Specificity of cAMP signalling pathways has shown that the intracellular targeting of the individual components confers a three-dimensional context to the signalling paradigms in which they can exquisitely control the specificity of the outcome of the signal. Pivotal to this paradigm is degradation of cAMP by sequestered PDEs (phosphodiesterases). cAMP rapidly diffuses within cells and, without the action of spatially confined PDE populations, cAMP gradients could not be formed and shaped within cells so as to regulate targeted effector proteins. Of particular importance in regulating compartmentalized cAMP signalling are isoforms of the PDE4 family, which are individually defined by unique N-terminal regions. We have developed and pioneered the concept that a major function of this N-terminal region is to confer intracellular targeting of particular PDE4 isoforms on specific signalling complexes and intracellular locations. The paradigm for this concept developed from our original studies on the PDE4A1 (RD1) isoform. The N-terminal region unique to PDE4A1 consists of two well-defined helical regions separated by a mobile hinge region. Helix-2 provides the core membrane-insertion module, with helix-1 facilitating membrane association and fidelity of targeting in living cells. The irreversible, Ca2+-dependent insertion of the N-terminal region of PDE4A1 into membranes provides ‘long-term’ memory of cell activation.

cAMP signalling is compartmentalized in cells

Generation of the ubiquitous second messenger cAMP in response to extracellular stimuli leads to numerous, diverse physiological outcomes [13]. In the early 1980s, the pioneering work of Brunton et al. [1,4] examined the stimulation of adenylate cyclase activity in cardiomyocytes via different G-protein-coupled receptors. The resultant differential activation of the RI and RII isoenzymes of PKA (protein kinase A; also known as cAMP-dependent protein kinase) in cardiomyocytes led to the concept of compartmentalization of cAMP signalling components [4]. Examining the specificity of cAMP signalling pathways has shown that the intracellular targeting of the individual components confers a three-dimensional context on the signalling paradigms in which they can exquisitely control the specificity of the outcome of the signal [13,5,6]. In developing this, Scott and co-workers' pioneering work furthered the appreciation that PKA subpopulations were tethered to anchor proteins, called AKAPs (PKA-anchoring proteins), that are able to detect cAMP gradients within cells [3,6]. Thus activation of Gs-coupled receptors stimulates specific adenylate cyclases that are targeted to discrete areas within the plasma membrane [7]. The resultant generation of cAMP is then ‘read’ by effector proteins, either targeted PKA, plasma membrane-bound CNGCs (cyclic nucleotide-gated ion channels) or localized EPACs (exchange proteins directly activated by cAMP) [2,3,5,6,8,9]. A myriad of studies has since verified the concept of spatial and temporal control of cAMP signalling pathways [2,3,6]. The development of fluorescence resonance energy transfer probes, which allow the visualization of cAMP gradients within living cells [10,11], and CNGC probes [7,9] has further increased our understanding of the importance of compartmentalization of cAMP signalling.

Targeting of cAMP PDEs (phosphodiesterases) underpins compartmentalized cAMP signalling

However, pivotal to this paradigm is degradation of cAMP by PDEs. cAMP rapidly diffuses within cells and, without PDE action, cAMP gradients could not be formed and shaped within cells so as to regulate targeted effector proteins [5,1215]. Indeed, PDEs clearly underpin compartmentalized cAMP signalling in cardiac myocytes, as shown through both selective inhibition of PDE families [10,16,17] and by a novel dominant-negative approach, targeting individual PDE4 isoforms [18,19].

There is a large, eight-member superfamily of PDEs able to hydrolyse cAMP [1,12]. Selective inhibitors have shown the PDE4 family to be of particular importance in regulating key cells associated with the immune system and in learning and memory, for example [12,13,15,2023]. Four genes encode the PDE4 family (A, B, C and D) and these generate over 20 different isoforms [12,13,15,20]. PDE4 isoforms are subcategorized based on the presence or absence of upstream conserved regions, called UCR1 and UCR2, which are sandwiched between the isoform-specific N-terminal region and the catalytic unit. Long forms have UCR1 and UCR2, short forms UCR2 only and super-short forms just a truncated UCR2. UCR1 and UCR2 interact with each other [24] and orchestrate the functional outcome of phosphorylation by ERK (extracellular-signal-regulated kinase) [25,26] and by PKA [2729].

Each PDE4 isoform has a signature N-terminal region that uniquely identifies it. We have developed and pioneered the concept that a major function of this region is to confer intracellular targeting on specific signalling complexes and intracellular locations [5,6,1215,20] so as to tailor compartmentalized signalling in cells through the selective expression of particular PDE4 isoforms. The paradigm for this concept developed from our original studies on the short PDE4A1 (RD1) isoform. Since then, N-terminal regions have been shown to play a defining role in allowing interaction of PDE4A4 and PDE4D4 with SRC family tyrosine kinases [3032], PDE4A4 with XAP2 (X-associated protein 2) [33], PDE4D5 with β-arrestin [34], PDE4D3 with mAKAP (muscle-specific AKAP) [35] and AKAP450 [36,37] and PDE4D5 with RACK1 (receptor for activated C-kinase 1) [3840], although an increasing body of evidence suggests that certain PDE4-interacting proteins also interact at a second site within the enzyme [33,34]. The current spectrum of species interacting with PDE4 isoforms is shown schematically in Figure 1.

The ‘PDE4 Interactome’

Figure 1
The ‘PDE4 Interactome’

The Figure shows the range of species that have been shown to interact with the indicated PDE4 family members, which are given in parentheses. There are four PDE4 genes that encode approx. 20 different isoforms, each of which has a unique N-terminal region. These PDE4 enzymes are subclassified into long, short and super-short forms based on whether they have both UCR1 and UCR2 (long), just UCR2 (short) and just a truncated UCR2 (super-short), which is determined by the point of alternative mRNA splicing. DISC1, disrupted in schizophrenia 1.

Figure 1
The ‘PDE4 Interactome’

The Figure shows the range of species that have been shown to interact with the indicated PDE4 family members, which are given in parentheses. There are four PDE4 genes that encode approx. 20 different isoforms, each of which has a unique N-terminal region. These PDE4 enzymes are subclassified into long, short and super-short forms based on whether they have both UCR1 and UCR2 (long), just UCR2 (short) and just a truncated UCR2 (super-short), which is determined by the point of alternative mRNA splicing. DISC1, disrupted in schizophrenia 1.

PDE4A1 has provided the paradigm for the targeting and role of the isoform's unique N-terminal region

N-terminal regions are encoded by distinct 5′-exons that are immediately preceded by an isoform-specific promoter [41,42]. PDE4A1 has a unique 25-amino-acid N-terminal region, which is encoded by a distinct 5′-exon located on Chr19p13.2 [43]. PDE4A1 is entirely membrane-associated, where it preferentially locates to the Golgi and vesicles trafficking from it [4346]. However, by the simple expedient of engineering the removal of this unique region, an entirely soluble species is formed that now locates to the cytosol and is clearly folded correctly as it is even more active than native PDE4A1 [44]. This pivotal study provided the paradigm for the PDE isoform diversity and the role of the N-terminal region in determining intracellular targeting and, thereby, underpinning compartmentalized cAMP signalling. Thus the identification of the importance of the N-terminal of PDE4A1 in targeting of the protein has paved the way for the realization that the unique N-terminal region of PDE4 isoforms in general is essential in directing intracellular localization and therefore in directing intracellular compartmentalization through targeting specific PDE4 isoforms [12,13,20]. This can be mediated by either protein–protein interactions or by protein–lipid interactions.

PDE4A1 is found predominantly in the Golgi and in vesicles underlying the plasma membrane in a number of cell types including COS1, COS7, follicular thyroid cells and SKNSH cells and, in brain, it is enriched in synaptosomes with particular propensity to fractions associated with post-synaptic densities [4346].

Helix-2 in the PDE4A1 unique N-terminal region provides a Ca2+-gated bilayer insertion module

1H-NMR analysis of the 25-amino-acid N-terminal region of PDE4A1 reveals a distinct structure comprising two α-helical domains separated by a flexible hinge [47]. Leu3–Cys11 form a regular amphipathic α-helix with the non-polar residues Leu3, Val4, Phe6 and Phe7 along one side of a helical cylinder. Cys11–Pro14 show a poorly defined backbone and side-chain conformation indicative of a highly mobile hinge region. Pro14–Arg25 form a second distinct helical region, with a high proportion of bulky hydrophobic residues forming a distinct, compact hydrophobic cluster on one face. The distinct nature of these three domains within the N-terminal region of PDE4A1 makes it tempting to speculate that each may have a role in targeting and regulating the enzyme, which appears to be the case.

Both GFP (green fluorescent protein) and CAT (chloramphenicol acetyltransferase) are found as soluble proteins when expressed as recombinant species in mammalian cells. However, when we made chimaeric species by linking the unique N-terminal region of PDE4A1 to either GFP or CAT, we generated entirely membrane-bound forms whose distribution in cells paralleled that of full-length PDE4A1. Thus all of the information required for membrane association and targeting is encompassed within the 25-residue N-terminal region unique to PDE4A1 [47,48].

Neither PDE4A1 nor these chimaeras can simply be released from membranes by altering the ionic strength or pH. Thus they are not peripheral membrane proteins associating with either lipids or proteins by ionic interactions. Instead, they require detergent for release, implying an association with the lipid bilayer itself. Either integration into lipid bilayers can occur co-translationally for transmembrane proteins or bilayer insertion of hydrophobic regions of mature proteins can occur. Neither helix, however, is sufficiently large enough to span a lipid bilayer, implying that co-translational insertion to make a transmembrane protein is unlikely. Two pieces of information indicate that a mature PDE4A1 can subsequently associate with lipid bilayers. Firstly, (1–25)-PDE4A1–CAT chimaera can be generated as soluble proteins in an in vitro transcription–translation system. However, this chimaera is converted into a fully membrane-bound species upon subsequent exposure to either membranes or phospholipid liposomes [4749]. Secondly, a 25-mer peptide of sequence identical with (1–25)-PDE4A1 inserts into lipid bilayers within 8 ms, as evidenced by stop-flow analysis [49]. Analysis of truncates shows that the bilayer insertion unit is provided by helix-2. Intriguingly, insertion is gated, requiring intracellular levels of Ca2+ to rise above basal in order to bind to Asp21 in helix-2. This elicits a conformational change, allowing insertion of residues found at one face of helix-2 into the bilayer (Figure 2). The core, essential insertion unit is provided by Trp19:Trp20. However, its effectiveness is aided by Leu16:Val17, which helps orientate the tryptophan pairing, and by Phe23, which provides additional hydrophobic interaction [49].

Insertion of helix-2 of the unique N-terminal region of PDE4A1 into the lipid bilayer

Figure 2
Insertion of helix-2 of the unique N-terminal region of PDE4A1 into the lipid bilayer

The top panel shows a schematic diagram of super-short PDE4A1 isoform and its 25-residue unique N-terminal region. The middle and bottom panels show a backbone and surface grid representation of the PDE4A1 N-terminal region as determined by 1H-NMR, indicating helix-1, helix-2 and the mobile hinge region and orientation of helix-2 so as to allow insertion of the core Trp19:Trp20 unit.

Figure 2
Insertion of helix-2 of the unique N-terminal region of PDE4A1 into the lipid bilayer

The top panel shows a schematic diagram of super-short PDE4A1 isoform and its 25-residue unique N-terminal region. The middle and bottom panels show a backbone and surface grid representation of the PDE4A1 N-terminal region as determined by 1H-NMR, indicating helix-1, helix-2 and the mobile hinge region and orientation of helix-2 so as to allow insertion of the core Trp19:Trp20 unit.

While (1–25)-PDE4A1 can insert into phosphatidylcholine bilayers, its effectiveness was greatly enhanced when PA (phosphatidic acid), but not other acidic phospholipids, was incorporated into the lipid vesicles. Thus PDE4A1 shows a marked preference, but not absolute requirement, for PA interaction. This appears to be due to PA being the only phospholipid having an overall charge of −2 at neutral pH, which allows it to participate in a charge network at the bilayer interracial region so as to yield overall neutrality [49]. This network involves Ca2+:Asp21(1−):PA(2−):Lys25(1+), located on the solvent-orientated surface of helix-2. We call this Ca2+-regulated bilayer insertion unit, which shows preference for interaction with PA, TAPAS1 (tryptophan-anchoring phosphatidic acid-selective binding domain 1) [49].

We have tried to find out whether other proteins potentially have the core bilayer insertion motif we have identified here as L(L/V)XWWXX(F) by interrogating databases. Table 1 shows examples of proteins that may possess this or related motifs. All those listed show the absolutely essential WW motif and the majority listed show an LXXWW motif, where, from NMR studies, we know that leucine interacts with the Trp19:Trp20 pairing [47]. Little, however, is known about the mode of membrane association or targeting of these species save for AKAP149, also known as D-AKAP1, and AKAP1. This is a dual-specificity AKAP, able to bind both PKA-RI and PKA-RII regulatory units [2,3,50]. A 15-residue bifunctional element has been identified within it that is responsible for targeting to endoplasmic reticulum and mitochondrial membranes [50]. Contained within this 15-residue region is the sequence LLGWWwfFs which contains all of the key elements of the bilayer insertion motif that we have identified in PDE4A1 (Table 1). Indeed, mutation within this sequence disrupts membrane association [50]. Thus other proteins, including the PKA-tethering AKAP149, may use the bilayer insertion motif we have uncovered in helix-2.

Table 1
Helix-2 bilayer insertion motif of PDE4A1

The core bilayer insertion motif of PDE4A1 is formed from Trp19:Trp20, whose efficiency of insertion is aided by Leu17:Val18. Subsequently, Phe23 is inserted into the bilayer. This list shows examples of membrane-associated and secreted proteins that show similarity (capital letters) as identified using a descriptor of [L], [L/V], [G], [W], [W], [X], [X], [F/Y/W], where X is another amino acid, to probe the Swiss-Prot database using the ScanSite resource (http://scansite.mit.edu/). ADAM, a disintegrin and metalloproteinase; ARL6, ADP-ribosylation factor-like 6. The key motif required for membrane insertion of helix-2, namely LXXWW, is shown in bold type.

PDE4A1 
Glucose-6-phosphate isomerase L W W 
SCS3 (suppressor of choline sensitivity 3) protein L W W 
Cytochrome c oxidase polypeptide III L W W 
Cytochrome c oxidase subunit IV isoform 2 L W W 
AKAP149 L W W 
Cytochrome b L W W 
Ancylostoma secreted protein precursor L W W 
Negative regulator of mitosis L W W 
Peroxisomal membrane protein PEX16 L W W 
Cytochrome P450 6a19 L W W 
Phosphatidate cytidylyltransferase L W W 
Geranylgeranyl transferase type II β-subunit L W W 
Inward rectifier potassium channel 4 L W W 
Crk-associated substrate (p130cas) L W W 
ARL6-interacting protein-1 (Aip-1) W W 
Apolipoprotein N-acyltransferase W W 
ADAM 29 precursor W W 
Putative ammonium transporter 3 W W 
PDE4A1 
Glucose-6-phosphate isomerase L W W 
SCS3 (suppressor of choline sensitivity 3) protein L W W 
Cytochrome c oxidase polypeptide III L W W 
Cytochrome c oxidase subunit IV isoform 2 L W W 
AKAP149 L W W 
Cytochrome b L W W 
Ancylostoma secreted protein precursor L W W 
Negative regulator of mitosis L W W 
Peroxisomal membrane protein PEX16 L W W 
Cytochrome P450 6a19 L W W 
Phosphatidate cytidylyltransferase L W W 
Geranylgeranyl transferase type II β-subunit L W W 
Inward rectifier potassium channel 4 L W W 
Crk-associated substrate (p130cas) L W W 
ARL6-interacting protein-1 (Aip-1) W W 
Apolipoprotein N-acyltransferase W W 
ADAM 29 precursor W W 
Putative ammonium transporter 3 W W 

We also used the regular expression [LV][LV]GWW[DE] as a motif to define the Ca2+-regulated insertion module that we have uncovered and performed searches on each available database (Swiss-Prot, Ensembl, TrEMBL and GenPept) for mammalian organisms. However, doing this we only identified PDE4A1, suggesting that it may be unique in this particular aspect of building a Ca2+-regulated ‘switch’ into the insertion module.

Helix-1 is needed for efficient membrane insertion and fidelity of targeting in living cells

Many proteins that interact with lipids to render them membrane-bound require two binding sites. For example, Ras requires post-translational modification to add farnesyl and methyl residues to its CAAX box in order for it to insert into membranes.

The dual helical structure of (1–25)-PDE4A1, separated by a mobile hinge region, implies functional relevance of helix-1 as well as helix-2. Consistent with this, we have recently shown (E. Huston, T.M. Houslay and M.D. Houslay, unpublished work) that helix-1 is essential for the efficient intracellular targeting of PDE4A1 in living cells. Our studies indicate that while helix-2 provides the core membrane insertion domain, the efficiency of membrane insertion and the retention of PDE4A1 to the Golgi is dependent on the presence of helix-1. Thus dual membrane anchors are incorporated into the unique N-terminal region of PDE4A1. These are a bilayer insertion unit located in helix-2 together with a presumed membrane-protein (Golgi?) association unit located in helix-1. These are held together by a mobile hinge region that, presumably, allows for a functionally effective orientation of these two units relative to each other in order to allow fidelity in membrane insertion and targeting (Figure 2). Consistent with these notions, a dual (helix-2:helix-2)GFP chimaera is indeed membrane-associated in living cells although, unlike (1–25)-PDE4A1–GFP, it is not uniquely Golgi-associated. In contrast with this, a dual (helix-1:helix-1)GFP chimaera is entirely soluble and cytosolic in cells. This shows that efficiency of membrane association of PDE4A1 in living cells requires two anchors; one involves helix-2, which provides the membrane insertion module and the other is provided by helix-1, which increases the efficiency of helix-2 action and fidelity of targeting to the Golgi. This we suggest may occur through an ability to interact with a Golgi-localized protein subsequent to membrane insertion of helix-1.

The Ca2+-dependent insertion of PDE4A1 into lipid bilayers is an essentially irreversible process, thereby it reflects a stable, ‘long-term’ memory of a cell having experienced ‘activation’ by a signal causing elevation in this second messenger (Ca2+). As PDE4A1 is an exclusively brain-associated isoform, it is tempting to consider that this event may be associated with memory processes.

Conclusion

A key challenge to understanding regulation of cell signalling in time and space is an appreciation of the molecular targeting of signalling proteins. This has importance for dissecting cell functioning and specificity of action of hormones, neurotransmitters, growth factors and cell adhesion molecules in health and disease as well as providing novel routes for designing therapeutics aimed at preventing intracellular protein targeting. The cAMP signalling pathway has provided the paradigm for compartmentalization. However, fundamental to the generation and shaping of gradients and ‘clouds’ of cAMP in cells is the action of tethered cAMP PDEs. Critical to compartmentalized signalling in many, if not all cells, are tethered subpopulations of PDE4 isoforms. Studies on PDE4A1 have provided a paradigm for our present consensus that specificity of PDE4 isoform targeting is intimately associated with the N-terminal regions found unique to each isoform. This poses possibilities of generating novel PDE4 ‘inhibitors’ by the design of reagents that disrupt targeting of specific PDE4 isoforms in cells, thereby removing them from their functionally relevant compartment in cells.

Compartmentalization of Cyclic AMP Signalling: Biochemical Society Focused Meeting held at King's College, Cambridge, U.K., 29–30 March 2006. Organized by D. Cooper (Cambridge, U.K.), M. Houslay (Glasgow) and M. Zaccolo (Padua, Italy). Edited by D. Cooper.

Abbreviations

     
  • CNGC

    cyclic nucleotide-gated ion channel

  •  
  • PKA

    protein kinase A

  •  
  • AKAP

    PKA-anchoring protein

  •  
  • CAT

    chloramphenicol acetyltransferase

  •  
  • GFP

    green fluorescent protein

  •  
  • PA

    phosphatidic acid

  •  
  • PDE

    phosphodiesterase

  •  
  • UCR

    upstream conserved region

This work was supported by grant G8604010 from the Medical Research Council to M.D.H.

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