Review
cAMP: Novel concepts in compartmentalised signalling

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Abstract

Cyclic adenosine 3,′5′-monophosphate (cAMP) is the archetypal second messenger produced at the membrane by adenylyl cyclase following activation of many different G protein-coupled receptor (GPCR) types. Although discovered over fifty years ago, the notion that cAMP responses were compartmentalised was born in the 1980s. Since then, modern molecular techniques have facilitated visualisation of cellular cAMP dynamics in real time and helped us to understand how a single, ubiquitous second messenger can direct receptor-specific functions in cells. The aim of this review is to highlight emerging ideas in the cAMP field that are currently developing the concept of compartmentalised cAMP signalling systems.

Highlights

► Targeted cAMP reporters have facilitated a greater understanding of the compartmentalised nature of cAMP signalling. ► Signalling via cAMP is not necessarily retarded following receptor internalisation. ► Epac signalling is compartmentalised by virtue of Epac scaffolding proteins.

Introduction

3′,5′-Cyclic adenosine monophosphate (cAMP) is the archetypal second messenger, produced at the cell membrane by adenylyl cyclase (AC) following G protein-coupled receptor (GPCR) ligation to activate a small number of cAMP effector proteins that trigger functional cellular processes [1]. Ever since the observation of Brunton and co-workers in the 1980s that a number of GPCR activators could elevate cellular cAMP to equivalent levels but at the same time produce distinct physiological outcomes [2], the idea that cAMP signalling is compartmentalised has gained credence. Unequivocal evidence that receptor-specific responses are underpinned by cAMP spatial microcompartmentation was obtained following development of genetically encoded cAMP reporters that used Förster resonance energy transfer (FRET) to measure cAMP dynamics in real time [3], [4], [5]. It is now clear that the precise location of proteins that manufacture, degrade and are activated by cAMP is crucial to maintain coherent downstream signalling events that are triggered by specific extracellular cues [6]. As phosphodiesterase (PDE) enzymes are the only route by which cAMP can be degraded in the cell [7], compartmentalisation of these proteins is particularly relevant to regulation of the magnitude and duration of cAMP-dependent events in defined compartments [8]. Current research using cAMP reporters targeted to different intracellular regions describes areas of high PDE expression as cAMP “sinks” that act to locally drain cAMP [9], [10]. This model is attractive as it can explain why many contiguous cAMP gradients can be formed simultaneously following a single GPCR activation. Presumably, the correct positioning of cAMP effectors such as cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epacs) within these gradients allow them to be exposed to cAMP concentrations above their threshold of activation to drive downstream signalling events only at times when cAMP is raised. Conversely, during times of low, basal cAMP, phosphodiesterase activity should prevent inappropriate activation of cAMP effectors.

Compartmentalised cAMP signalling has been the subject of a number of recent reviews looking at the role of phosphodiesterases [7], A-kinase anchoring proteins (AKAPs) [11], [12], Epac [13], adenylyl cyclases [14], [15] and cAMP itself via cAMP reporters [4], [5]. The aim of this review, however, is to cover emerging concepts in cAMP signalling that involve compartmentalised responses. Recent noteworthy advances that have extended and contradicted established tenets of the cAMP field include the concept of sustained cAMP signalling from internalised receptors, compartmentalisation of Epac-directed signals and real time direct visualisation of cAMP gradients within intact cardiac myocytes.

Section snippets

New advances in measuring cAMP gradients in the heart

The concept of intracellular compartmentalisation of cAMP arose almost two decades ago from seminal studies performed in cardiac myocytes. Activation of β-adrenergic and prostaglandin receptors was found to result in similar increases in cellular cAMP, however, whereas β-adrenergic stimulation coupled to myocyte contraction and PKA phosphorylation of downstream effectors such as troponin I, prostaglandin receptor stimulation did not [16], [17]. These puzzling differences could only be explained

Macromolecular signalling complexes: The mAKAP/ryanodine receptor complex as a paradigm for compartmentalised cardiac cAMP signalling

As an important second messenger that regulates multiple pathways within the cardiac myocyte, cAMP signals must be tightly controlled and integrated with those from other second messengers such as calcium. The formation of macromolecular signalling complexes, or signalling ‘nodes’, by scaffold proteins allows co-ordination of cAMP and other upstream signals with downstream effectors, by incorporating components of different signalling pathways and signal terminating enzymes. One such

Persistent cAMP signalling from internalised GPCRs

Recently, one of the classical paradigms of cAMP signalling has been challenged by a number of reports which propose that cAMP production may occur not only at the membrane immediately after GPCR stimulation but also in a sustained manner following receptor internalisation (reviewed in [67], [68]). Internalisation of receptors is the end stage of the desensitisation process in which cell surface receptors show a decreasing reaction to constant agonist challenge. The desensitisation process is

Compartmentalised Epac signalling

As outlined above, spatial organisation of the cAMP signal transduction pathway by anchoring and adaptor proteins is crucial for maintaining the fidelity of cellular responses that are triggered by this second messenger. Certainly, for PKA, it has been appreciated for some time that discrete cellular localisation via AKAPs underpins the precise manner by which this promiscuous kinase selects only a fraction of its possible substrates for phosphorylation following increases in cellular cAMP [80]

Conclusion

The concept of compartmentalised cAMP signalling is now widely accepted, thanks to studies employing FRET sensors, which have allowed direct visualisation of cAMP gradients within live cells. Cellular compartmentalisation of cAMP is achieved by the scaffolding of PKA within macromolecular complexes, which bring PKA close to specific downstream effectors. These complexes also include cAMP-degrading phosphodiesterases, signal-terminating phosphatases and adenylyl cyclases. Emerging evidence

Acknowledgements

G.S.B. is supported by grants from the Medical Research (UK; G0600765) and Fondation Leducq (06CVD02). H.V.E. is supported by a grant from the BBSRC.

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