Localised GPCR signalling as revealed by FRET biosensors
Introduction
Compartmentalised signalling — the regulation of signalling in space and time — is a mechanism for cells to distinguish between the activation of distinct receptors to generate highly-specific responses (reviewed in Ref. [1,2]). Since the first reports of the use of intramolecular FRET biosensors to monitor compartmentalised G protein-coupled receptor (GPCR) signalling [3, 4, 5], their use has rapidly expanded, revealing that many active GPCRs can also be found in discrete sub cellular compartments (reviewed in Refs. [6,7•]). Here, the evidence for localised GPCR signalling as revealed by FRET biosensors is discussed with a focus on reports in the last two years. Compartmentalisation of cAMP remains the focus of most studies. For example, many elegant studies have clearly demonstrated compartmentalisation of cAMP stimulated by the β2-adrenoceptor in cardiac myocytes, and the loss of this in heart failure. This focus on cAMP compartmentalisation is likely due to two main factors: first, that Gαs and Gαi/o-coupled receptors (which account for two thirds of all GPCRs with approved drugs [8]) have been classically defined by their ability to stimulate or inhibit cAMP levels, respectively; and second, there are many well-characterised FRET biosensors available to detect cAMP (Figure 1) that have been targeted to many sub cellular locations (Figure 2). Importantly, the challenge to link compartmentalised GPCR signalling with major physiological and pathophysiological processes has been addressed with advances in two areas: compartmentalised cAMP signalling of the β2-adrenoceptor has been linked to excitation-contraction coupling of cardiac myocytes; and inhibition of endosomal signalling of the tachykinin NK1 receptor, calcitonin CGRP receptor and the proteinase-activated receptor, PAR2, has been demonstrated to increase therapeutic efficacy of receptor antagonists. Finally, some elegant solutions to the limitations of the use of FRET biosensors are highlighted.
Section snippets
Localised GPCR signalling from distinct plasma membrane microdomains
The classic example of GPCR signal compartmentalisation is the control of cAMP signalling in cardiac myocytes; these cells express β-adrenoceptors and prostanoid receptors, all of which increase cAMP (reviewed in Ref. [9]). However, the physiological effects of activation of each of the receptors are different, and this has been attributed to compartmentalisation of the cAMP signal (reviewed in Ref. [9]). Compartmentalisation of cAMP has now been directly demonstrated using a cAMP FRET
Localised signalling controlled by GPCRs at intracellular locations
There are many examples of compartmentalised signalling detected by FRET biosensors that have been linked to GPCR internalisation using endocytic inhibitors and/or by correlating the timing of this signalling with receptor internalisation [19, 20, 21, 22, 23, 24]. Other elegant studies have combined FRET biosensors with detailed image analysis to show localised Cdc42 activity within invadosomes [25], and distinct control of GPCR signalling in the cilia, dendrites, synaptic boutons and soma of
Compartmentalised β-adrenoceptor signalling in cardiac myocytes
The challenge for the study of compartmentalised signalling is to directly demonstrate its relevance at the level of the whole organism or during pathogenesis. Localised cAMP produced by the β2-adrenoceptor has been well-studied using FRET biosensors in cardiac myocytes where it is important for excitation-contraction coupling (reviewed in Ref. [45]). In these cells, the assembly of a β2-adrenoceptor-protein complex at the T-tubules restricts cAMP to a very localised region; this organisation
Localised signalling from GPCRs in endosomes regulates pain transmission
We recently demonstrated the therapeutic relevance of targeting localised GPCR signalling within endosomes to increase the therapeutic efficacy of antagonists in models of pain [55••,56,57] (Figure 4). For the tachykinin NK1 receptor, activation by substance P caused a transient increase in plasma membrane localised cAMP and cytosolic ERK, dependent on receptor localisation at the plasma membrane [55••]. After receptor internalisation to early endosomes, the NK1 receptor instead caused a
Perspective
FRET biosensors have been an extremely powerful method with which to unravel the importance of compartmentalised signalling in the control of cellular processes. Much of the evidence for signalling of internalised GPCRs initially relied on measuring a change in signal following the inhibition of endocytosis by chemical means or by the over expression of dominant negative proteins essential to this process. This has been an undoubtedly effective approach; however, it will also impact on the
Conflict of interest statement
Nothing declared.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
MLH is a National Health and Medical Research Council of Australia (NHMRC) RD Wright Fellow (1061687).
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