Arrestin times for compartmentalised cAMP signalling and phosphodiesterase-4 enzymes

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Various methods reveal that cyclic AMP (cAMP) signalling in cells is compartmentalised. These methods use FRET probes based upon either protein kinase A (PKA) or EPAC, cAMP-gated ion channels, or the selective activation of AKAP-anchored PKA isoforms. The basis of compartmentalisation involves point sources of cAMP generation within sub-domains of the plasma membrane coupled to degradation by spatially segregated, anchored forms of cAMP phosphodiesterases. cAMP-specific phosphodiesterase-4 (PDE4) isoforms play a central role in determining compartmentalisation, as exemplified in cardiac myocytes and T cells. The PKA phosphorylation status of the β2-adrenoreceptor, and hence its ability to switch its signalling from Gs to Gi and thus to activate ERK, is regulated dynamically by the agonist-stimulated recruitment of PDE4 to the receptor in complex with β-arrestin. The co-receptor CD28 enhances signalling through the T-cell receptor by recruiting a PDE4/β-arrestin complex, which then attenuates PKA phosphorylation of Csk.

Introduction

The cAMP signalling pathway controls a diverse range of cellular processes [1, 2]. Indeed, not only did cAMP provide the paradigm for the second messenger concept, but the pioneering work of Brunton and colleagues [3] on cardiac myocyte cAMP signalling provided the paradigm for signalling compartmentalisation. They showed that while various receptors stimulated cAMP accumulation in cardiac myocytes, the different receptors caused different physiological outcomes [3]. They suggested that this could be explained by compartmentalised changes in cAMP effecting the selective activation of cAMP-dependent protein kinase A (PKA) isoforms. However, difficulties in visualizing cAMP, and thereby in obtaining spatial readouts of cAMP levels in cells, compromised progress in this field. By contrast, studies on other intracellular messenger systems, namely Ca2+, tyrosyl kinases and inositol phospholipids, have unequivocally established signal compartmentalisation as crucial to normal cellular functioning.

This review highlights the pivotal role played by PDE4 cAMP-specific phosphodiesterases in underpinning compartmentalised cAMP signalling in cells.

Section snippets

Probes that detect spatial and temporal changes in intracellular cAMP

Recently, however, probes have been developed that detect and define spatial and temporal changes in intracellular cAMP in living cells. One powerful approach [4] has utilized a genetically engineered FRET probe in which donor/acceptor GFP forms are linked to the catalytic (C) and binding (R) subunits of PKA. Thus cAMP binding to the R-subunit causes it to dissociate from the C-subunit, with consequential loss of FRET. Using this approach, striking, spatially distinct changes in cAMP levels in

Phosphodiesterases at the heart of generating cAMP gradients in cells

Undoubtedly a major contribution to our appreciation of compartmentalised cAMP signalling is the demonstration that the PKA RII isoform is anchored at specific intracellular sites through binding to AKAPs (PKA [A-Kinase] anchor proteins) [2, 10]. These are a widely divergent set of signalling scaffold proteins that bind to the RII dimerization interface and thereby target PKA to distinct intracellular sites. Additionally, AKAPs invariably recruit specific PKA targets, allowing PKA to ‘read’

β-arrestin interaction with PDE4

Arrestins are signalling scaffold proteins that play a vital role in the desensitisation of many G-protein-coupled receptors (GPCRs) [37, 38]. Their primary role is to initiate receptor desensitisation by translocating from the cytosol to bind activated GPCRs at the plasma membrane. Recruited arrestin physically blocks interaction between GPCRs and their cellular effectors. The paradigm for this is provided by the β2-adrenoreceptor, which, upon agonist challenge, couples to Gs to activate

Functional significance of PDE4 translocation to the β2-adrenoreceptor

To determine the functional significance of agonist-induced, β-arrestin-mediated PDE4 translocation to the membrane, a catalytically inactive, ‘dominant negative’ PDE4 construct was engineered [41••]. Ectopic overexpression of this species displaced endogenous, active PDE4s from β-arrestin, with the result that agonist challenge failed to induce increased cAMP degradation at the β2-adrenoreceptor. This resulted in the selective amplification of agonist-induced PKA activity at the plasma

PDE4D5: the preferred partner for β-arrestin

Analyses done on various cell types identified the PDE4D5 isoform as preferentially recruited to the β2-adrenoreceptor by isoprenaline and preferentially co-immunoprecipitated with β-arrestin [40••]. This is because the unique N-terminal region of PDE4D5 has a binding site for β-arrestin in addition to that located within the conserved PDE4 catalytic domain.

β-arrestin also has two sites involved in binding PDE4D5 [40••]. One, which contains pairs of lysine residues essential for interaction, is

CD28-mediated recruitment of β-arrestin/PDE4 potentiates T-cell signalling

The ligated T-cell receptor (TCR) is located within microdomains rich in cholesterol and sphingolipids, called lipid rafts. Upon TCR engagement, Lck becomes activated, resulting in the tyrosyl phosphorylation of ITAM (immunotyrosine-based activation motif) domains within CD3, a process that is critical for full T-cell activation [2].

Cyclic AMP signalling in T cells is tightly regulated, with stimulation of the TCR leading to a transient rise in cAMP. One consequence of this is that the

Conclusions

Cellular targeting of PDE4 cAMP-specific phosphodiesterases, conferred by isoform specific N-terminal regions, underpins the shaping of localized cAMP gradients necessary for compartmentalised signal transduction. That PDE4s can complex to, and translocate with, members of the β-arrestin family sheds new perspectives on the dynamic possibilities of this paradigm. Thus anchored PDE4s may be recruited dynamically to different areas of the cell to coordinate with other signalling pathways and to

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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