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Genetically Encoded FRET Biosensors to Illuminate Compartmentalised GPCR Signalling
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Review
Genetically Encoded FRET Biosensors to Illuminate Compartmentalised GPCR Signalling Michelle L. Halls1,* and Meritxell Canals1,* Genetically encoded Förster resonance energy transfer (FRET) biosensors have been instrumental to our understanding of how intracellular signalling is organised and regulated within cells. In the last decade, the toolbox, dynamic range and applications of these sensors have expanded beyond basic cell biology applications. In particular, FRET biosensors have shed light onto the mechanisms that control the intracellular organisation of G protein-coupled receptor (GPCR) signalling and have allowed the visualisation of signalling events with unprecedented temporal and spatial resolution. Here we review the use of these sensors in the GPCR field and how it has already provided invaluable advances towards our understanding of the complexity of GPCR signalling.
Trends Genetically encoded FRET sensors have been instrumental to understand compartmentalised signalling. FRET-based biosensors offer increased spatial and temporal resolution over traditional cell-based signalling assays and allow the discrimination between the responses of different cell populations. The presence of GPCRs and key signalling effectors in subcellular microdomains governs the assembly of signalling complexes that generate compartmentalised signalling and is a key determinant of signal selectivity.
G Protein-Coupled Receptors With more than 800 members, G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors in the human genome. They participate in virtually every physiological process and are the target of more than one-third of current medicines. Functionally, GPCRs transduce a wide range of extracellular stimuli (ranging from light and small ions to bigger molecules and peptides) into various intracellular signalling pathways via coupling to heterotrimeric G proteins or to other GPCR-interacting proteins. The fact that different receptors that activate common signalling pathways in the same cell exert divergent physiological effects suggests a high degree of organisation and regulation of intracellular signalling. Spatiotemporal compartmentalisation – the restriction of second messengers in space and time – provides a mechanism whereby GPCRs can direct the assembly of focused ‘platforms’ for specific signalling. These signalling platforms facilitate second messenger production, the organisation and scaffolding of effectors, and co-ordination of regulatory elements. In this context, genetically encoded Förster resonance energy transfer (FRET) biosensors have proved as invaluable tools for visualising GPCR signalling dynamics with high spatial and temporal resolution in living systems as they circumvent the need for cell lysis and allow for real-time monitoring of signalling events. Here, we will provide an overview of FRET-based biosensors and review how the use of these sensors in the GPCR field has already provided significant breakthroughs in our understanding of the complexity and distinct physiological consequences of GPCR signalling. (For a review of the use of bioluminescence resonance energy transferbased biosensors to investigate GPCR signalling and trafficking, see Bouvier et al. in this issue.)
Compartmentalisation of Intracellular Signalling The idea of compartmentalised signalling, while initially proposed by the cAMP field [1], was first directly demonstrated for calcium signalling due to the availability of high-affinity calcium-
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The spatial and temporal control of compartmentalised signalling defines the physiological outcomes of GPCR activation.
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Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia
*Correspondence: [email protected] (M.L. Halls) and [email protected] (M. Canals).
https://doi.org/10.1016/j.tips.2017.09.005 © 2017 Elsevier Ltd. All rights reserved.
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sensitive fluorescent dyes, such as fura-2. These initial imaging studies revealed sparks, puffs and blinks of calcium within the cell (reviewed in [2,3]), and demonstrated compartmentalised signalling at the most basic level – high levels of a second messenger at the site of production, with a steep decline in concentration to ambient levels in the rest of the cell. The more recent development of genetically encoded biosensors to detect changes in cAMP levels and kinase activity has further refined our ideas of signal compartmentalisation. While the restricted localisation of the source of a second messenger can give rise to localised ‘hot spots’ of signalling in bulk areas of the cell, mechanisms that control the lifetime or the diffusion of such second messenger can create more specialised signalling hubs that have greater spatial and temporal flexibility. For example, cAMP compartmentalisation is often controlled by the close proximity of the enzymes that break down cAMP [phosphodiesterases (PDEs)] to the enzymes that produce it [adenylyl cyclases (ACs)] to create highly localised elevations of the second messenger [4–7]. Subsequent manipulation of these biosensors to target them to discrete cellular domains (e.g., plasma membrane vs. cytosol) or to individual proteins (e.g., ACs, PDEs) has demonstrated the importance of scaffolding proteins for assembling focused signalling hubs [1,8,9]. For cAMP signalling, the most studied and iconic scaffolding proteins are A-kinase anchoring proteins (AKAPs); originally identified for their ability to bind and regulate protein kinase A (PKA), AKAPs are now recognised as multimodal scaffolds that control the compartmentalisation of cAMP signalling by scaffolding activators [e.g., ACs, protein kinase C (PKC)], regulators (e.g., PDEs) and effectors (e.g., PKA, Epac, ion channels) [2,3,10,11]. This ensures high fidelity of the second messenger signal, with the signalling hubs therefore dictating not only the nature of the cAMP signal, but also its cellular consequences. It is now well recognised that compartmentalisation of signalling is often dependent on the formation of higher-order protein complexes to allow the efficient and specific scaffolding of proteins that activate and regulate key second messengers [4–7,12,13].
FRET Biosensors for Single-Cell and Subcellular Resolution of Signalling Events Most biosensors that are currently available rely on FRET. In this approach, a peptide chain containing the ‘sensor’ of the second messenger (e.g., second messenger binding site or target peptide) is fused to a complementary pair of fluorophores with overlapping spectra. Energy transfer between these two fluorophores depends on their close proximity and alignment. Binding of a second messenger, or modification of the target peptide, triggers a conformational change of the sensor peptide chain that alters the distance between these two fluorophores, thus changing the FRET signal (Figure 1). Typically, cyan fluorescent protein and YFP are used as the FRET pair, although biosensors utilising GFP and red fluorescent protein are becoming more common (which also allows for multiplexing of complementary signalling biosensors), as are the use of variants engineered to be less sensitive to fluctuations in pH (e.g., mCitrine [14]), which is particularly important for the use of these biosensors in primary cells such as neurons. Importantly, in ratiometric FRET biosensors the effect on the FRET signal due to changes in expression levels between experiments or even cellular compartments is eliminated, as the two fluorescent proteins are attached to the same sensor, and therefore are expressed as a single polypeptide. Finally, it is important to highlight that different FRET biosensors that detect the same second messenger can have a different dynamic range (Box 1). Ratiometric biosensors that directly detect second messengers (e.g., the Epac2-based biosensor that detects cAMP [15] or the cGES-DE5 sensor that detects cGMP [16]) undergo a conformational change upon the binding of the second messenger, leading to a change in the FRET signal. Ratiometric FRET biosensors to detect kinase activity – for example, A kinase
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Figure 1. FRET Biosensor Strategies. FRET biosensors rely on a conformational change as a result of (A) binding of a second messenger (e.g., cAMP) to a second messenger-sensitive-binding domain (e.g., cAMP-binding domain), (B) phosphorylation of a substrate peptide with a consensus target sequence for specific kinases that is recognised by a phospho-binding domain (e.g., EKAR sensor [18]) or (C) exchange of GDP for GTP that is recognised by another binding domain (e.g., RaichuRac1 sensor [21]). [18_TD$IF]Abbreviations: CFP, cyan fluorescent protein; EKAR, extracellular signal-regulated kinase activity reporter; ERK, extracellular signal-regulated kinase; FRET, Förster resonance energy transfer.
activity reporter (AKAR) for PKA; extracellular signal-regulated kinase activity reporter (EKAR) for extracellular signal-regulated kinase (ERK); or C kinase activity reporter (CKAR) for PKC [17–20] – have slightly different mechanisms of action. While the principle of conformational change within the biosensor is still utilised, this change is induced following phosphorylation of a target sequence by the corresponding kinase, which facilitates an interaction between the phosphorylated target sequence and an attached binding protein that is sensitive to such
Box 1. Dynamic Range of Fluorescent Biosensors FRET biosensors are a highly sensitive technique for measuring intracellular signalling, and can detect smaller responses than conventional, population-based signalling assays. However, this increased sensitivity comes with limitations in their dynamic range; a by-product of sensitive detection is often a decreased upper detection limit. It is therefore important to determine the absolute sensitivity and dynamic range of a particular biosensor in each system. Different FRET biosensors for the same target can have differences in their dynamic range. For example, two FRET biosensors that detect cAMP, Epac2-camps [15] and ICUE2 [37], actually detect different concentration ranges of cAMP. The sensitivity of the biosensors differs by an order of magnitude (Epac2-camps, EC50 of 0.92 mM [15]; ICUE2, EC50 of 12.5 mM [37]). As such, while ICUE2 can detect an order of magnitude higher peak cAMP than Epac2-camps, Epac2-camps is more suited to detecting very small increases in cAMP. Although these are important considerations to take into account when choosing an appropriate FRET biosensor for single-cell imaging studies, the different dynamic range of sensors for the same second messenger also expands the utility of these tools. It is also important to note that modifications of the same biosensor to enable targeting to distinct cellular domains can impact biosensor sensitivity. GCaMP2 is a genetically encoded high-affinity Ca2+ biosensor [38]. It contains a circularly permuted eGFP, flanked by calmodulin (a Ca2+-binding protein) and a calmodulin-binding peptide, M13. Increases in Ca2+ drive an interaction between Ca2+/calmodulin and the M13 peptide, leading to a conformational change in the biosensor and an increase in GFP fluorescence. GCaMP2, originally expressed within the cytosol, has been targeted to various intracellular domains and proteins, including to the plasma membrane by palmitoylation/myristoylation [9], to the N terminus of AC2 and AC8 [9] and to the a-subunit of the Na2+ pump [11]. Addition of the targeting sequences to GCaMP2 causes small changes in the sensitivity of the biosensor to Ca2+ [9,11,38]. Again, this highlights the importance of understanding and assessing the dynamic range of each biosensor. Similar changes in potency have been observed following targeting of the cAMP FRET biosensor, Epac2-camps [15]. The original Epac2-camps (expressed within the cytosol) has an EC50 for cAMP of 0.4 mM, and this sensitivity was decreased following targeting to the plasma membrane (pmEpac2-camps, EC50 of 1.7 mM) or to the N terminus of AC8 (Epac2-AC8, EC50 of 2.2 mM) [22]. Although these are relatively small changes (i.e., GCaMP2-AC8 drops the sensitivity to Ca2+ by 1.5-fold, and Epac2-AC8 drops sensitivity to cAMP by fourfold), it does require caution when interpreting data obtained from the same biosensor targeted to a distinct location.
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phosphorylation. In this case, the FRET biosensor can be considered a substrate for the second messenger in question. The Rho G protein FRET biosensors (e.g., Raichu-Rac1, Raichu-Cdc42 [21]) utilise yet another slightly different mechanism of conformational change. In this case, the biosensor contains the Rho G protein of interest, and the exchange of GDP/GTP [by guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity] allows the GTP-bound protein to interact with an adjacent binding partner, causing the conformational change to alter FRET within the biosensor. Importantly, all the aforementioned conformational changes are reversible, such that dynamic increases and decreases in the second messenger can be accurately measured (Figure 1). Many of these biosensors have been subsequently modified to incorporate subcellular targeting sequences – for example, the original cAMP biosensors were expressed within the cytosol of the cell, but have since been targeted to the plasma membrane [8], to proteins including ACs [8,9,22] and PDEs [10], and intracellular organelles such as the nucleus and mitochondria [23]. The comparison of FRET signals at different sites within the cell, in response to the same stimulus, has been instrumental in advancing our understanding of how compartmentalised signals are controlled (Box 1).
Advantages of Single-Cell Resolution of Signalling Events In traditional cell-based signalling assays, tens to hundreds of thousands of cells are seeded in multiwell plates, incubated with a stimulus and then the stimulation is ‘stopped’ at a predetermined time typically requiring cell lysis. The second messenger of interest is then extracted from the samples and measured by antibody, radioimmunoassay, immunoblotting, etc. Continuous measurements are obviously not possible in such assays, with time-course approximations often limited in resolution to minutes. These measurements detect, by default, the average response of a cell population, so that they can be considered population (as opposed to single-cell)-based signalling assays. Live cell imaging of FRET biosensors is noninvasive and nondestructive, and is only limited by the capture speed of the camera, thus providing increased temporal resolution over more traditional cell-based signalling assays. However, the greatest advantage of these sensors is the ability to discriminate between subpopulations of cells. This is important as a large variation in signalling responses to a stimulus can be observed within a single-cell population and is evident not only in cultures of isolated primary cells (which often consist of a mixed cell populations) but also in cell lines with endogenous receptor expression (likely dependent on the relative level of expression of a particular receptor at the single-cell level). The ability of FRET biosensors to discriminate between subpopulations of cells provides an additional level of information that would otherwise be lost in population-based signalling assays, where the total response reflects a composite of many individual responses [24]. This additional resolution can be utilised in a number of ways: for example, by assessing the ability of an inhibitor to change relative populations exhibiting different responses, that is, from transient to sustained [25] (Figure 2), or by poststaining of a primary cell culture with heterogeneous cell composition and analysis of the different cell types or of only the cells that express the receptor of interest.
Compartmentalised GPCR Signalling Revealed Using FRET Biosensors Subcellularly targeted FRET biosensors have proved extremely useful for the study of GPCR signalling. Moreover, the increased resolution provided by real-time detection of signalling events using sensors specifically targeted to different cellular compartments has challenged some of the classical views of GPCR signalling. For example, the high sensitivity of FRET biosensors to detect signal transduction and second messenger levels has allowed the detection of previously unappreciated signals. Halls and Cooper [26] used the cAMP Epac2-camps FRET biosensor targeted to the plasma membrane or the cytosol to investigate
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Figure 2. FRET Biosensors Allow Discrimination of Responses in Different Subpopulations of Cells. The ability to assess signalling at a single-cell level provides the resolution to separate cellular populations according to their responses. In the example, the response of the MOR agonist morphine in dorsal root ganglia (DRG) neurons expressing the cytosolic ERK FRET biosensor (cytoEKAR) was measured in the absence (A) or presence (B) of a PKC inhibitor. Analysis of single-cell responses shows a shift of the relative population of cells responding in a transient fashion to this agonist [25]. [182_TD$IF]Abbreviations: EKAR, extracellular signal-regulated kinase activity reporter; ERK, extracellular signal-regulated kinase; FRET, Förster resonance energy transfer; MOR, mu-opioid receptor; PKC, protein kinase C.
compartmentalised cAMP signalling of the hormone relaxin. These experiments unveiled that the relaxin receptor, RXFP1, forms a preassembled signalosome that provides a unique regulatory mechanism for the extremely low levels of relaxin found in circulation (Figure 3A). This constitutively assembled RXFP1-signalosome is formed by both stimulatory (AKAP79 and AC2) and regulatory (b-arrestin2, PDE4D3 and PKA) components that interact with the receptor in two defined regions of the C-terminal tail and allow the activation of the receptor by subpicomolar concentrations of peptide. FRET biosensors have also been instrumental to reveal the importance signal compartmentalisation for the generation of specific cellular responses upon GPCR activation. Nikolaev et al. [27] generated a transgenic mouse with the cAMP FRET biosensor HCN2-camps and visualised the differences in cAMP signalling elicited by b1- and b2-adrenoceptors (ARs) in isolated adult cardiac myocytes. While the b1ARs and b2ARs are the main AR subtypes controlling
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Figure 3. Illuminating GPCR Intracellular Signalling. Genetically encoded FRET biosensors have allowed the detection of signalling events with unprecedented temporal and spatial resolution. (A) The plasma membrane-targeted cAMP FRET sensor (pmEpac2) allowed the detection of responses to subpicomolar concentrations of relaxin mediated by its receptor, RXFP1 [26]. (B) Substance P activation of the NK1R induces a nuclear ERK response (nucEKAR) that is dependent on receptor internalisation as it is blocked by the clathrin-inhibitor Pitstop 2 but not its inactive analogue [34]. (C) Inhibition of the Gbg subunits of the G protein with bARKct changes the nuclear ERK profile (nucEKAR) of the mu-opioid receptor (MOR) when activated by morphine [25]. [182_TD$IF]Abbreviations: DMSO, dimethyl sulfoxide; EKAR, extracellular signal-regulated kinase activity reporter; ERK, extracellular signal-regulated kinase; FRET, Förster resonance energy transfer; GPCR, G protein-coupled receptor; NK1R, neurokinin 1 receptor.
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cardiac response, only b1AR stimulation seems to induce cardiomyocyte hypertrophy and apoptosis. The use of the cAMP FRET sensor allowed the visualisation of key differences in the propagation of the cAMP signal by the two receptor subtypes. While b1ARs induce farreaching cAMP signals that propagate over distances including multiple sarcomeres, the cAMP signal evoked by the b2AR is far more localised and does not propagate throughout the cell. Genetically encoded biosensors have also been used to assess the relevance of GPCR localisation within the cell for its downstream signalling. Indeed, GPCRs have now been described to signal from different regions of the plasma membrane as well as from other subcellular compartments such as the endosomes, the nucleus or even the mitochondria. In this context, several groups have used biosensors in combination with pharmacological and genetic strategies to assess the importance of changes in GPCR location for the generation of specific cellular responses. Vincent et al. [28] showed that the intracellular metabotropic glutamate receptor, mGluR5, in spinal neurons plays a critical role in neuropathic pain. They showed that in spinal dorsal horn neurons, mGluR5 is located on nuclear membranes and that nuclear expression of this receptor increases upon nerve injury. Using a genetically encoded calcium indicator specifically targeted to the cell nucleus (pCMV-NL-S-R-GECO), the authors showed that stimulation of the intracellular mGluR5 leads to sustained nuclear calcium responses. Blockade of these responses with permeant antagonists or decreasing the levels of intracellular glutamate reduces neuropathic pain behaviours and Fos expression. Genetically encoded RET biosensors have also provided spatial and temporal resolution that biochemical measurements of GPCR signalling previously lacked. This major advantage has been instrumental for one of the concepts that has revolutionised the GPCR field in the last decade: that some internalised GPCRs remain active and exhibit sustained G-protein-dependent signalling. As the list of GPCRs that exhibit sustained endosomal signalling is still growing, here we choose to highlight some examples that have employed biosensors to investigate this phenomenon. The FRET sensors most widely used to investigate endosomal signalling by GPCRs are those that detect cAMP changes. This is mainly due to the fact that most receptors identified to date that signal once internalised are coupled to Gas G proteins and therefore, activate AC. In 2009, two groups showed the importance of receptor internalisation for the generation of sustained cAMP signals. Calebiro et al. [29] generated a transgenic mouse with ubiquitous expression of the fluorescent sensor for cAMP CAG-Epac1-camps and studied cAMP responses to thyroidstimulating hormone (TSH) in native thyroid follicles isolated from these mice. In this system, using endocytic inhibitors as well as subcellular fractionation approaches, they demonstrated that TSH receptor internalisation is responsible for the sustained cAMP signalling observed upon receptor stimulation. Furthermore, they also demonstrated that such cAMP signalling elicited by internalised receptors is responsible for the depolymerisation of actin in response to TSH in mouse thyroid cells, hence suggesting that signalling from endocytosed receptors has important functional consequences. In another study using the same cAMP biosensor, Ferrandon et al. [30] showed that the parathyroid hormone receptor (PTHR) behaves in a similar fashion as the TSH receptor. Using PTH-derived peptides they demonstrated that long-acting ligands interact tightly with the receptor in a conformationally dependent manner. The receptor is then locked into a prolonged active state inducing sustained receptor–G-protein coupling and sustained G-protein activation. They also showed that the ternary ligand–receptor–Gprotein complex is preserved during internalisation and persists over time to appear as a key structure in the prolonged downstream signalling of PTH-like hormone.
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Generation of cAMP by the b2AR occurs partly within the endocytic compartments and, importantly, the localisation of cAMP pools has key downstream consequences [31]. Although these studies did not use FRET-based but instead a luciferase-based sensors (GloSensor) to delineate these mechanisms, they represented a key turning point in our understanding of GPCR signalling. In 2013, Irannejad et al. [32] generated conformational biosensors to detect agonist-activated b2AR and activated Gas protein to demonstrate the existence of an active b2AR-Gas complex within the endosomes. Later the same group showed that the full repertoire of cAMP-dependent transcriptional responses requires b2AR endocytosis [31]. To demonstrate that cells are able to discriminate the location of cAMP generation when initiating a signalling response, they generated an elegant optogenetic strategy based on a bacteria-derived AC, which allowed cAMP production to be induced by light [bacterial photoactivated adenylyl cyclase (bPAC)]. By specifically targeting and activating bPAC at the plasma membrane, the endosomes or the cytosol, they showed that localised cAMP production generates distinct transcriptional responses. Interestingly, Jean-Alphonse et al. [33] have recently proposed a new mechanism through which b2ARs promote the formation of cAMP in endosomes. Using intramolecular and intermolecular FRET sensors the authors propose that b2AR activation, via isoproterenol, enhances and prolongs signalling by the PTHR in response to PTH(1–34) by releasing noncatalytic Gbg subunits. This augmentation of the pool of free Gbg promotes the assembly and functional activity of signalling complexes composed of PTHR, Gbg, Gas and AC2, which persist in a functional state in endosomes. Whether this is the only mechanism of b2AR-mediated endosomal signalling remains to be addressed. An example of a non-Gas-coupled receptor that exhibits signalling once internalised is the neurokinin 1 receptor (NK1R). Jensen et al. [34] have recently used FRET biosensors to detect cytosolic and plasma membrane cAMP (Epac2-camps), cytosolic and plasma membrane PKC activation (CKAR), and cytosolic and nuclear ERK activation (EKAR) to study the link between NK1R endocytosis and signalling in subcellular compartments with high spatiotemporal fidelity. Activation of the Gaq/11-coupled NK1R by the neuropeptide substance P exhibits a repertoire of compartmentalised signalling responses and some of these responses are dependent on receptor endocytosis. Moreover, the endosomal NK1R signalling platform is essential for sustained pain transmission. The use of genetic and pharmacological tools to prevent receptor internalisation as well as the generation of endosomally targeted antagonists for the NK1R demonstrated the dependence of nuclear ERK, cytosolic cAMP and cytosolic PKC on receptor endocytosis (Figure 3B). Moreover, these results were translated to spinal cord slices and to animal models of inflammatory and noninflammatory pain to demonstrate the importance of internalised NK1R signalling for (patho)physiological processes. Although, as shown earlier, some GPCRs exhibit endosomal signalling, for other GPCRs, compartmentalised signalling does not arise from endocytic compartments but, rather, from different locations ‘within’ the plasma membrane. We have recently used cAMP (Epac2camps), ERK (EKAR) and PKC (CKAR) biosensors to delineate the spatiotemporal dynamics of activation of the mu-opioid receptor (MOR) in model cell lines and in primary cultures of dorsal root ganglion neurons [25]. These results provide a new mechanism to explain the differential signalling between two different MOR ligands as they suggest that events controlling lateral mobility within the plasma membrane, rather than internalisation, govern MOR signalling profiles. DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin) activation of MOR does not activate PKC, but triggered fast receptor translocation within the plasma membrane. This translocation precedes trafficking to clathrin-containing domains and internalisation, and is dependent on receptor phosphorylation (as MOR phosphorylation mutants show altered plasma membrane distribution). It is this MOR translocation, and not internalisation, that determines the transient cytosolic ERK profile and the activation of nuclear ERK. By contrast, morphine activates plasma membrane-localised PKC, via Gbg subunits of the G protein, which prevents
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receptor translocation within the plasma membrane. This results in sustained cytosolic ERK and no nuclear ERK activity. Inhibition of Gbg or PKC allows the morphine-activated MOR to translocate within the plasma membrane and transforms its spatiotemporal signalling profile (Figure 3C). Importantly, this new signalling profile mimics that of the internalising ligand DAMGO (i.e., transient cytosolic and nuclear ERK) but occurs in the absence of b-arrestin recruitment and without receptor internalisation. Finally, the combination of GPCRs as sensing proteins with genetically encoded fluorescent reporters provides an elegant alternative to monitor hormone release in situ and in vivo. For example, FRET sensors have been used to detect and measure the release of neurotransmitters in the brain. CNiFERs are clonal cell lines engineered to express a Gaq-coupled GPCR, which triggers an increase in intracellular calcium, and a genetically encoded FRET-based calcium sensor [35,36]. Thus, the system transforms specific neurotransmitter receptor binding to a change in FRET, providing a real-time optical readout of local neurotransmitter activity. This approach bypasses a major obstacle in neuroscience: the inability to detect the release of some monoaminergic transmitters in vivo with sufficient chemical specificity, and spatial and temporal resolution.
Outstanding Questions Technological Challenges Can we measure multiple GPCRmediated responses by multiplexing FRET biosensors? Can we use FRET biosensors in living animals? Molecular Mechanisms of Compartmentalised Signalling What are the general and GPCR-specific mechanisms controlling the dynamics of subcellular responses? What are the components of the receptor-mediated platforms that allow the generation of compartmentalised signalling?
Concluding Remarks The generation of an expanding toolbox of genetically encoded FRET sensors and of transgenic mice expressing such sensors (Box 2) represents the next step in the use of FRET biosensors for advancing our understanding of the spatiotemporal regulation of signal transduction in relevant biological contexts (see Outstanding Questions). Together with the fast advances in imaging approaches, these sensors will illuminate the intricacies of intracellular signalling to unprecedented resolution. FRET biosensors have been instrumental in understanding how the importance of the location of GPCRs and their signalling effectors can control biological responses. Consequently, understanding the spatiotemporal control of GPCR signalling will
How does GPCR trafficking affect compartmentalised signalling? Physiological Relevance of Compartmentalised Signalling Is the compartmentalised signalling elicited by a specific GPCR cell type[184_TD$IF][0dependent? Is compartmentalised altered upon disease?
signalling
Box 2. Transgenic Mice Expressing FRET Biosensors The study of the physiological relevance of signal compartmentalisation requires the expression of FRET sensors in native systems. This represents a substantial hurdle particularly in cells that are not easy to transfect/nucleofect. While a strategy to circumvent this issue is to use viral expression systems, several groups have started generating transgenic mice that will be of undoubted utility to understand the relevance of spatiotemporal signalling for physiology and disease. The generation in 2009 of a transgenic mouse with ubiquitous expression of the highly sensitive FRET-based cAMP biosensor Epac1-camps [29] revealed the control of cAMP signalling from endocytosed TSH receptors in native thyroid follicles [29] and of the somatostatin receptor in live pituitary [39]. The Matsuda laboratory has generated transgenic mice that express biosensors for cytosolic and nuclear ERK and PKA and demonstrated their activity in various cell types and tissues including peritoneal macrophages, skin and small intestine. These mouse lines have been used more recently to investigate the dynamic modulation of PKA and ERK in the striatum [40], and to visualise ERK signal propagation in vivo in mouse skin [41] during thrombus formation [42] and during leukocyte activation [43]. The Raichu-Rac biosensor allows to visualise activation of the small G protein Rac, which is an essential controller of actin cytoskeletal dynamics. The Rac-FRET transgenic mouse has revealed the tight spatiotemporal control of Rac signalling during neutrophil chemotaxis, tissue homeostasis, disease initiation and disease progression [44]. Although this sensor has yet to be used in the context of GPCR signalling, it is very likely to prove of invaluable significance to understand how GPCRs control cell morphology and migration. The ability to study GPCR signalling in native systems using transgenic mice expressing FRET biosensors in specific tissues or cell populations represents one of the most exciting avenues for future research into the spatiotemporal resolution of receptor activity. It is therefore very likely that, in the next years, the number of ‘reporter’ mice expressing sensors of different signalling effectors will increase and provide fascinating insights into GPCR biology.
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also impact on our current interpretation of novel concepts of GPCR pharmacology and on the development of new therapeutic strategies to target this family of receptors. For example, the concept of biased agonism (whereby different receptor ligands binding to the same receptor in an identical cellular background to selectively activate a distinct subset of intracellular responses) should now be extended to incorporate the ‘location’ and ‘duration’ of such responses. Moreover, the classical view of GPCR desensitisation, that is, the switching off receptor signalling at the plasma membrane, will also need revisiting and investigation of the mechanisms switching off receptors in other subcellular compartments will need to be investigated. Finally, recognition that GPCRs can signal from multiple intracellular locations will provide novel strategies that may improve the effectiveness of GPCR-based drug development. Indeed, selective targeting of GPCR antagonists to endosomes has been already proven to effectively block pain transmission, providing the proof of concept of the importance of not only targeting the right receptor, but also targeting it in the right location [34]. The understanding of the when and where of GPCR signalling will allow for the specific targeting of these important drug targets, at the right time and in the right place. Thus, FRET biosensors will contribute to the development of novel strategies of targeting this important family of receptors. Acknowledgements Work in the authors’ labs is supported by a Monash Fellowship to M.C., a National Health and Medical Research Council (NHMRC) RD Wright Fellowship to M.L.H. (1061687); NHMRC Project Grants (1121029, 1083054) to M.C. and M.L.H.; and by the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology.
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21. Itoh, R.E. et al. (2002) Activation of Rac and Cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes inthemembraneof livingcells. Mol.Cell.Biol.22, 6582–6591 22. Wachten, S. et al. (2010) Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells. J. Cell Sci. 123, 95–106
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Review
Genetically Encoded FRET Biosensors to Illuminate Compartmentalised GPCR Signalling Michelle L. Halls1,* and Meritxell Canals1,* Genetically encoded Förster resonance energy transfer (FRET) biosensors have been instrumental to our understanding of how intracellular signalling is organised and regulated within cells. In the last decade, the toolbox, dynamic range and applications of these sensors have expanded beyond basic cell biology applications. In particular, FRET biosensors have shed light onto the mechanisms that control the intracellular organisation of G protein-coupled receptor (GPCR) signalling and have allowed the visualisation of signalling events with unprecedented temporal and spatial resolution. Here we review the use of these sensors in the GPCR field and how it has already provided invaluable advances towards our understanding of the complexity of GPCR signalling.
Trends Genetically encoded FRET sensors have been instrumental to understand compartmentalised signalling. FRET-based biosensors offer increased spatial and temporal resolution over traditional cell-based signalling assays and allow the discrimination between the responses of different cell populations. The presence of GPCRs and key signalling effectors in subcellular microdomains governs the assembly of signalling complexes that generate compartmentalised signalling and is a key determinant of signal selectivity.
G Protein-Coupled Receptors With more than 800 members, G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors in the human genome. They participate in virtually every physiological process and are the target of more than one-third of current medicines. Functionally, GPCRs transduce a wide range of extracellular stimuli (ranging from light and small ions to bigger molecules and peptides) into various intracellular signalling pathways via coupling to heterotrimeric G proteins or to other GPCR-interacting proteins. The fact that different receptors that activate common signalling pathways in the same cell exert divergent physiological effects suggests a high degree of organisation and regulation of intracellular signalling. Spatiotemporal compartmentalisation – the restriction of second messengers in space and time – provides a mechanism whereby GPCRs can direct the assembly of focused ‘platforms’ for specific signalling. These signalling platforms facilitate second messenger production, the organisation and scaffolding of effectors, and co-ordination of regulatory elements. In this context, genetically encoded Förster resonance energy transfer (FRET) biosensors have proved as invaluable tools for visualising GPCR signalling dynamics with high spatial and temporal resolution in living systems as they circumvent the need for cell lysis and allow for real-time monitoring of signalling events. Here, we will provide an overview of FRET-based biosensors and review how the use of these sensors in the GPCR field has already provided significant breakthroughs in our understanding of the complexity and distinct physiological consequences of GPCR signalling. (For a review of the use of bioluminescence resonance energy transferbased biosensors to investigate GPCR signalling and trafficking, see Bouvier et al. in this issue.)
Compartmentalisation of Intracellular Signalling The idea of compartmentalised signalling, while initially proposed by the cAMP field [1], was first directly demonstrated for calcium signalling due to the availability of high-affinity calcium-
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The spatial and temporal control of compartmentalised signalling defines the physiological outcomes of GPCR activation.
1
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia
*Correspondence: [email protected] (M.L. Halls) and [email protected] (M. Canals).
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sensitive fluorescent dyes, such as fura-2. These initial imaging studies revealed sparks, puffs and blinks of calcium within the cell (reviewed in [2,3]), and demonstrated compartmentalised signalling at the most basic level – high levels of a second messenger at the site of production, with a steep decline in concentration to ambient levels in the rest of the cell. The more recent development of genetically encoded biosensors to detect changes in cAMP levels and kinase activity has further refined our ideas of signal compartmentalisation. While the restricted localisation of the source of a second messenger can give rise to localised ‘hot spots’ of signalling in bulk areas of the cell, mechanisms that control the lifetime or the diffusion of such second messenger can create more specialised signalling hubs that have greater spatial and temporal flexibility. For example, cAMP compartmentalisation is often controlled by the close proximity of the enzymes that break down cAMP [phosphodiesterases (PDEs)] to the enzymes that produce it [adenylyl cyclases (ACs)] to create highly localised elevations of the second messenger [4–7]. Subsequent manipulation of these biosensors to target them to discrete cellular domains (e.g., plasma membrane vs. cytosol) or to individual proteins (e.g., ACs, PDEs) has demonstrated the importance of scaffolding proteins for assembling focused signalling hubs [1,8,9]. For cAMP signalling, the most studied and iconic scaffolding proteins are A-kinase anchoring proteins (AKAPs); originally identified for their ability to bind and regulate protein kinase A (PKA), AKAPs are now recognised as multimodal scaffolds that control the compartmentalisation of cAMP signalling by scaffolding activators [e.g., ACs, protein kinase C (PKC)], regulators (e.g., PDEs) and effectors (e.g., PKA, Epac, ion channels) [2,3,10,11]. This ensures high fidelity of the second messenger signal, with the signalling hubs therefore dictating not only the nature of the cAMP signal, but also its cellular consequences. It is now well recognised that compartmentalisation of signalling is often dependent on the formation of higher-order protein complexes to allow the efficient and specific scaffolding of proteins that activate and regulate key second messengers [4–7,12,13].
FRET Biosensors for Single-Cell and Subcellular Resolution of Signalling Events Most biosensors that are currently available rely on FRET. In this approach, a peptide chain containing the ‘sensor’ of the second messenger (e.g., second messenger binding site or target peptide) is fused to a complementary pair of fluorophores with overlapping spectra. Energy transfer between these two fluorophores depends on their close proximity and alignment. Binding of a second messenger, or modification of the target peptide, triggers a conformational change of the sensor peptide chain that alters the distance between these two fluorophores, thus changing the FRET signal (Figure 1). Typically, cyan fluorescent protein and YFP are used as the FRET pair, although biosensors utilising GFP and red fluorescent protein are becoming more common (which also allows for multiplexing of complementary signalling biosensors), as are the use of variants engineered to be less sensitive to fluctuations in pH (e.g., mCitrine [14]), which is particularly important for the use of these biosensors in primary cells such as neurons. Importantly, in ratiometric FRET biosensors the effect on the FRET signal due to changes in expression levels between experiments or even cellular compartments is eliminated, as the two fluorescent proteins are attached to the same sensor, and therefore are expressed as a single polypeptide. Finally, it is important to highlight that different FRET biosensors that detect the same second messenger can have a different dynamic range (Box 1). Ratiometric biosensors that directly detect second messengers (e.g., the Epac2-based biosensor that detects cAMP [15] or the cGES-DE5 sensor that detects cGMP [16]) undergo a conformational change upon the binding of the second messenger, leading to a change in the FRET signal. Ratiometric FRET biosensors to detect kinase activity – for example, A kinase
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Figure 1. FRET Biosensor Strategies. FRET biosensors rely on a conformational change as a result of (A) binding of a second messenger (e.g., cAMP) to a second messenger-sensitive-binding domain (e.g., cAMP-binding domain), (B) phosphorylation of a substrate peptide with a consensus target sequence for specific kinases that is recognised by a phospho-binding domain (e.g., EKAR sensor [18]) or (C) exchange of GDP for GTP that is recognised by another binding domain (e.g., RaichuRac1 sensor [21]). [18_TD$IF]Abbreviations: CFP, cyan fluorescent protein; EKAR, extracellular signal-regulated kinase activity reporter; ERK, extracellular signal-regulated kinase; FRET, Förster resonance energy transfer.
activity reporter (AKAR) for PKA; extracellular signal-regulated kinase activity reporter (EKAR) for extracellular signal-regulated kinase (ERK); or C kinase activity reporter (CKAR) for PKC [17–20] – have slightly different mechanisms of action. While the principle of conformational change within the biosensor is still utilised, this change is induced following phosphorylation of a target sequence by the corresponding kinase, which facilitates an interaction between the phosphorylated target sequence and an attached binding protein that is sensitive to such
Box 1. Dynamic Range of Fluorescent Biosensors FRET biosensors are a highly sensitive technique for measuring intracellular signalling, and can detect smaller responses than conventional, population-based signalling assays. However, this increased sensitivity comes with limitations in their dynamic range; a by-product of sensitive detection is often a decreased upper detection limit. It is therefore important to determine the absolute sensitivity and dynamic range of a particular biosensor in each system. Different FRET biosensors for the same target can have differences in their dynamic range. For example, two FRET biosensors that detect cAMP, Epac2-camps [15] and ICUE2 [37], actually detect different concentration ranges of cAMP. The sensitivity of the biosensors differs by an order of magnitude (Epac2-camps, EC50 of 0.92 mM [15]; ICUE2, EC50 of 12.5 mM [37]). As such, while ICUE2 can detect an order of magnitude higher peak cAMP than Epac2-camps, Epac2-camps is more suited to detecting very small increases in cAMP. Although these are important considerations to take into account when choosing an appropriate FRET biosensor for single-cell imaging studies, the different dynamic range of sensors for the same second messenger also expands the utility of these tools. It is also important to note that modifications of the same biosensor to enable targeting to distinct cellular domains can impact biosensor sensitivity. GCaMP2 is a genetically encoded high-affinity Ca2+ biosensor [38]. It contains a circularly permuted eGFP, flanked by calmodulin (a Ca2+-binding protein) and a calmodulin-binding peptide, M13. Increases in Ca2+ drive an interaction between Ca2+/calmodulin and the M13 peptide, leading to a conformational change in the biosensor and an increase in GFP fluorescence. GCaMP2, originally expressed within the cytosol, has been targeted to various intracellular domains and proteins, including to the plasma membrane by palmitoylation/myristoylation [9], to the N terminus of AC2 and AC8 [9] and to the a-subunit of the Na2+ pump [11]. Addition of the targeting sequences to GCaMP2 causes small changes in the sensitivity of the biosensor to Ca2+ [9,11,38]. Again, this highlights the importance of understanding and assessing the dynamic range of each biosensor. Similar changes in potency have been observed following targeting of the cAMP FRET biosensor, Epac2-camps [15]. The original Epac2-camps (expressed within the cytosol) has an EC50 for cAMP of 0.4 mM, and this sensitivity was decreased following targeting to the plasma membrane (pmEpac2-camps, EC50 of 1.7 mM) or to the N terminus of AC8 (Epac2-AC8, EC50 of 2.2 mM) [22]. Although these are relatively small changes (i.e., GCaMP2-AC8 drops the sensitivity to Ca2+ by 1.5-fold, and Epac2-AC8 drops sensitivity to cAMP by fourfold), it does require caution when interpreting data obtained from the same biosensor targeted to a distinct location.
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phosphorylation. In this case, the FRET biosensor can be considered a substrate for the second messenger in question. The Rho G protein FRET biosensors (e.g., Raichu-Rac1, Raichu-Cdc42 [21]) utilise yet another slightly different mechanism of conformational change. In this case, the biosensor contains the Rho G protein of interest, and the exchange of GDP/GTP [by guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity] allows the GTP-bound protein to interact with an adjacent binding partner, causing the conformational change to alter FRET within the biosensor. Importantly, all the aforementioned conformational changes are reversible, such that dynamic increases and decreases in the second messenger can be accurately measured (Figure 1). Many of these biosensors have been subsequently modified to incorporate subcellular targeting sequences – for example, the original cAMP biosensors were expressed within the cytosol of the cell, but have since been targeted to the plasma membrane [8], to proteins including ACs [8,9,22] and PDEs [10], and intracellular organelles such as the nucleus and mitochondria [23]. The comparison of FRET signals at different sites within the cell, in response to the same stimulus, has been instrumental in advancing our understanding of how compartmentalised signals are controlled (Box 1).
Advantages of Single-Cell Resolution of Signalling Events In traditional cell-based signalling assays, tens to hundreds of thousands of cells are seeded in multiwell plates, incubated with a stimulus and then the stimulation is ‘stopped’ at a predetermined time typically requiring cell lysis. The second messenger of interest is then extracted from the samples and measured by antibody, radioimmunoassay, immunoblotting, etc. Continuous measurements are obviously not possible in such assays, with time-course approximations often limited in resolution to minutes. These measurements detect, by default, the average response of a cell population, so that they can be considered population (as opposed to single-cell)-based signalling assays. Live cell imaging of FRET biosensors is noninvasive and nondestructive, and is only limited by the capture speed of the camera, thus providing increased temporal resolution over more traditional cell-based signalling assays. However, the greatest advantage of these sensors is the ability to discriminate between subpopulations of cells. This is important as a large variation in signalling responses to a stimulus can be observed within a single-cell population and is evident not only in cultures of isolated primary cells (which often consist of a mixed cell populations) but also in cell lines with endogenous receptor expression (likely dependent on the relative level of expression of a particular receptor at the single-cell level). The ability of FRET biosensors to discriminate between subpopulations of cells provides an additional level of information that would otherwise be lost in population-based signalling assays, where the total response reflects a composite of many individual responses [24]. This additional resolution can be utilised in a number of ways: for example, by assessing the ability of an inhibitor to change relative populations exhibiting different responses, that is, from transient to sustained [25] (Figure 2), or by poststaining of a primary cell culture with heterogeneous cell composition and analysis of the different cell types or of only the cells that express the receptor of interest.
Compartmentalised GPCR Signalling Revealed Using FRET Biosensors Subcellularly targeted FRET biosensors have proved extremely useful for the study of GPCR signalling. Moreover, the increased resolution provided by real-time detection of signalling events using sensors specifically targeted to different cellular compartments has challenged some of the classical views of GPCR signalling. For example, the high sensitivity of FRET biosensors to detect signal transduction and second messenger levels has allowed the detection of previously unappreciated signals. Halls and Cooper [26] used the cAMP Epac2-camps FRET biosensor targeted to the plasma membrane or the cytosol to investigate
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Figure 2. FRET Biosensors Allow Discrimination of Responses in Different Subpopulations of Cells. The ability to assess signalling at a single-cell level provides the resolution to separate cellular populations according to their responses. In the example, the response of the MOR agonist morphine in dorsal root ganglia (DRG) neurons expressing the cytosolic ERK FRET biosensor (cytoEKAR) was measured in the absence (A) or presence (B) of a PKC inhibitor. Analysis of single-cell responses shows a shift of the relative population of cells responding in a transient fashion to this agonist [25]. [182_TD$IF]Abbreviations: EKAR, extracellular signal-regulated kinase activity reporter; ERK, extracellular signal-regulated kinase; FRET, Förster resonance energy transfer; MOR, mu-opioid receptor; PKC, protein kinase C.
compartmentalised cAMP signalling of the hormone relaxin. These experiments unveiled that the relaxin receptor, RXFP1, forms a preassembled signalosome that provides a unique regulatory mechanism for the extremely low levels of relaxin found in circulation (Figure 3A). This constitutively assembled RXFP1-signalosome is formed by both stimulatory (AKAP79 and AC2) and regulatory (b-arrestin2, PDE4D3 and PKA) components that interact with the receptor in two defined regions of the C-terminal tail and allow the activation of the receptor by subpicomolar concentrations of peptide. FRET biosensors have also been instrumental to reveal the importance signal compartmentalisation for the generation of specific cellular responses upon GPCR activation. Nikolaev et al. [27] generated a transgenic mouse with the cAMP FRET biosensor HCN2-camps and visualised the differences in cAMP signalling elicited by b1- and b2-adrenoceptors (ARs) in isolated adult cardiac myocytes. While the b1ARs and b2ARs are the main AR subtypes controlling
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Figure 3. Illuminating GPCR Intracellular Signalling. Genetically encoded FRET biosensors have allowed the detection of signalling events with unprecedented temporal and spatial resolution. (A) The plasma membrane-targeted cAMP FRET sensor (pmEpac2) allowed the detection of responses to subpicomolar concentrations of relaxin mediated by its receptor, RXFP1 [26]. (B) Substance P activation of the NK1R induces a nuclear ERK response (nucEKAR) that is dependent on receptor internalisation as it is blocked by the clathrin-inhibitor Pitstop 2 but not its inactive analogue [34]. (C) Inhibition of the Gbg subunits of the G protein with bARKct changes the nuclear ERK profile (nucEKAR) of the mu-opioid receptor (MOR) when activated by morphine [25]. [182_TD$IF]Abbreviations: DMSO, dimethyl sulfoxide; EKAR, extracellular signal-regulated kinase activity reporter; ERK, extracellular signal-regulated kinase; FRET, Förster resonance energy transfer; GPCR, G protein-coupled receptor; NK1R, neurokinin 1 receptor.
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cardiac response, only b1AR stimulation seems to induce cardiomyocyte hypertrophy and apoptosis. The use of the cAMP FRET sensor allowed the visualisation of key differences in the propagation of the cAMP signal by the two receptor subtypes. While b1ARs induce farreaching cAMP signals that propagate over distances including multiple sarcomeres, the cAMP signal evoked by the b2AR is far more localised and does not propagate throughout the cell. Genetically encoded biosensors have also been used to assess the relevance of GPCR localisation within the cell for its downstream signalling. Indeed, GPCRs have now been described to signal from different regions of the plasma membrane as well as from other subcellular compartments such as the endosomes, the nucleus or even the mitochondria. In this context, several groups have used biosensors in combination with pharmacological and genetic strategies to assess the importance of changes in GPCR location for the generation of specific cellular responses. Vincent et al. [28] showed that the intracellular metabotropic glutamate receptor, mGluR5, in spinal neurons plays a critical role in neuropathic pain. They showed that in spinal dorsal horn neurons, mGluR5 is located on nuclear membranes and that nuclear expression of this receptor increases upon nerve injury. Using a genetically encoded calcium indicator specifically targeted to the cell nucleus (pCMV-NL-S-R-GECO), the authors showed that stimulation of the intracellular mGluR5 leads to sustained nuclear calcium responses. Blockade of these responses with permeant antagonists or decreasing the levels of intracellular glutamate reduces neuropathic pain behaviours and Fos expression. Genetically encoded RET biosensors have also provided spatial and temporal resolution that biochemical measurements of GPCR signalling previously lacked. This major advantage has been instrumental for one of the concepts that has revolutionised the GPCR field in the last decade: that some internalised GPCRs remain active and exhibit sustained G-protein-dependent signalling. As the list of GPCRs that exhibit sustained endosomal signalling is still growing, here we choose to highlight some examples that have employed biosensors to investigate this phenomenon. The FRET sensors most widely used to investigate endosomal signalling by GPCRs are those that detect cAMP changes. This is mainly due to the fact that most receptors identified to date that signal once internalised are coupled to Gas G proteins and therefore, activate AC. In 2009, two groups showed the importance of receptor internalisation for the generation of sustained cAMP signals. Calebiro et al. [29] generated a transgenic mouse with ubiquitous expression of the fluorescent sensor for cAMP CAG-Epac1-camps and studied cAMP responses to thyroidstimulating hormone (TSH) in native thyroid follicles isolated from these mice. In this system, using endocytic inhibitors as well as subcellular fractionation approaches, they demonstrated that TSH receptor internalisation is responsible for the sustained cAMP signalling observed upon receptor stimulation. Furthermore, they also demonstrated that such cAMP signalling elicited by internalised receptors is responsible for the depolymerisation of actin in response to TSH in mouse thyroid cells, hence suggesting that signalling from endocytosed receptors has important functional consequences. In another study using the same cAMP biosensor, Ferrandon et al. [30] showed that the parathyroid hormone receptor (PTHR) behaves in a similar fashion as the TSH receptor. Using PTH-derived peptides they demonstrated that long-acting ligands interact tightly with the receptor in a conformationally dependent manner. The receptor is then locked into a prolonged active state inducing sustained receptor–G-protein coupling and sustained G-protein activation. They also showed that the ternary ligand–receptor–Gprotein complex is preserved during internalisation and persists over time to appear as a key structure in the prolonged downstream signalling of PTH-like hormone.
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Generation of cAMP by the b2AR occurs partly within the endocytic compartments and, importantly, the localisation of cAMP pools has key downstream consequences [31]. Although these studies did not use FRET-based but instead a luciferase-based sensors (GloSensor) to delineate these mechanisms, they represented a key turning point in our understanding of GPCR signalling. In 2013, Irannejad et al. [32] generated conformational biosensors to detect agonist-activated b2AR and activated Gas protein to demonstrate the existence of an active b2AR-Gas complex within the endosomes. Later the same group showed that the full repertoire of cAMP-dependent transcriptional responses requires b2AR endocytosis [31]. To demonstrate that cells are able to discriminate the location of cAMP generation when initiating a signalling response, they generated an elegant optogenetic strategy based on a bacteria-derived AC, which allowed cAMP production to be induced by light [bacterial photoactivated adenylyl cyclase (bPAC)]. By specifically targeting and activating bPAC at the plasma membrane, the endosomes or the cytosol, they showed that localised cAMP production generates distinct transcriptional responses. Interestingly, Jean-Alphonse et al. [33] have recently proposed a new mechanism through which b2ARs promote the formation of cAMP in endosomes. Using intramolecular and intermolecular FRET sensors the authors propose that b2AR activation, via isoproterenol, enhances and prolongs signalling by the PTHR in response to PTH(1–34) by releasing noncatalytic Gbg subunits. This augmentation of the pool of free Gbg promotes the assembly and functional activity of signalling complexes composed of PTHR, Gbg, Gas and AC2, which persist in a functional state in endosomes. Whether this is the only mechanism of b2AR-mediated endosomal signalling remains to be addressed. An example of a non-Gas-coupled receptor that exhibits signalling once internalised is the neurokinin 1 receptor (NK1R). Jensen et al. [34] have recently used FRET biosensors to detect cytosolic and plasma membrane cAMP (Epac2-camps), cytosolic and plasma membrane PKC activation (CKAR), and cytosolic and nuclear ERK activation (EKAR) to study the link between NK1R endocytosis and signalling in subcellular compartments with high spatiotemporal fidelity. Activation of the Gaq/11-coupled NK1R by the neuropeptide substance P exhibits a repertoire of compartmentalised signalling responses and some of these responses are dependent on receptor endocytosis. Moreover, the endosomal NK1R signalling platform is essential for sustained pain transmission. The use of genetic and pharmacological tools to prevent receptor internalisation as well as the generation of endosomally targeted antagonists for the NK1R demonstrated the dependence of nuclear ERK, cytosolic cAMP and cytosolic PKC on receptor endocytosis (Figure 3B). Moreover, these results were translated to spinal cord slices and to animal models of inflammatory and noninflammatory pain to demonstrate the importance of internalised NK1R signalling for (patho)physiological processes. Although, as shown earlier, some GPCRs exhibit endosomal signalling, for other GPCRs, compartmentalised signalling does not arise from endocytic compartments but, rather, from different locations ‘within’ the plasma membrane. We have recently used cAMP (Epac2camps), ERK (EKAR) and PKC (CKAR) biosensors to delineate the spatiotemporal dynamics of activation of the mu-opioid receptor (MOR) in model cell lines and in primary cultures of dorsal root ganglion neurons [25]. These results provide a new mechanism to explain the differential signalling between two different MOR ligands as they suggest that events controlling lateral mobility within the plasma membrane, rather than internalisation, govern MOR signalling profiles. DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin) activation of MOR does not activate PKC, but triggered fast receptor translocation within the plasma membrane. This translocation precedes trafficking to clathrin-containing domains and internalisation, and is dependent on receptor phosphorylation (as MOR phosphorylation mutants show altered plasma membrane distribution). It is this MOR translocation, and not internalisation, that determines the transient cytosolic ERK profile and the activation of nuclear ERK. By contrast, morphine activates plasma membrane-localised PKC, via Gbg subunits of the G protein, which prevents
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receptor translocation within the plasma membrane. This results in sustained cytosolic ERK and no nuclear ERK activity. Inhibition of Gbg or PKC allows the morphine-activated MOR to translocate within the plasma membrane and transforms its spatiotemporal signalling profile (Figure 3C). Importantly, this new signalling profile mimics that of the internalising ligand DAMGO (i.e., transient cytosolic and nuclear ERK) but occurs in the absence of b-arrestin recruitment and without receptor internalisation. Finally, the combination of GPCRs as sensing proteins with genetically encoded fluorescent reporters provides an elegant alternative to monitor hormone release in situ and in vivo. For example, FRET sensors have been used to detect and measure the release of neurotransmitters in the brain. CNiFERs are clonal cell lines engineered to express a Gaq-coupled GPCR, which triggers an increase in intracellular calcium, and a genetically encoded FRET-based calcium sensor [35,36]. Thus, the system transforms specific neurotransmitter receptor binding to a change in FRET, providing a real-time optical readout of local neurotransmitter activity. This approach bypasses a major obstacle in neuroscience: the inability to detect the release of some monoaminergic transmitters in vivo with sufficient chemical specificity, and spatial and temporal resolution.
Outstanding Questions Technological Challenges Can we measure multiple GPCRmediated responses by multiplexing FRET biosensors? Can we use FRET biosensors in living animals? Molecular Mechanisms of Compartmentalised Signalling What are the general and GPCR-specific mechanisms controlling the dynamics of subcellular responses? What are the components of the receptor-mediated platforms that allow the generation of compartmentalised signalling?
Concluding Remarks The generation of an expanding toolbox of genetically encoded FRET sensors and of transgenic mice expressing such sensors (Box 2) represents the next step in the use of FRET biosensors for advancing our understanding of the spatiotemporal regulation of signal transduction in relevant biological contexts (see Outstanding Questions). Together with the fast advances in imaging approaches, these sensors will illuminate the intricacies of intracellular signalling to unprecedented resolution. FRET biosensors have been instrumental in understanding how the importance of the location of GPCRs and their signalling effectors can control biological responses. Consequently, understanding the spatiotemporal control of GPCR signalling will
How does GPCR trafficking affect compartmentalised signalling? Physiological Relevance of Compartmentalised Signalling Is the compartmentalised signalling elicited by a specific GPCR cell type[184_TD$IF][0dependent? Is compartmentalised altered upon disease?
signalling
Box 2. Transgenic Mice Expressing FRET Biosensors The study of the physiological relevance of signal compartmentalisation requires the expression of FRET sensors in native systems. This represents a substantial hurdle particularly in cells that are not easy to transfect/nucleofect. While a strategy to circumvent this issue is to use viral expression systems, several groups have started generating transgenic mice that will be of undoubted utility to understand the relevance of spatiotemporal signalling for physiology and disease. The generation in 2009 of a transgenic mouse with ubiquitous expression of the highly sensitive FRET-based cAMP biosensor Epac1-camps [29] revealed the control of cAMP signalling from endocytosed TSH receptors in native thyroid follicles [29] and of the somatostatin receptor in live pituitary [39]. The Matsuda laboratory has generated transgenic mice that express biosensors for cytosolic and nuclear ERK and PKA and demonstrated their activity in various cell types and tissues including peritoneal macrophages, skin and small intestine. These mouse lines have been used more recently to investigate the dynamic modulation of PKA and ERK in the striatum [40], and to visualise ERK signal propagation in vivo in mouse skin [41] during thrombus formation [42] and during leukocyte activation [43]. The Raichu-Rac biosensor allows to visualise activation of the small G protein Rac, which is an essential controller of actin cytoskeletal dynamics. The Rac-FRET transgenic mouse has revealed the tight spatiotemporal control of Rac signalling during neutrophil chemotaxis, tissue homeostasis, disease initiation and disease progression [44]. Although this sensor has yet to be used in the context of GPCR signalling, it is very likely to prove of invaluable significance to understand how GPCRs control cell morphology and migration. The ability to study GPCR signalling in native systems using transgenic mice expressing FRET biosensors in specific tissues or cell populations represents one of the most exciting avenues for future research into the spatiotemporal resolution of receptor activity. It is therefore very likely that, in the next years, the number of ‘reporter’ mice expressing sensors of different signalling effectors will increase and provide fascinating insights into GPCR biology.
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also impact on our current interpretation of novel concepts of GPCR pharmacology and on the development of new therapeutic strategies to target this family of receptors. For example, the concept of biased agonism (whereby different receptor ligands binding to the same receptor in an identical cellular background to selectively activate a distinct subset of intracellular responses) should now be extended to incorporate the ‘location’ and ‘duration’ of such responses. Moreover, the classical view of GPCR desensitisation, that is, the switching off receptor signalling at the plasma membrane, will also need revisiting and investigation of the mechanisms switching off receptors in other subcellular compartments will need to be investigated. Finally, recognition that GPCRs can signal from multiple intracellular locations will provide novel strategies that may improve the effectiveness of GPCR-based drug development. Indeed, selective targeting of GPCR antagonists to endosomes has been already proven to effectively block pain transmission, providing the proof of concept of the importance of not only targeting the right receptor, but also targeting it in the right location [34]. The understanding of the when and where of GPCR signalling will allow for the specific targeting of these important drug targets, at the right time and in the right place. Thus, FRET biosensors will contribute to the development of novel strategies of targeting this important family of receptors. Acknowledgements Work in the authors’ labs is supported by a Monash Fellowship to M.C., a National Health and Medical Research Council (NHMRC) RD Wright Fellowship to M.L.H. (1061687); NHMRC Project Grants (1121029, 1083054) to M.C. and M.L.H.; and by the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology.
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