type enzyme (100%), when assayed in the presence of 1 AM cAMP as

substrate. (n= 6, separate determinations) Values are given as means with

errors as S.D.


(S)-(+)-rolipram exhibiting an EC50 value of 3.9F 0.9 AMcompared to that of 0.48F 0.09 AM, for (R)-(�)-rolipram

(meanF S.D.; n = 4 experiments).

Remarkably, in contrast to the action of rolipram and

these two other PDE4 selective inhibitors, another pair of

PDE4 selective inhibitors, namely ArifloR [11,39,40] and

RP73401 [41], are completely unable to cause foci forma-

tion in PDE4A4–GFP (data not shown). Indeed, as with

forskolin (Fig. 6c), both ArifloR and RP73401 are able to

antagonise the formation of foci by rolipram and actually to

disperse foci pre-formed by rolipram (Fig. 7c). This occurs

in a dose-dependent fashion with IC50 values of 2.6F 0.4

AM for ArifloR and 15F 2 nM for RP73401 when 5 AMrolipram is employed to generate foci (meanF S.D.; n = 3

experiment values). Such values reflect the different affin-

ities of these two inhibitor compounds for inhibiting the

catalytic activity of PDE4A4 [25].

We observed that treatment of CHO cells with either

ArifloR or RP73401 causes an increase in total GFP

fluorescence to a similar extent to that seen using rolipram.

Thus, for example, we show here the dose-dependent

increase in the total cellular fluorescence from cells express-

ing the PDE4A4–GFP chimera treated with either RP73401

or rolipram (Fig. 8a). In both cases, the maximum level of

increase of the total GFP signal is 32F 7% over a 16-

h incubation period (Fig. 8a), although at no concentration

of RP73401 did we observe any accretion foci produced.

This indicates that any increase in the amount of cellular

PDE4A4–GFP caused by the effects of treatment with

certain PDE4 inhibitors does not necessarily translate into

foci formation. In other words, formation of accretion foci

with rolipram is not simply a cellular response to increased

levels of PDE4A4 product, as it is not seen with RP73401.

Additionally, the facile reversibility of foci formation, by

simply washing out rolipram, under conditions where little

or no change in total PDE4A4–GFP levels occur, also

militates against foci generation being due to inappropriate

processing.

We also record a further difference in the action of these

two PDE4 selective inhibitors. This arose from experiments

where we first treated cells with either rolipram or RP73401

for 10 h, washed them free of inhibitor and then left them in

inhibitor-free medium for 2.5 h to allow the foci to disappear

in the rolipram-treated cells. After this, we challenged both

groups of cells with 5 AM rolipram and followed the rate of

(re)formation of accretion foci. While this rate was very


much faster in the rolipram-pretreated cells, the RP73401-

pretreated cells respond with a rate of foci formation that was

equivalent to that of naı̈ve cells (Fig. 8b). Thus, while both

rolipram and RP73401 cause a similar increase in the total

GFP signal over 10 h, through increasing the expression

levels of PDE4A4–GFP, it is only pretreatment with roli-

pram that serves to sensitize the cells to rolipram-induced

foci formation. This indicates that sensitisation is not due to

any general PDE4 inhibitor-induced increase in PDE4A4–

GFP expression, which is presumably mediated through an

increase in intracellular cyclic AMP. Instead, sensitisation is

specifically associated with certain PDE4 selective inhibi-

tors, such as rolipram, which are able to elicit a discrete sig-

nalling event consequent upon binding to PDE4A4.

3.4. Generation of PDE4A4 mutants that are resistant to

foci formation by rolipram

It has been suggested that PDE4 isoforms can exist in

distinct conformational states [13,37,42] that can be dis-

criminated by their differential sensitivity to inhibition by

the (R)-(�) and (S)-(+) enantiomers of rolipram; specifically

a heightened sensitivity to the (R)-(�), compared to (S)-(+),

enantiomer. The novel process that we have identified here,

involving PDE4A4, clearly shows discrimination in the

ability to effect foci generation by these two enantiomers

of rolipram (Fig. 7b). We suggest that the binding of

rolipram to the PDE4A4 catalytic site may engender a con-

formational change that is required for the accretion of

PDE4A4 to foci. Such a condition implies that it might be

possible to employ mutagenesis to effect alterations in the

catalytic site of PDE4A4 that serve to ablate the accretion of

PDE4A4 to foci. An exhaustive mutagenesis procedure is

not tenable, however. This is not only because the catalytic

unit is large, but also because functional assessment of mu-

tants requires that clonal cell lines stably expressing them be

generated, each of which constitutes a very considerable

undertaking. Nevertheless, we set out to try and address the

principle that foci formation results from a distinct confor-

mational change consequent on the binding of rolipram to

the active site rather than by serving merely to effect in-

hibition of cAMP hydrolysis. To do this, we studied muta-

tion of a few key residues, which rational consideration of

the enzyme’s structure suggested might play a role in the

proposed conformational change induced by rolipram.

Although no crystal structure is currently available for

any full-length PDE4 enzyme, that of the PDE4B catalytic

unit has been derived [43]. As this region is highly con-

served (>90%) throughout the PDE4 family [3], the

PDE4B2 catalytic unit structure is likely to provide a good

Fig. 7. The action of various other PDE4 selective inhibitors on the rolipram

induced accretion foci of PDE4A4–GFP in CHO cells. (a) Structures of the

indicated PDE4 selective inhibitors; (b) dose-effect analyses of the

formation of accretion foci of PDE4A4–GFP in CHO cells treated with

the indicated compounds Ro20–1724, RS25344, (R)-(�)-rolipram and (S)-

(+)-rolipram. MeanF S.D. (n= 4); (c) dose-effect analyses of the inhibition

of formation of rolipram-induced accretion foci of PDE4A4–GFP in CHO

cells treated with the indicated concentrations of either ArifloR (open

circles) or RP73401 (filled circles). For the ArifloR competition experi-

ment, all cells were co-incubated with 5 AM rolipram. In the RP73401

competition, cells were also co-incubated with 5 AM rolipram. MeanF S.D.

(n= 3 separate experiments).

Fig. 8. Rolipram and RP73401 on PDE4A4–GFP expression in CHO cells. (a) Dose–response curves for the increase in total GFP in CHO/PDE4A4–GFP

cells following treatment with racemic rolipram (filled circles) or RP73401 (open circles). Cells were incubated for 16 h with the inhibitors and then read for

total GFP. There is no correction for cell number in these measurements. Values are represented as percentage increase above the lowest concentration tested,

being ‘100%’ at 0.03 AM rolipram and 0.003 AM RP73401. There are no accretion foci formed in cells at these lowest doses of inhibitor, and none are formed

at any successively higher dose of RP73401. Each value expressed as meanF S.D. (n= 4). In (b) are shown CHO cells expressing PDE4A4–GFP with images

taken at the indicated times after the re-addition of rolipram (10 AM), 3 h after washout of prior inhibitor treatments. In the first panel of images (i, ii, iii), cells

had previously been exposed to RP73401 (3 AM). No accretion foci formed with this treatment, but PDE4A4–GFP increased per cell (as shown in Fig. 8a).

They were then washed and incubated in fresh medium without rolipram for 3 h at 37 jC. In the second panel of images (iv, v, vi), cells were pretreated with

rolipram (10 AM) for 16 h to induce accretion foci. They were then washed and incubated in fresh medium without rolipram for 3 h at 37 jC so that all

rolipram-induced foci of PDE4A4–GFP had dispersed. Cells were returned to incubator conditions between images.



model for that of all four subfamilies. The PDE4 catalytic

centre [43] features a binuclear Zn2 +–Mg2 + motif at one

end of a deep substrate-binding cleft (Fig. 9). The Zn2 +

centre is located more deeply in the cleft and is tightly

engaged by four direct ligand interactions from protein

residues. One of these residues, corresponding to Asp474

in PDE4A4, together with a hydroxide ion, acts as a

bridging ligand between the two metal centres. The Mg2 +

coordination shell is completed by solvent ligands, which

are networked through hydrogen bonds to four adjacent

residues, corresponding to His473, Glu503, His506 and

Thr544 in PDE4A4. These residues, which grip the Mg2 +

ion, are contributed by the N- and C-terminal ends of

helices-10 and -11 in the PDE4B crystal structure and by

the loop between helices-7 and -8. The sequence spanning

the 7/8-loop through helix-11 exhibits 92% residue identity

between PDE4A and PDE4B and where differences are

present, these are only conservative substitutions. The

catalytic centre thus comprises a tightly bound Zn2 + ion

coupled to a more loosely held Mg2 + centre. It has been

suggested [44,45] that conformational changes in the PDE4

catalytic unit that can be detected through changes in

binding affinity for rolipram may be effected by alterations

in liganding of the Mg2 + ion. Thus, amino acids serving to

ligand Mg2 +, either directly or indirectly through bound

H2O, may provide a potential route whereby the binding of

rolipram in the active site might ‘transmit’ conformational

changes that trigger the series of events leading to foci

generation. In this regard, His506 is of interest because it is

closely linked to the bound Mg2 + ion through a hydrogen

bond from one of its imidazole ring nitrogens to one of the

Mg2 + water ligands and because it is located on helix-10,

which is partially surface-exposed (Fig. 9a–c). Not only

this, but on the other side of the His506 imidazole ring, the

NH group H-bonds to the main chain amide carbonyl O

atom of Val475 (Fig. 9b), a residue located on the loop

between helices-7 and -8, which is also partially surface-

exposed (Fig. 9c) and which contributes the bridging metal

ligand, Asp474. This network has the potential to provide a

means for ‘inside-out’ signalling, where conformational

changes occurring in the active site could be transmitted

to the molecule surface. To investigate this possibility, we

set out to perturb the catalytic site at the position of His506

in such a way that we hoped might affect rolipram-induced

Fig. 9. The PDE4 catalytic site and ‘inside-out’ signalling elicited by rolipram bin

core catalytic unit with the Mg2 + ion held between helices-10 and -11. cAMP is ma

the model of Xu et al. [43]; the cleft is defined by the Zn2 +–Mg2 + motif and key

detail of the Mg2 + binding environment viewed from the N-terminus of helix-10; H

residues in PDE4A4 are H506 and V475. The location of the surface exposed H3

PDE4A4 are H505 and D476. (c) Two views of the PDE4 core catalytic unit with

helix-10 (red) and 7/8-loop (green) are marked; H505 on helix-10 is highlighted

behind the blue surface with access (orthogonal to the view) as indicated by the arr

the floor of a pronounced surface cleft. (d) Model for the purported interactions wit

(e) Dimerisation interface of the PDE4 core catalytic unit. The structure of the

highlighting one molecule in yellow and one in white. The white molecule has

orthogonal to helix-10. The colour coding on the white molecule is the same as th

(purple).

foci formation while retaining catalytic activity. In doing

this, we reasoned that Asn might serve as a surrogate for a

neutral His residue (Fig. 9d) and, therefore, that His506Asn

mutation might still support catalytic activity but would

subtly alter the catalytic site sufficiently to perturb a

potential rolipram-induced conformational change mediated

through the Mg2 + ion and His506 on helix-10. Here we

show that the His506Asn mutant form of PDE4A4–GFP is

clearly catalytically active, albeit this is reduced to around

half that exhibited by the wild-type enzyme (Table 1). In

addition, as we had hoped, the sensitivity of the His506Asn

mutant to activation by Mg2 + is attenuated by around eight-

fold, relative to the wild-type enzyme (Table 1). Strikingly,

however, in dramatic contrast to the wild-type enzyme, the

His506Asn mutant singularly fails to form accretion foci in

cells challenged with rolipram (5 up to 100 AM, 10–48 h;

data not shown), while still exhibiting a similar sensitivity to

wild-type PDE4A4 in its ability to be inhibited by rolipram

(Table 1).

Given that the NH group of the His506 imidazole ring H-

bonds to the main chain amide carbonyl O atom of Val475

(Fig. 9b), we set out to evaluate the potential importance of

Val475 in foci formation. In doing so, we decided to mutate

Val475 to a charged aspartate residue on the basis that the

change in polarity of the residue at this position might

sufficiently perturb the main chain to affect the hydrogen-

bonded interaction of Val475 with His506. We see here that

the Val475Asp mutant is also catalytically active, albeit with

an activity some 23F 4% (meanF S.D.; n = 6) that of the

wild-type enzyme (Table 1). Consistent with the proposed

‘coupling’ of the interaction of Val475 with the Mg2 +-

interacting His506, we noted a dramatic decrease in sensi-

tivity of the Val475Asp mutant to activation by Mg2 + of

some 11-fold (Table 1). Furthermore, as with the His506Asn

mutant, the Val475Asp mutant singularly fails to form

accretion foci in cells challenged with rolipram (5 up to

100 AM, 10–48 h; data not shown), despite exhibiting

similar sensitivity to wild-type PDE4A4 in its ability to be

inhibited by rolipram (Table 1).

We also evaluated His505, a surface-exposed residue

adjacent to His506 on helix-10 which, although not liganded

to the bound Mg2 +, forms a salt bridge to the side chain of

Asp476, which is adjacent to Val475 and thus provides

another link between helix-10 and the loop between heli-

ding. (a) Shows the crystal structure and three subdomains of the PDE4B2

nually positioned into the active site and inhibitor-binding cleft according to

residues marked (equivalent PDE4A4 numbers in parenthesis). (b) Shows

307 is hydrogen bonded to Mg2 +-bound water and V276; the corresponding

06, and its interaction with D277 is also highlighted; equivalent residues in

surface rendering taken orthogonal to axis of helix-10. The exposed regions

(blue). The catalytic cleft (obscured from view) is located approximately

ow. View (ii), as observed along the indicated ‘view’ arrow, reveals H505 in

h mutation of the histidine at position 307 (H506 in PDE4A4) to asparagine.

dimeric PDE4D core catalytic unit [53] is shown with surface rendering

a similar orientation to view (i) in image (c) and is viewed approximately

at in image (c) except that the surface of helix-9 is additionally highlighted

Fig. 9.



ces-7 and -8 (Fig. 9b). Consistent with the suggested impor-

tance of such a link, the His505Asnmutant form again fails to

form accretion foci in cells challenged with rolipram (5 up to

100 AM, 10–48 h; data not shown). However, His505Asn is

catalytically functional, with an activity that is around half

that of wild-type PDE4A4. Intriguingly, it is not only still

inhibited by rolipram in a manner not too dissimilar from that

of wild-type PDE4A4 (Table 1), but also shows but little

difference in its sensitivity to activation by Mg2 + (Table 1).

This suggests that ablation of rolipram-induced foci genera-

tion is not directly related to attenuated sensitivity to Mg2 +.

Specificity for PDE4A4 in foci formation by rolipram

indicates a role for the unique N-terminal region of this

isoform in the response. It is possible that ‘inside-out’

signalling achieved by the rolipram-induced conformational

change in the catalytic unit exerts itself by altering an

interaction of the catalytic unit with the N-terminal region

of PDE4A4. We sought to provide an initial evaluation of

this by trying to disrupt the interaction between the N-

terminal portion of PDE4A4 with the catalytic unit. To do

this, we focussed on LR2, the 25 amino acid region that

connects UCR2, and through it both UCR1 and the unique

N-terminal of PDE4A4, to the catalytic unit. We have

previously shown [25] that the Db-LR2-PDE4A4 construct,

which has a small eight amino acid (APRPRPSQ) deletion

in LR2, is catalytically active. However, Db-LR2-PDE4A4

causes a conformational change in PDE4A4 that ablates the

ability of LYN SH3 interaction to alter the kinetics of

rolipram inhibition of this isoform [25], making examina-

tion of this mutant particularly pertinent. We show here that

Db-LR2-PDE4A4–GFP exhibits an attenuated ability to

form foci in response to challenge with rolipram (Fig. 10).

That it is not ablated is consistent with our demonstration

that PDE4A5, the rat homologue of PDE4A4 can form foci

(Fig. 3d) but does not possess the proline and arginine-rich

region deleted in the Db-LR2-PDE4A4–GFP construct

[25]. This suggests that the appropriate orientation of the

Fig. 10. Attenuated foci formation seen upon disruption of LR2.

Experiments were done as described in Fig. 6a. Each value is the

meanF S.D. (n= 6). Data are shown for wild-type PDE4A4-chimera (filled

circles) and the Db-LR2-PDE4A4–GFP chimera (open triangles).

unique N-terminal portion of PDE4A4 to the catalytic unit is

required for efficient foci formation to ensue.

4. 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 fash-

ion. 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 cells [23]. Of course,

as with any tool used in investigative biology, it is not

without its potential problems. We appreciate that the data

presented in this study may well appear to be provocative as

the effects reported are simply so very striking. Interesting-

ly, the formation of similar, striking cytoplasmic foci has

recently been reported in the identification of cytoplasmic

processing bodies, which among other things accumulate

translationally repressed mRNAs, as evaluated in studies

using both GFP chimera and native protein [46–48]. We

would contend that the foci formation detected here reflects

a specific event that is associated with and triggered by the

PDE4A4 component rather than some simple artefact asso-

ciated with either GFP or the various other protein tags used

to generate chimeras with PDE4A4. Our reasons for think-

ing this are that (i) foci formation can simply be reversed by

removal of rolipram, with subsequent rolipram re-addition

causing re-formation of foci; (ii) rolipram does not cause the

redistribution of GFP itself; (iii) rolipram induces foci

formation in PDE4A4 tagged with various other proteins

and with wild-type PDE4A4; (iv) various single point

mutations in the PDE4A4 catalytic unit specifically ablate

foci formation while still allowing rolipram to inhibit

PDE4A4 catalytic activity; (v) foci formation shows spec-

ificity for PDE4A4 and its homologue, PDE4A5, rather than

all PDE4 species; (vi) certain PDE4 inhibitors, exemplified

by Ariflo and RP73401, not only are unable to cause

accretion foci to form, but will both disperse rolipram pre-

formed foci and compete out the foci-forming action of

rolipram in a dose-dependent fashion; (vii) elevation of

intracellular cAMP levels achieved through forskolin-stim-

ulation of adenylyl cyclase competes out foci formation,

presumably by displacing rolipram from the active site of

PDE4A4 and (viii) foci formation shows discrimination

between the two enantiomeric forms of rolipram, with (R)-

(�)-rolipram preferred over (S)-(+)-rolipram. Thus, not only

is PDE4A4 essential for foci formation but rolipram is

needed in the active site of PDE4A4 for it to occur (Fig. 11).

Various investigators [3,36,37,41,44] have suggested that

whereas rolipram, Ro20–1724 and RS25344 appear able to

‘sense’ so-called high affinity (HARBS) and low affinity

(LARBS) PDE4 conformers, this is not seemingly true for

Fig. 11. Schematic for the generation and dispersal of accretion foci of PDE4A4–GFP in cells. A model summarising key aspects of the changes that lead up to

rolipram-induced foci generation in PDE4A4. Binding of rolipram to the catalytic unit is envisaged to cause a conformational change that is transmitted to the

surface of the molecule by ‘inside-out’ signalling. This change in conformation, together with the unique N-terminal region of PDE4A4 promotes the

aggregation of this isoform. Such a process requires active protein synthesis, which may be to provide either directly or indirectly interacting protein(s) needed

for foci formation. We note that at low concentrations of PDE4A4, this process may be curtailed at the level of either dimerisation or lower value oligomers.

Mass action would then indicate that the formation of larger foci would be promoted at higher expression levels of PDE4A4 or in localised regions of cells

where PDE4A4 expression is compartmentalised.


ArifloR and RP73401, which have been suggested primarily

interact with low affinity conformers [40,41]. Our data

extend this notion by suggesting that inhibitors such as

rolipram, Ro20–1724 and RS25344, on binding within the

catalytic site of PDE4A4, are themselves able to stabilise a

very particular conformational state of the catalytic unit of

this isoform. That various other PDE4 selective inhibitors,

as exemplified here by ArifloR and RP73401, cannot do

this suggests that a specific region of the active site is

involved. Here we detect this by the accretion of PDE4A4

chimeras into foci.

Specificity of this effect for PDE4A4 [30] and its rodent

homologue, PDE4A5 [49] implies that the N-terminal

region, which is unique to these isoforms, is intimately

involved in foci formation along with the PDE4 catalytic

unit, where rolipram binds in order to trigger this response.

That not all PDE4 selective inhibitors elicit such foci

formation suggests that the particular mode of binding of

certain of these inhibitors to the catalytic unit causes a

distinct conformational change. Indeed our discrete set of

mutations within the catalytic unit shows that it is possible

to ablate the ability of rolipram to form foci while retaining

its ability to inhibit catalytic activity. Such observations are

consistent with the notion that enzyme inhibition and foci

formation are not directly connected; as is evident from the

identification of non-foci-forming inhibitors. A consequence

of this proposal (Fig. 11) is that by simply competing out

rolipram from the catalytic site, then both elevated cAMP

levels and non-foci-forming PDE4 inhibitors would be able

to antagonise rolipram-induced foci formation and to dis-

perse rolipram pre-formed foci, as indeed is shown here.

Given the important role of PDE4 in cellular signalling

pathways and the enzyme’s potential therapeutic signifi-

cance, we would hope that the novel distinction between

PDE4 inhibitors, revealed by their ability to induce foci

formation, and the proposed conformational differences in

the enzyme associated with their binding might provoke

structural studies of PDE4A4 done in the presence of

compounds that either do or do not cause foci formation.

Together, with the indicated involvement of the enzyme’s

N-terminus, we consider that the binding of particular

inhibitors, such as rolipram, to the active site might induce

specific conformational changes by movement in the metal

centres, which is relayed through the ligand residues and

propagated to the exterior of the core catalytic unit. We call

this action, where rolipram binding to the catalytic site

effects a signalling action transmitted to the molecule

surface, ‘inside-out’ signalling. In this regard, other than

the catalytic site itself, the surfaces of the 7/8-loop, N-

terminus of helix-10 and C-terminus of helix-11 constitute

the most proximal externally exposed regions of the enzy-

me’s core catalytic unit to the Mg2 + centre (Fig. 9c), and

provide the most direct route for transmitting, to the

molecule surface, conformational changes consequent upon

rolipram binding at the active site. Although clearly pro-

scribed, our single point mutation studies, which identify

residues whose mutation ablates foci formation while still

allowing rolipram to cause inhibition of catalytic activity,


hint at the possible importance of this route. These identify

His506, which is hydrogen bonded through one of its

imidazole ring nitrogen atoms to a water molecule ligand

on the Mg2 + ion in the catalytic site. Also His505, which

although surface-exposed on helix-10, interacts with the

loop between helices-7 and -8, as does His506 (Fig. 9c).

This loop, like helix-10, contributes a Mg2 +-binding residue

(Asp474) and forms part of the exposed surface of the PDE4

catalytic site (Fig. 9b). Consistent with the suggested

importance of the 7/8-loop in relaying the presence of bound

rolipram to the protein surface, the Val475Asp mutant,

while again catalytically active and inhibited similarly to

wild-type PDE4A4 by rolipram, failed to form foci in

rolipram-challenged cells. This may be due to the disruption

of interaction with His506, and thereby subtle interference

in the positioning and/or movement of helix-10 and the 7/8-

loop, although without precise structural data, we cannot

exclude a more general disruption of the catalytic unit by

this mutation. However, that this mutant PDE4A4 is both

active and sensitive to inhibition by rolipram might indicate

that a subtle perturbation has been achieved. It is intriguing

to note that His505 is found exposed at the base of a dramatic

groove that runs across the surface of the 7/8-loop and

around the circumference of helix-10 (Fig. 9c), which might

provide a docking site for an interacting protein. Alterna-

tively, it might accommodate either the PDE4A4 unique N-

terminal region or the regulatory UCR1/2 modules, thereby

providing a link between the catalytic unit and the unique N-

terminal region of PDE4A4. Certainly, the unique N-termi-

nal portion of PDE4A4 appears to be essential for foci

generation since other PDE4 isoforms (PDE4B2, PDE4C2,

PDE4D3 and PDE4D5) fail to form accretion foci upon

rolipram challenge. Moreover, perturbation of its linkage to

the catalytic unit by deletion of a small portion within the

region between UCR2 and the catalytic unit (LR2; [25]), as

seen in the Db-LR2-PDE4A4–GFP chimera, clearly makes

foci formation somewhat less efficient. Our investigations on

this deletion construct also indicate that ligation of an SH3-

domain-containing protein to the proline and arginine-rich

region LR2 of PDE4A4 [25] is not required for rolipram-

induced foci formation to ensue.

The molecular basis of this redistribution process, and

whether this action of rolipram exploits a natural regulatory

process triggered either by the binding of an as yet uniden-

tified ligand or a post-translational modification remains to

be ascertained. However, while it is clear that foci genera-

tion is not driven by either PKA action or increased cAMP

levels, active protein synthesis is required. Indeed when

cells are washed free of rolipram to disperse foci, their

rechallenge with rolipram causes foci to reform more

rapidly than in naı̈ve cells (Fig. 5). This implies that the

requirement for protein synthesis is to induce a protein(s)

essential for foci formation and this is clearly specific for

rolipram since pretreatment with non-foci-forming PDE4

inhibitors does not impart such memory (Fig. 5). Thus,

rolipram-elicited ‘inside-out’ signalling might trigger the

induction of specific proteins required for foci formation.

However, defining any such action poses a considerable

technical challenge that lies beyond the scope of this study.

Unfortunately, in the various cultured cells, we have

examined so far, endogenously expressed PDE4A4 is found

at such low protein levels that it militates against its analysis

in immunofluorescence studies. Thus, either technological

advances or the identification of a suitable cell type are

required in order to be able to evaluate whether endoge-

nously expressed PDE4A4 undergoes rolipram-mediated

relocalisation to foci. It is conceivable that at the low levels

of native expression of PDE4A4 and in the absence of

additional protein tags the reversible aggregation may be

less acute, even perhaps restricted to a facilitation of

dimerisation. Dimerisation has been suggested to occur for

certain PDE4 isoforms [50–52] and, indeed, a dimerisation

interface has been identified in a recent X-ray crystal

structure of the PDE4D core catalytic unit complexed with

the inhibitor, zardaverine [53]. The dimer interface region

identified in this study is highly conserved across all PDE4

isoforms and involves residues on the surface-exposed

regions of helices-9 and -10, which are close to both

His505 and the region that we have identified in PDE4A4

in connection with the ‘inside-out’ signalling of rolipram

(Fig. 9e). An important question, then, is whether rolipram

and related inhibitors that promote foci formation stabilise

the dimeric interface and whether, following the induction

of protein synthesis, dimeric PDE4A4 units further aggre-

gate to form foci (Fig. 11). Clearly, in this study, cells have

been designed to overexpress PDE4A4 in order to detect

and analyse it, thus any change in intracellular distribution

of the native enzyme may then be far subtler. However, if

rolipram-induced relocalisation of PDE4A4 should occur

natively, then it may have functional consequences that

extend further than simply the spatial re-organisation of

cAMP degradation caused, for example, through any ac-

companying translocation of PDE4A4-interacting proteins.

Thus, particular PDE4 selective inhibitors, such as rolipram,

may exert effects on cells through PDE4A4 that extend

beyond simple catalytic inhibition and any consequent

elevation of cAMP levels (Fig. 11). Such cAMP-indepen-

dent effects exerted by this ‘inside-out’ signalling may not

only take the form of the induction of specific proteins but

also extend to additional functional consequences caused by

the cellular relocalisation of any PDE4A4-associated pro-

teins to foci. Even without unequivoval confirmation of this

possibility, it is clear that specific inhibitor-dependent con-

formational changes in the structure of PDE4A4 must be

occurring. The technology described here offers a novel and

sensitive means of sub-categorising PDE4 inhibitors and

probing for conformational changes in PDE4A4 induced by

inhibitor binding. In this regard, we have pinpointed a core

group of networked residues on helix-10 and the 7/8-loop of

the core catalytic unit that may provide part of a conforma-

tional relay, communicating the presence of a bound inhib-

itor from the catalytic site and its bound Mg2 + centre to the


protein surface. Whether the functional outcome of the

induced conformational changes in PDE4A4-associated foci

formation relates to the side effects seen with various PDE4

inhibitors and which have compromised their therapeutic

deployment or whether it provides added therapeutic op-

portunity remains to be seen. Of course foci formation may

reflect the activation of a normal process involved in

regulating PDE4A4 that happens, as we show here, also

to be activated by rolipram. We trust that the analyses

presented here will serve to stimulate studies aimed at

addressing these various questions.

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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Occupancy of the catalytic site of the PDE4A4 cyclic AMP phosphodiesterase by rolipram triggers the...

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