Colette Denis
email: <colette.denis@inserm.fr

Aude Sauli

Gol Ne Galandrin

Probing heterotrimeric G protein activation: applications to biased ligands

Keywords: Animals, Drug Discovery, methods, Heterotrimeric GTP-Binding Proteins, metabolism, Humans, Ligands, Receptor Cross-Talk, Receptors, G-Protein-Coupled, agonists, metabolism, Signal Transduction GPCRs, G protein, biased agonist, ligand-directed trafficking, ligand efficacy, G protein sensors, signaling pathways

Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of ligand-directed trafficking has led to a conceptual revolution in pharmacological theory, thus opening " " new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting activation of specific signals and thus generating textured responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligand responses. However, considering that a single receptor has pleiotropic signalling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even if some G protein-independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discuss them in the context of G protein biased -ligands.

INTRODUCTION

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptors and are virtually involved in the regulation of all physiological processes. They represent therefore a primary target for modern drugs development with potential application in all clinical fields. It has been estimated that some 30 50 of clinically available drugs target the function of GPCR family -%

members

. These receptors propagate highly diverse extracellular signals into the cell interior by interacting with a broad range of [1 -3 ] intracellular proteins . However, coupling with -trimeric G proteins remains the common hallmark of all GPCR family members and [START_REF] Ritter | Fine-tuning of GPCR activity by receptor-interacting proteins[END_REF] αβγ these proteins constitute one of the earliest plasma membrane transducers, relaying information from the cell surface receptor to others intracellular signaling molecules. Recently, GPCRs were found not to work linearly as simple on/off switches, triggering the full signaling machinery downstream the receptor, but rather as filters capable of activating a subset of specific effectors and fine tuning cellular responses and associated physiological responses. This concept, known as ligand-directed trafficking or biased-agonism , emphasizes

" " " "
that receptors are capable of generating textured responses to ligands . On the other hand, the efficacy of ligands acting on GPCRs may [START_REF] Kenakin | Agonist-receptor efficacy. II. Agonist trafficking of receptor signals[END_REF] be different depending upon the cellular effector considered , . In keeping with these different concepts, it follows that the choice of [6 7 ] cellular effectors to measure receptor activation is crucial and that different signaling pathways should be considered in order to appreciate the real texture of ligand effects. However, pleiotropic and crosstalk signaling between GPCRs makes functional selectivity of ligands difficult to decode. One alternative to bypass this problem might be to look at the initial step following agonist binding to receptors at the level of the plasma membrane: i.e. G protein activation. This review will focus on the heterotrimeric G proteins with a specific emphasis on the different tools available to evaluate receptor-mediated G protein activation.

HETEROTRIMERICG PROTEINS STRUCTURE

The discovery of heterotrimeric G proteins relaying information from receptors inserted in plasma membrane to intracellular effectors revolutionized our view of how ligands functions. Alfred G. Gilman and Martin Rodbell were jointly awarded the Nobel Prize in Physiology or Medicine in 1994, , .

for their discovery of G-proteins and the role of these proteins in signal transduction in cells and post-translational processing, leading to up to 23 different G subunits isoforms. Even if theoretically more than one thousand of α distinct heterotrimers may exist, it has been shown that all combinations may not be relevant in signal transduction . Moreover, the [START_REF] Hynes | Live cell imaging of Gs and the beta2-adrenergic receptor demonstrates that both alphas and beta1gamma7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the beta2-adrenergic receptor[END_REF] nature of the heterotrimer depends on the cell type . [START_REF] Dupre | The role of Gbetagamma subunits in the organization, assembly, and function of GPCR signaling complexes[END_REF] The G subunit ( ) is composed of an intrinsic GTPase domain involved in GTP binding and hydrolysis but also in interactions α Fig. [START_REF] Hopkins | The druggable genome[END_REF] with G subunits, receptor and effectors . This domain is characterized by three flexible loops identified as switches I, II and III and βγ [START_REF] Oldham | Heterotrimeric G protein activation by G-protein-coupled receptors[END_REF] regulates G protein activation through very subtle conformational rearrangements. The subunit exhibits an additional helical domain α connected to the GTPase domain by the flexible linker 1 acting as a lid over the nucleotide binding pocket , . All G subunits [14 15 ] α (except G ) are palmitoylated and/or myristoylated at their N-terminus allowing anchor age to the plasma membrane. the endoplasmic reticulum to the plasma membrane . As G subunits lack lipid modifications, G subunits act as chaperones for G [START_REF] Michaelson | Membrane trafficking of heterotrimeric G proteins via the endoplasmic reticulum and Golgi[END_REF] β γ β targeting to the plasma membrane.

Several receptor regions contact surfaces of all three subunits , ( ). Both N-and C-terminal regions of G subunits have [13 19 ] Fig. [START_REF] Hopkins | The druggable genome[END_REF] α been implicated in receptor interaction. However, the C-terminus plays a crucial interaction point since derived peptides can directly compete for the coupling of the G protein with the receptor (See section 6.1). G subunits enhance receptor-G interaction but can also βγ α directly interact with the receptor through their C-terminal regions. The receptor regions involved in these interactions localized to the intracellular loops and the C-terminal tail. Basic amino acids sequences in both the N-terminal and C-terminal part of the third intracellular loop appear particularly important. The C-terminal tail of the receptor also determines important interactions with G subunit. It is of note β that today, we still do not understand the molecular basis for the selectivity of G protein coupling to the receptor.

THE HETEROTRIMERIC G PROTEIN ACTIVATION CYCLE

According to classical dogma, the heterotrimeric G protein activation cycle operates as follows ( ). In the absence of receptor Fig. [START_REF] Vassilatis | The G protein-coupled receptor repertoires of human and mouse[END_REF] stimulation, G and G remain associated in a GDP-bound, inactive form physically dissociated from the receptor. Agonist binding to the α βγ receptor initiates conformational changes allowing coupling with G . This interaction initiates G protein activation which then enters the αβγ GTPase cycle , , . The activated receptor acts as a guanine nucleotide exchange factor (GEF), promoting a conformational " " [8 17 20 ] change in G and ensuing GDP release. GTP, in much higher concentrations than GDP in the cytosol, then binds to G , switching its α α conformation to the active state. In the traditional view of heterotrimeric G protein activation, GDP/GTP exchange drives the dissociation of G from G and the receptor ( The dissociated G -GTP and G can activate different effectors and signaling cascades (ion channels, enzymes ). Termination of the

α βγ …
signal is facilitated by the inherent GTPase catalytic activity of G which hydrolyses GTP to GDP, and allows reassociation of G with G α α β

. Then, the G protein initiates a new cycle. To date, although we distinguish different G subunits isoforms functionally, they all share a γ α similar mechanism of activation.

GDP/GTP exchange and GTP hydrolysis represent two limiting steps in the G protein activation cycle. They are tightly regulated by accessory proteins which accelerate or impede these events by modulating kinetic constants and differ according to the G isoform. These α numerous regulatory proteins were reviewed by Sato . and may act as: et al [START_REF] Sato | Accessory proteins for G proteins: partners in signaling[END_REF] GEFs (guanine nucleotide exchange factors), such as AGS1, Ric-8, GAP-43 for example. These regulators interact with G , likely in a α subtype-specific manner, and stimulate the exchange of GDP for GTP to accelerate the generation of the active form.

GDIs (guanosine nucleotide dissociation inhibitors), such as AGS3, AGS4, AGS5, RGS12, RGS14. In contrast to GEFs, GDIs stabilize G in an inactive GDP-bound conformation. Most of these proteins possess a 19 30 amino acids conserved motif named GPR (G α protein regulatory) or GoLoco ( G -Loco interaction) motif which specifically interacts with G subunits and is directly involved in

" α i/o " α i/o
prevention of GDP dissociation but also in G -G reassociation. In fact, these proteins exhibit dual functions: they may hinder signaling α βγ through G by stabilizing the GDP-bound conformation, but they may also sustain G -dependent activation by inhibition of G

α i/o βγ βγ
association with GDP-G .

α [START_REF] Siderovski | The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits[END_REF] GAPs (GTPase-activating proteins) which antagonize GEFs activity and accelerate GTP hydrolysis back to GDP, thus favoring the G protein resting state and termination of G protein signaling. They act allosterically to stabilize the transition state occurring during GTP hydrolysis and promote reassociation of G and G . Some G protein regulated effectors can also exhibit GAP activity such as

α βγ - 3 20
phospholipase C-activated by G and p115RhoGEF activated by G . Among GAPs, RGSs (regulators of G protein signaling) represent

β q α 13
the largest family with more than 20 members identified so far .

[25 ]

DIFFERENT CLASSES OF HETEROTRIMERIC G PROTEINS FOR ACTIVATION OF DIFFERENT EFFECTORS

G proteins are classified into four families, based on t he homology of the primary sequence of the G subunit and to some extent, the α selectivity of effectors activation (reviewed in , ) ( ).

[26 27 ] Fig.(3)
The G s family is composed of four isoforms, produced by alternative splicing, with a ubiquitous distribution, and G which has a α α olf more restricted expression in olfactory neurons. These G proteins directly stimulate transmembrane adenylyl cyclases (AC) leading to the production of cAMP. These proteins were also shown to stimulate GTPase activity of tubulin and Src tyrosine kinase. They are substrate

for ADP-ribosylation mediated by cholera toxin responsible for inhibition of GTPase activity and the permanent activation of G .

α s
The G family comprises the ubiquitous G , G , G but also G and G which are predominantly expressed by neurons and GTPase signaling through RhoGEF activation and control cell cytoskeletal remodeling, thus regulating key biological processes such as cell migration. They can also activate a large panel of different effectors such as radixin, A-kinase anchoring proteins, phospholipases D, protein phosphatase

α i/o α i1 α i2 α i3 α oA α oB neuroendocrine cells.
G complexes, initially thought to only favor G anchorage to the plasma membrane and to stabilize the inactive state of G , are now βγ α α known to mediate many functional responses on their own following GPCRs activation (reviewed in ). Many effectors can be activated [START_REF] Dupre | The role of Gbetagamma subunits in the organization, assembly, and function of GPCR signaling complexes[END_REF] by interaction with G subunits such as K channels (GIRK 1,2,4), phospholipases C-, adenylyl cyclase (II,IV,VII), Src kinases, while βγ + β others will be inhibited such as, adenylyl cyclase I, Ca channels (N, P/Q, R types). The G dimer composition seems to be essential in 2 + βγ dictating the specificity of both receptors and the effectors for their coupling to the G protein . Although G is always depicted as an [START_REF] Mcintire | Structural determinants involved in the formation and activation of G protein betagamma dimers[END_REF] βγ inseparable dimer, several works have suggested the existence of G or G monomer activity (reviewed in ) and recently it was β γ [START_REF] Mcintire | Structural determinants involved in the formation and activation of G protein betagamma dimers[END_REF] demonstrated that G can activate pheromone pathway in the absence of the G subunit .

INDIRECT ASSAYS TO ASSESS RECEPTOR-MEDIATED G PROTEIN ACTIVATION

GPCRs ligands are usually classified according to their receptor specificity and intrinsic activity. Originally, this classification was essentially based on the effect of ligands on the activity of only the primary effector pathway, usually involving second messenger generation, which is generally assigned to the receptor of interest. According to their relative efficacy compared with the physiological agonist, ligands were identified as partial or full agonists when able to induce a fraction or a full response respectively, whereas neutral antagonists were believed to be devoid of effect and inverse agonists allow the inhibition of constitutive activity.

G -G activation s i

Both G and G proteins predominantly act by activating or inhibiting adenylyl cyclase respectively and thus regulating intracellular s i

ATP conversion into cAMP (

). The direct measurement of adenylyl cyclase activity is possible using -P ATP as the enzyme Fig. (3)

[α 32 ]

substrate

. However, most investigators usually determine intracellular cAMP levels. The intracellular cAMP concentration is [START_REF] Nakai | Assay for adenylate cyclase and cyclic nucleotide phosphodiesterases and the preparation of high specific activity 32-P-labeled substrates[END_REF] regulated by the balance between production rate by adenylyl cyclases and degradation rate by phosphodiesterases. Historically, cAMP was the first second messenger quantified in living cells and its measure has been widely used to test ligands acting on G -and G -coupled s i receptors. The early development of cAMP antibodies allowed the development of Radioimmunoassays (RIA), Enzyme [START_REF] Steiner | Radioimmunoassay for the measurement of adenosine 3 ,5 -cyclic phosphate ′ ′[END_REF] Immunoassays (EIA), Chemiluminescent Immunoassays (CLIA) and more recently Homogeneous Time Resolved Fluorescence (HTRF) assay, motivated by the desire to move away from the use of radioactivity. These methods are now very sensitive (detection of less than one femtomole) and adapted to a homogenous format allowing their use in the context of ligand screening in pharmaceutical industry.

However, they cannot follow kinetics of cAMP levels fluctuations in living cells as they consist essentially of static measurements after cell lysis and based on the accumulation of cAMP in the presence of a phosphodiesterase blocker so to increase cAMP levels and enhance detection sensitivity. Another disadvantage of current cAMP assays to assess ligand efficacy may be the global cAMP levels evaluation of the cell since cAMP signal was shown to be compartmentalized within the cell and local responses may thus be diluted in the general background . During the last few years, fluorescent-based sensors evaluating spatiotemporal resolution of cAMP signals in living cells [START_REF] Nikolaev | Molecular basis of partial agonism at the neurotransmitter alpha2A-adrenergic receptor and Gi-protein heterotrimer[END_REF] have been extensively developed . All of these approaches rely on the use of genetically encoded fluorescent reporters using cAMP [START_REF] Willoughby | Live-cell imaging of cAMP dynamics[END_REF] binding properties of cAMP downstream effectors, protein kinase A (PKA) and Epac (exchange proteins directly activated by cAMP).

After cell transfection of the biosensor, accurate monitoring and visualizing of the cAMP dynamics in the different cell compartments is possible by measuring FRET (fluorescence resonance energy transfer). Sensing of cAMP can rely on conformational changes of the biosensor when the reporter is solely based on the use of the cAMP binding domain of the effector as for the Epac-based biosensor or [START_REF] Dipilato | Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments[END_REF] on the behavior of the entire effector as for protein kinase A sensor measuring dissociation of its regulatory and catalytic subunits which occurs after cAMP binding . Similar strategies were applied using BRET-based biosensors based on the use of luminescence [34 ] [35 ] probes which offer better sensitivity and wide dynamic range of detection. Such probes are suitable tools for screening of new ligands for GPCRs. However, despite their high sensitivity, all these techniques remain indirect readouts of the G protein activation since their focus on the evaluation of cAMP downstream signaling. More recently, Pantel described a more direct cAMP detection system using a et al.

genetically modified luciferase whose activity is restricted to the cAMP binding domain of RII B subunit of the PKA (pGloSensor ,

β ™ Promega)
. When coexpressed with the melanocortin-4 receptor, this cAMP-luciferase probe demonstrated very high sensitivity [START_REF] Pantel | Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay[END_REF] allowing detection of inverse agonist activity of ligands in the absence of PDE inhibitor. Despite promise, this latter method requires use of stably transfected cells for high efficiency as it was poorly reproducible intransient transfected cells (unpublished data from our lab).

Another recent technology for direct cAMP detection using luminescence as a read-out and based on enzyme fragment complementation technology (Discoverex, CA) offers great utility and sensitivity for a large panel of both G -and G -coupled GPCRs with large range s i dynamics for inhibitory signals (unpublished data from our lab).

Another specific difficulty to evaluate ligand efficacy at the level of cAMP signal emanates from G -coupled receptors. In this case, i adenylyl cyclase must be obligatorily pre-stimulated, generally using forskolin, to increase cAMP concentration in order to detect the inhibition of the enzyme in a second step. Despite wide use, this strategy is at risk of biased interpretations since forskolin and G bind α s different adenylyl cyclase regions and therefore induce different conformations of the enzyme catalytic core . In fibroblast cells [START_REF] Tesmer | Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gsalpha[END_REF] overexpressing PTX-insensitive G proteins, Ghahremani . have shown that the dopamine G -coupled D2S receptor may inhibit the

α i/o et al i activity of AC through distinct G proteins .
In fact, when adenylyl cyclase is stimulated with forskolin, D2S-induced inhibition of the

α i [38 ]
enzyme is mediated by G , while following activation by PGE1, the receptor inhibits AC through G . This suggests that G and G

α i2 α i3 α i2 α i3
demonstrate specificity for different conformational states of adenylyl cyclase. Furthermore, differences may also arise from activation specificities among adenyl cyclase isoforms. It was shown that forskolin preferentially activates AC over AC , AC , or AC , while G

I II V VI α s
stiumlates AC more efficiently than AC , AC , or AC . Today, evaluating ligand efficacy at G -coupled receptors still remains a II I V VI [START_REF] Sutkowski | Regulation of forskolin interactions with type I, II, V, and VI adenylyl cyclases by Gs alpha[END_REF] i challenging task and especially for the detection of weak efficacies, as available cAMP assays provide generally too low dynamic ranges of inhibition detection. the first chimeric G protein was designed by substituting three amino acids of G by the corresponding residues of G . This G

α q α i2 [40 ] α q-i2
was functionally expressed and induced PLC activation (G effector) through the stimulation of G -coupled receptors, demonstrating that α q i the chimera keeps G specificity . Due to its ability to associate with multiple receptors without high selectivity , G was then i [START_REF] Conklin | Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha[END_REF] [41 ] α 16 thought to be the optimal G protein backbone for the generation of G protein chimera to obtain a truly universal G protein adaptor stimulated by all the GPCRs. However, several G -linked receptors and also some G -linked receptors are unable to activate PLC via G i s α 16

.

To optimize the G so to enlarge its binding capacity to a maximum of receptors, different chimeras were constructed by incorporating . Another chimera has been generated based on the mutation of a critical amino acid i s q [START_REF] Liu | Galpha(16/z) chimeras efficiently link a wide range of G protein-coupled receptors to calcium mobilization[END_REF] located in the linker region connecting the GTPase and the helical domain of the G protein. Hence, the mutant acquires the capacity to α q transmit G -and G -linked GPCR signals . Further additional mutations in the C-terminus of G combined with the linker mutation i s [START_REF] Heydorn | Identification of a novel site within G protein alpha subunits important for specificity of receptor-G protein interaction[END_REF] α q led to an optimized universal G protein chimera now functional for all the three G protein families . Even if these artificial chimeras " " [START_REF] Kostenis | A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity[END_REF] are far from the reality of natural G proteins, they remain very attractive tools to screen for ligands at GPCRs especially for orphan GPCRs , , at least at first instance.

G activation q

To test ligands efficacy on G -coupled receptors, different assays have been developed to measure inositol phosphate or calcium q/11 concentrations as reflects of PLC activity ( ). Original methodologies for determination of PLC activity used artificial phospholipid Fig. [START_REF] Jacoby | The 7 TM G-protein-coupled receptor target family[END_REF] vesicles containing H -inositol PIP and enzyme activity was measured by following the amount of H -inositol triphosphate (IP )

[ 3 ] 2 [ 3 ]
released into the aqueous solution . This technique was improved by Mullinax . who developed labeled phospholipids bound on [START_REF] Vivo | Assays for G-protein regulation of phospholipase C activity[END_REF] et al microplates . However, this technique is only suitable for cell extracts or permeabilized cells. More classically used for the [START_REF] Mullinax | Monitoring Inositol-Specific Phospholipase C Activity Using a Phospholipid FlashPlate(R)[END_REF] investigation of G -coupled receptors is the monitoring of inositol phosphate derivative production. The radiolabelled precursor, H q/11

[ 3 ]
inositol, is incorporated into intact cells as H -phosphatidylinositol 4,5-bisphosphate. Upon agonist binding to the receptor, PI P myo -

[ 3 ] 4,5 2
are hydrolyzed by PLC into H -IP and DAG. LiCl must be added to prevent dephosphorylation of IP and to increase sensitivity. The cAMP, specific IP antibodies allowed development of several specific and sensitive immunoassays. Other assays using IP binding 3 3 proteins competition can also be used and were proposed for high-throughput screening of GPCR ligands . However, IP production is developed and offer both high sensitivity and assay window. This sensitive technique already allowed the detection of inverse agonist activity at mGlu5 receptor and is readily adaptable to high-throughput screening assays to screen for ligands efficacy.

[55 ]

A common alternative to explore ligand efficacy at G -coupled-receptor is the evaluation of calcium mobilization. In response to PLC q activation, IP produced in the cytoplasm, binds to endoplasmic reticulum IP receptors thus liberating calcium from internal stores. different criteria to select the suitable probe were recently reviewed with advantages and limitations discussed for each probe . These [START_REF] Paredes | Chemical calcium indicators[END_REF] indicators are very powerful tools, easy to use and to calibrate, and suitable for cell imaging. However, they present some limitations to their use. First, they act as Ca buffers and can therefore influence Ca levels and kinetics and, second, their cellular localization per se -sensitive photoprotein isolated from the jellyfish . In the presence of Ca , the photoprotein undergoes a Aequorea Victoria [START_REF] Shimomura | Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea[END_REF] 2 + conformational change allowing oxidation of its substrate coelenterazine into coelenteramide. Upon relaxation, this product goes from an excited state to the ground state and emits a flash blue light (469 nm). The great advantage of aequorin is that it can be targeted to several intracellular cell compartments (nucleus, endoplasmic reticulum (ER), Golgi apparatus, mitochondrial matrix, mitochondrial intermembrane space, plasma membrane) by direct fusion of specific targeting sequences, thus offering the possibility of subcellular Ca 2 + measurements. Generally, Gq-coupled receptors mediated Ca signals are often monitored using mitochondrial-aequorin, because ER has . However, when compared with fluorescent dyes, this approach, which necessites a transfection step, is not easy to calibrate and not ] sensitive enough for cell imaging since one molecule of aequorin will produce only a single photon. To overcome some of these limitations, others Ca -sensitive fluorescent proteins have been developed, such as the cameleon conformational FRET sensors. These binding peptide from myosin light-chain kinase). Upon Ca binding, calmodulin forms a compact complex with the M13 domain and this 2 + intramolecular rearrangement modifies FRET between the fluorescent proteins . These probes have been further improved and present [START_REF] Miyawaki | Fluorescent indicators for Ca2 based on green fluorescent proteins and calmodulin +[END_REF] now expanded dynamic range. Others FRET-based Ca indicators, such as pericams and camgaroos, have also been developed (reviewed 2 + in ) and are amenable for high throughput screening in drug discovery .

[60 ]

[61 ]

G activation 12/13

The evaluation of G activation still remains problematic, essentially because we still have not identified their specific direct 12/13 effectors as compared with the other G protein families. Since they were found to regulate actin cytoskeleton remodeling, G proteins 12/13 have been essentially studied in the context of cell proliferation, migration and morphology where they have been shown to regulate many diverse effectors ( ). Thus, given their biological action, they elicit the interest of a large number of research groups especially for Fig. [START_REF] Jacoby | The 7 TM G-protein-coupled receptor target family[END_REF] the chemokine receptors for which they play major role in regulating chemotaxis process. However, quantitative measurement of G /G 12 13 activation remains a challenging task today. Usually, evaluation of G activation is based on measurement of downstream effectors. The evaluation of ligand efficacy.

All together, these strategies involving measurement of effector activity and/or second messenger production have been greatly improved during the last few years and are valuable for the study of the effects of GPCR ligands. However, they suffer from some general limitations. First, they are distal events following interaction of the ligand with the GPCR and may be subjected to amplification and compartmentalization which cannot always be readily appreciated. Second, most receptors generally simultaneously activate different G protein isoforms. Therefore, assessment of receptor activity at the level of second messenger increases the occurrence of cross -talks thus making evaluation of ligand efficacy complicated. That is, signaling becomes more complicated to analyze when one looks farther from the initiating event of receptor activation at the plasma membrane. One strategy to overcome these limitations would be examining the initial step of receptor activation common to all GPCR families which is the direct activation of heterotrimeric G proteins. Indeed, G proteins represent the only signaling relay common to all GPCRs and their activation is the first step consequent to receptor stimulation.

For these reasons, an accurate method to depict the intrinsic activity of ligands would be to determine unequivocally their activation profile on different G protein isoforms. Several methods have been developed to evaluate G protein activation which are more or less adapted to accurate ligand efficacy assessment.

DIRECT ASSAYSTO ASSESS RECEPTOR-MEDIATED G PROTEIN ACTIVATION

General pharmacological tools

When available, pharmacological inhibitors targeting specific G protein families provide powerful tools to study the involvement of these proteins in GPCR signal transduction by preventing associated downstream signaling.

Pertussis toxin (PTX), formerly called Islet Activating Protein (IAP), isolated from , catalyses ADP-ribosylation Bordetella pertussis of all G subunits which subsequently remain locked in their inactive state, thus unable to activate its effectors. This mechanism prevents

α i/o
G proteins from functionally interacting with GPCRs. PTX have been largely used to characterize involvement of G proteins in

i/o i/o
receptor signaling in many cellular models. Recently, the role of G protein was studied using transgenic mice expressing the PTX i/o in vivo catalytic subunit specifically in pancreatic islet . Mastoparan, a peptide toxin from wasp venom, can also interfere primarily with G [START_REF] Regard | Probing cell type-specific functions of Gi in vivo identifies GPCR regulators of insulin secretion[END_REF] i/o proteins . It promotes dissociation of GDP and accelerates GTP binding on G subunits thus mimicking an agonist-bound receptor.

[65 ] α i/o

A mastoparan derivative, mastoparan-S, was described to selectively activate G . Cholera toxin from , targets

α s [START_REF] Sukumar | A Gs-selective analog of the receptor-mimetic peptide mastoparan binds to Gs alpha in a kinked helical conformation[END_REF] Vibrio cholerae specifically intracellular G proteins and induces their constitutive activation by permanent ADP-ribosylation . The toxin was quite s [START_REF] Van Dop | Amino acid sequence of retinal transducin at the site ADP-ribosylated by cholera toxin[END_REF] useful in the purification and characterization of G proteins . The search for specific G inhibitors led to the identification of suramin, s [START_REF] Northup | Purification of the regulatory component of adenylate cyclase[END_REF] s an anti-helminthic drug, and its derivatives that directly interact with G proteins and interfere with GTP binding on both G and G s i/o proteins . Some suramin analogues, NF449 and NF503, appear to be specific for G and to block the coupling of -adrenergic [START_REF] Freissmuth | Suramin analogues as subtype-selective G protein inhibitors[END_REF] α s β receptors to G in S49 cyc -cells .

s [START_REF] Hohenegger | Gsalpha-selective G protein antagonists[END_REF] The G -dependent signaling pathway can also be modulated by recently discovered molecules: toxin (PMT) q Pasteurella multocida and YM-254890. PMT is a bacterial toxin activating G proteins, but the molecular mechanism underlying G protein activation is q/11 unknown . However, PMT was also suggested to activate others signaling pathways such as G and Rho proteins , .

[71 ] [74 ] α q α 11 α 14 analysis of the X-ray crystal structure of the G YM-254890 complex showed that YM-254890 binds specifically to the linker domain

α q βγ-
connecting the helical from the GTPase domain of G , thus preventing flexibility during the G activation process . More recently,

α q α [75 ]
Ayoub have described another small molecule BIM-46187, as a non-specific and ubiquitous inhibitor of receptor-G protein signaling et al.

through selective binding to the G subunit . This pan-inhibitor of GPCR signaling might be useful to dissect G protein -dependent α [START_REF] Ayoub | Inhibition of heterotrimeric G protein signaling by a small molecule acting on Galpha subunit[END_REF] and -independent signaling pathways.

An alternative approach for the identification of selective G protein-dependent pathways is the use of synthetic peptides mimicking the COOH-terminus of the different G subunits which compete with G subunit binding to the receptor and thus inhibit G protein dependant α α signaling. In fact, the COOH-terminal part of G subunits is critical for both the interaction with their cognate receptors and the specificity α of each G isoform. These G protein inhibitors were first used in permeabilized cells where they were able to block the stimulation of α adenylyl cyclase mediated by -adrenergic receptors . The technique was further extended with the generation of minigene plasmid β [START_REF] Rasenick | Synthetic peptides as probes for G protein function. Carboxyl-terminal G alpha s peptides mimic Gs and evoke high affinity agonist binding to beta-adrenergic receptors[END_REF] vectors encoding the C-terminal peptide sequence of most G subunits facilitating their expression in living cells by transfection or α infection . However, since the peptides act as competitive inhibitors and thus must be expressed in the cell at high concentrations for [START_REF] Gilchrist | G alpha minigenes expressing C-terminal peptides serve as specific inhibitors of thrombin-mediated endothelial activation[END_REF] high efficiency, one has to be cautious on the results interpretations as they will largely depend on the transfection/expression efficacy of the peptides . Obviously, this will also vary between cell lines as they usually have different G proteins complements . By [START_REF] Gilchrist | G alpha COOH-terminal minigene vectors dissect heterotrimeric G protein signaling[END_REF] [80 ]

opposition to chemical G protein inhibitors, which only allows discrimination between G protein families, this strategy further permits the dissection of G protein isoforms involved in receptor -mediating signaling.

Participation of specific G proteins in signal transduction has been extensively elucidated using G protein-deficient mouse in vivo models allowing classical or conditional inactivation of the genes encoding the different G protein subunits. Several knockout mice models lacking expression of one or two G subunits or G subunits have been generated and the consequences of the genetic disruption on α βγ physiology and physiopathology have been extensively reviewed . Another similar strategy is the knockdown of G and G subunits

[81 ] α β through small interfering RNA, adapted to studies . Although these genetic strategies provide high specificity of inactivation in vitro [START_REF] Krumins | Targeted knockdown of G protein subunits selectively prevents receptor-mediated modulation of effectors and reveals complex changes in non-targeted signaling proteins[END_REF] between the different and closely related G protein subunits, they can be subject to compensatory mechanisms by modifications of the expression levels of other endogenous G proteins, thus interfering with the results interpretation.

Although all these methods are quite useful, they still remain qualitative in that they just help in the dissection of molecular mechanisms underlying G protein-dependent receptor signaling. However, they cannot replace direct measurement of the protein activity as a quantitative assessment of G protein activation. The most common technique to directly measure G proteins activation following agonist stimulation is the S GTP S binding assay

[ 35 ] γ
monitoring the nucleotide exchange process in membranes extracts, which was first described by Hilf .

. This radioactive GTP et al [START_REF] Hilf | Muscarinic acetylcholine receptor-stimulated binding of guanosine 5 -O-(3-thiotriphosphate) to guanine-nucleotide-binding proteins in ′ cardiac membranes[END_REF] analog binds the G subunit following activation but resists GTPase hydrolysis, thus stabilizing the G in the active form and preventing G α α protein activation cycle arrest. Therefore, G -S GTP S subunits accumulate and radioactivity can be counted following filtration

α [ 35 ] γ
procedures to separate bound from free radioactivity. As S GTP S cannot cross plasma membranes, the assay is restricted to cell

[ 35 ] γ
membrane preparations or permeabilized cells . The method was also used in tissue sections and autoradiography allowing anatomical [START_REF] Wieland | Analysis of receptor-G protein interactions in permeabilized cells[END_REF] localization of activated G proteins . The pharmacology of a large panel of GPCRs ligands has been largely investigated by the use of [START_REF] Sim | In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5 -gamma-35S thio -triphosphate ′ [ [ ] ] binding[END_REF] this method (reviewed in ), thus allowing accurate characterization of their G protein potency and efficacy. The S GTP S binding

[86 ] [ 35 ] γ
method provides a sensitive tool to characterize constitutive activity of receptors through identification of inverse agonists but also to [START_REF] Newman-Tancredi | Agonist and inverse agonist efficacy at human recombinant serotonin 5-HT1A receptors as a function of receptor:G-protein stoichiometry[END_REF] evaluate antagonist activity by the shift of agonist-induced dose-response curves (pA value determination). While it proves to be a 2 powerful assay to evaluate G -coupled receptor activation, a major pitfall of this method comes from its low sensitivity to analyze i/o receptors coupled to others G protein families, essentially due to a poor signal to background ratio. Indeed, PTX-sensitive G proteins generate optimal results most probably because of their generally higher expression levels in most mammalian cells and their greater nucleotide exchange rate. Even though suitable for the measurement of endogenous G protein activity, a large number of S GTP S stoichiometry may profoundly influence ligand pharmacology, for example, the relative potency of agonists as discussed by Kenakin in the light of the concept of ligand-selective receptor conformations . This has led to the conception of receptor-G fusion proteins, forcing

[90 ] α a 1:1 expression ratio, which will be discussed below. Beside potency modulation, G protein subtype overexpresssion may modify ligand efficacy at G proteins as well and thus reveal protean agonism of ligands as shown for -adrenergic receptor .

α 2A [START_REF] Pauwels | Evidence for protean agonism of RX 831003 at alpha 2A-adrenoceptors by co-expression with different G alpha protein subunits[END_REF] Even if S GTP S binding accurately measures direct G protein activity, it cannot provide information about the subtypes specificity

[ 35 ] γ
of the activated G protein and therefore has been further improved. Thus, the existence of selective antisera for the different G subunits α allows immunocapture and thus enrichment of G proteins of interest following S GTP S binding , . Immunoprecipitation of the

[ 35 ] γ [92 93 ]
G protein can also be coupled to scintillation proximity assays (SPA) to eliminate unbound radioactivity separation steps and may be applied to high-throughput screening , . In this method, G protein immunoprecipitation is followed by a second immunoprecipitation of the radioactive G protein immuno-complex using specific beads containing scintillant and coated with a non specific anti-IgG. When S is in close proximity to the scintillant, it generates a luminescent signal detected by a microplate scintillation counter. However, the problem of antibody specificity and immune-capture efficiency remains an impediment to these assays. Other modifications of the initial S GTP S assay use non-radioactive GTP analogs. Among them, Europium-labeled GTP appears to be an further validation because of their non negligible hydrolysis rate by G subunits .

α i/o [99 ]
The receptor-G protein (R-G) fusion strategy was developed during the 90 s to force a 1:1 stoichiometry efficient coupling of a given ' receptor to a specific G protein subunit and has subsequently been applied to a large number of receptors . This technique (potency and efficacy) depending on the G -protein subunits fused to the -receptor . This approach also proved useful in the

α β 2 [104 ]
context of protean agonism revealing for instance that one dopamine receptor ligand could behave as an agonist or an antagonist depending on the D2R-G -subunit pair considered . Beyond G protein activity, the use of R-G fusions was an ingenious and well-adapted G protein families (G , G , G ). Another concern is the non-dynamic measurement of R-G activity since the G subunit is s q/11 12/13 α irreversibly fused to the receptor and thus could give rise to artefactual interpretations. Finally, an additional problem comes from the fusion of the G protein to the C-terminus of the receptor by itself that can impair the proper trafficking of the receptor to the plasma membrane and/or its pharmacological properties (ligand binding/activation process). Also, an accurate characterization of the R-G fusion must precede its subsequent use.

Plasmon Waveguide Resonance

Plasmon waveguide resonance (PWR) spectroscopy is an optical approach derived from Surface Plasmon Resonance and developed by Salamon s group to allow the study of membrane-associated proteins. coated by a thicker silica layer deposited onto the surface of a glass prism. Laser excitation generates an evanescent electromagnetic field localized at the outer surface of the silica. Resonance excitation generated depends on the angle of incidence of the laser beam and is modulated by molecules present at the outer surface. The protein of interest is inserted in a single lipid bilayer at the interface between the silica film of the resonator and an aqueous buffer compartment in which molecules can be added . PWR allows real-time

[109 ]
measurement of molecule or protein binding to a specific receptor inserted in the lipid bilayer with high sensitivity in the absence of radioactive or fluorescent label. This technique has been used to characterize kinetics and thermodynamics of conformational events associated with the binding of ligands and of G proteins on the -opioid receptor (DOR) and provided new insights into the function of δ these molecules . These studies demonstrated that receptor-G protein interactions are quite selective depending both on the

[110 ]
ligand-bound states of the receptor but also on the G protein isoforms . Interestingly, by coupling PWR studies of receptor-G protein [110 ] interaction with GTP S binding assay examining the G protein activation state, a disconnection between the two events was demonstrated γ which highlights the existence of a non-active precoupled state of the receptor. Thus, PWR has been demonstrated as a powerful approach to measure selectivity and activity of GPCR ligands towards the different G protein isoforms. This was possible by the use and insertion of different purified G protein isoforms in reconstituted membrane systems containing the receptor and thus allows an acute control of the expression of each protein partners . On the other hand, this method is limited because it is based on artificial cell system [111 ] reconstitution which does not reproduce the real cellular environment, especially regulations by other cellular proteins which could participate and modify ligand-receptor-G protein relationships. Moreover, PWR requires receptor and G proteins purification steps, thus dramatically impairing its use for large -scale screens. Finally, this approach is hardly accessible to non-specialist researchers, as illustrated by the limited number of GPCR studied to date , .

[110 112 -114 ] "RETvolution : monitoring real-time G protein activation in living cells

"

The field of cell biology has been subjected to a real shake-up with the introduction of non invasive biophysical RET-based approaches, allowing for the first time, quantification of intracellular signaling events dynamics in real-time and in living cells. The evaluation of receptor-mediated G protein activation did not escape from view using these technologies.

The RET principle

The Resonance Energy Transfer (RET) biophysical principle was discovered and published by Theodor F rster in 1940s

, but its

ö [115 ]
ultimate impact is still evolving. Basically, RET relies on non-radiative energy transfer between an energy donor and an acceptor molecule occurring under highly restrictive distance parameters ( ). When donor and acceptor are in close proximity (distance < 100 ), the Fig. (4A) Å energy generated by donor excitation is then transferred to the acceptor through a non radiative process of resonance, which in turned becomes excited and emits at a different wavelength from that of the donor. Given its distance dependence, RET is highly suitable to monitor protein-protein interactions in living cells between two partners tagged with different RET partners following transfection.

Interestingly, the efficiency of RET is inversely proportional to the sixth power of the distance between donor and acceptor dipoles, thus allowing accurate measurement of relatively small variations in distance or orientation between the two RET partners. Thus, RET is suitable not only to measure intermolecular events (protein-protein interactions) but also allows monitoring of intramolecular events like protein conformational changes. Another parameter limiting RET efficiency and fluctuating between energy donor/acceptor couples is the spectral overlap between the emission wavelength of the donor and the excitation wavelength of the acceptor, since acceptor excitation is only dependent on the donor emission. Depending on the nature of the energy donor, RET will be defined as: i/FRET (Fluorescent Resonance Energy Transfer) when using a fluorescent donor and excited by an external energy source (laser or arc lamp), or ii/BRET (Bioluminescent Resonance Energy Transfer) when using an enzymatic donor ( luciferase) and excited by oxidation of its Renilla coelenterazine substrate (

). Both FRET and BRET use a fluorescent energy acceptor. Because of its fluorescent nature, FRET Fig. (4B) allows subcellular localization of RET events. However, on the other end, extrinsic donor excitation by a light source has several limitations due to direct excitation of the fluorophore acceptor or photobleaching of the FRET partners and, in the cell context, cell autofluorescence . In this context, BRET is a better choice because of the enzyme-dependent activation of the donor, thus allowing a

[116 ]

better signal to noise ratio .

[116 ]

Originally, BRET-based approaches used two BRET generations called BRET and BRET based on the use of different luciferases,

Renilla

(Rluc8), demonstrating equal or even higher luminescence intensity compared with BRET . Rluc8-BRET2 is so sensitive that it can 1 [119 ] be even used for microscopic detection of BRET in living cells but also in living animals . The main limiting aspects of the use of

[120 ]
BRET or FRET approaches are the necessity to transfect cells for introduction of RET-tagged proteins, and the molecular weight of the protein fused to the proteins of interest (27 36 kDa). Thus, it remains essential to control for the biological properties (cell localization, functionality) of the fusion proteins before subsequent RET experiments.

Sensing G protein activation using RET

In the traditional view of heterotrimeric protein activation (see section 2), the active receptor initiates sequential events based on multiple protein-protein interactions and/or conformational changes: i/receptor-G GDP-protein interaction, ii/G -GDP/GTP exchange,

Indirect measurement of G protein activation: receptor-G protein interactions

G protein activation can be assessed indirectly by RET by monitoring interactions in real time between receptors and G proteins subunits in living cells ( ). For this purpose, BRET or FRET donors and acceptors are fused on the C-terminus of the receptor and FRET sensitivity over BRET (see RET principle) may probably account for these discrepancies. In all studies, agonist stimulation of the receptor promoted a rapid (milliseconds) modification of the RET signal between several GPCRs and either G or G proteins.

s i/o
Interestingly, depending on the insertion site of the RET partners within the G protein subunit, agonist-stimulation can induce either an increase or a decrease in RET, thus demonstrating that the RET in fact monitors conformational rearrangements within preformed R-G complexes (or occurring during the receptor-G protein interaction step). This notion is supported by a study where three different BRET probes within the G subunit were used to monitor its interaction with the -adrenergic receptor . Similar results were obtained α i1 α 2A [START_REF] Gales | Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes[END_REF] when measuring the interaction between G protein subunits and the -opioid receptor . Receptor-G protein RET-biosensors

δ [123 ]
monitoring conformational rearrangements occurring during the activation process are highly prone to pharmacological characterization of ligands. Two comparable studies performed on -adrenergic and -adrenergic receptors demonstrated that agonist stimulation

β 2 α 2A
promoted a concentration-dependent increase in RET between receptor and G in good agreement with second messenger responses , increase between the G /G coupled -adrenergic receptor and G was only detected in presence of G and G but not with G or G s i

β 2 γ 2 α s α i α q α 11
while a FRET increase between the G -coupled protease-activated receptor and G was only detected in the presence of G but not activation . The capability of the R-G BRET assay to probe ligand-induced structural rearrangements in preexisting receptor-G protein [START_REF] Gales | Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes[END_REF] complexes and leading to changes in the distance between the receptor carboxyl tail and the G protein subunits has profound impact in the field of biased agonists. Actually, if different ligands promote distinct R -G conformational changes through a unique receptor, this could highly suggest that they engage different signaling outputs. Similar approaches allowed characterization of ligand-biased MAPK signaling through the -adrenergic receptor .

β 1 [124 ]

Direct measurement of G protein activation: G protein subunits interactions

According to the classical model of heterotrimeric protein activation, receptor-mediated G -GDP/GTP exchange triggers the α dissociation of the G -GTP from the G dimer and the receptor. Obviously, measuring RET between G and G subunits was identified RET between G and G have been interpreted as evidence of receptor-promoted dissociation of the G protein complex. Although loss of α RET is consistent with dissociation, it can also reflect conformational rearrangements that promote an increase in the distance between the two RET partners. Consistent with this latter hypothesis, and as observed for receptor-G protein subunits interactions, the use of RET probes at different positions within the G complex could lead to either an increase or a decrease in RET signals RET monitoring receptor-G protein or G protein subunit interactions represents a quite promising approach in the future to dissect GPCRs ligand efficacies most proximal to the receptor. Even if this strategy has only been described for a few G RET probes, it could be α easily enlarged to all the other G subunits for all G proteins family and could thus help unravel potential ligand-biased activity at specific α sets of G protein subunits that remains elusive. Another benefit of RET-based assays over other assays is the acute temporal appreciation of signaling events, allowing detection of ligand selectivity at the level of kinetics. Commonly, ligand selectivity is quite apparent in concentration-response curves; however, differences between agonists can only be detectable by kinetics analysis as already reported for β -arrestin translocation . Finally, RET-based assays offer the possibility to visualize spatial organization of the signaling and so to [134 ] dissect biased activity of ligands in terms of signal compartmentalization.

G PROTEIN ACTIVATION AND BIASED AGONISM

Originally, GPCRs were thought to function necessarily through rapid activation of heterotrimeric G proteins, thus propagating the different intracellular signaling pathways. In the last few years, it seems that GPCRs could activate distinct G protein-dependent and -independent transduction pathways and that GPCR ligands, namely biased-ligands, can selectively favour activation of only a subset of the pathways activated by a given receptor. Although GPCRs can modulate a large variety of distinct signaling pathways, classification of biased-ligands was restricted to two groups depending on their ability to activate two main transduction pathways : i/ G

[135 ]

protein-biased ligands which promote G protein activation without -arrestin recruitment and ii/ -arrestin-biased ligands which recruit activates the G pathway after binding to the G -coupled oxytocin receptor without any G -mediated signal transduction and very little i q/i q receptor desensitization, thereby leading to the selective inhibition of cell growth . In opposition to G protein biased activity, the vast majority of biased ligands identified so far exhibits exclusive -arrestin activity for a number of receptors , , including the AT1 appreciation of G protein biased-ligands. It is interesting to note that most of the work describing -arrestin-selective signaling never β evaluated potential involvement of the G protein component in this pathway, essentially as they failed to primarily identify G protein activity using classical direct assays (which does not mean there is not). Indeed, this could be easily performed by blunting G protein expression and/or activity using siRNA strategy or toxin/chemical inhibitors as mentioned above. -arrestin siRNA strategies were often β used to evaluate implication of this protein in a signaling pathway.

Thus, evaluation of biased activity is not an easy task given the high diversity of GPCRs signaling and the molecular crosstalk which can occur between the different signaling pathways. Restricting evaluation to the G protein and the -arrestin components appears exclude β the full array of signaling pathways linked to a given receptor and their interconnections. G protein and -arrestin are good examples as, β originally, these two proteins were tightly connected given the canonical role of -arrestin in dampening G protein signaling during β desensitization but they also demonstrate independent signaling as shown by the selective G protein or -arrestin biased activity of

[160 ] β ligands. Another difficulty comes from the insufficient sensitivity of the different assays to evaluate activity of the different signaling components which will depend on the ligand efficacy. This highlights the necessity to accurately monitor the various signal transduction pathways in order not to underestimate ligand efficacy. One possibility is to multiplex different assays to evaluate activation of specific effectors. This is currently being done to evaluate -arrestin-dependant pathways but is still missing for G protein signaling, for which only β one assay is generally performed. However, even with accurate -arrestin and G protein assays, multiplexing assays which will examine β different vantage points in the signalosome, from the initial signaling event of receptor/G protein activation at the plasma membrane to the more downstream signaling events inside the cell, will probably be the best way to fully characterize the efficacy of a given ligand completely. Rho. Then, active Rho-GTP can activate various proteins responsible for cytoskeleton regulation.

Fig. 4

Resonance Energy transfer (RET).

RET is a non radiative energy transfer which occurs between an energy donor and an energy acceptor (A) over a restricted distance. When the energy donor is excited and is in close proximity (< 100 ) to the donor, the energy released is then Å transferred by resonance ( ) to the energy acceptor, which in turns becomes excited and emits at a different wavelength to that of the donor. R

Depending on the nature of the energy donor we distinguish two RET: i/FRET (Fluorescence Resonance Energy Transfer) with a (B) fluorescent energy donor excited by external light and ii/BRET (Bioluminescence Resonance Energy Transfer) using an enzymatic energy donor ( Luciferase) excited by degradation of its substrate (coelenterazine). Renilla

α i1 α i1 R γ 2 R γ 2
rearrangements would thus create an exit route for the guanine nucleotide and thus measure directly the activation state of the G protein.

"" [8 9 ]

 9 G proteins are heterotrimeric proteins consisting of G , G and G subunits tightly associated and bound to the inner face of the cell α β γ plasma membrane ( ), where they predominantly relay receptor activation. To date, 16 different G subunit encoding genes, 5 G Fig.(1) α βsubunit genes and 12 G subunit genes have been described in humans . Additional variants can be generated by alternative splicing

Fig

  Fig. (1)

α 16 variable 16 [

 1616 length of G (G family) or G sequences into the C terminus of G and were highly efficient to mediate both G and G further analysis revealed that the G 16-z chimera is able to transmit signal from virtually all G 42 43 ] α protein-linked receptors (G -, G -and the G )

  [47 48 ]

[ 3 ] 3 3 3 [ 3

 3333 mass of soluble IP is a quantitative readout of receptor activation. H -IP is quantified following purification on anion-exchange ] 3 chromatography or by HPLC , . HPLC also allows quantification of the production of the other inositol phosphates . As for [51 52 ] [53 ]

  to its extremely short half life making it difficult to accurately quantify. By comparison, IP , a downstream metabolite 1 of IP , is stable in the presence of LiCl providing a better read out of G -coupled receptors. Thus, IP -based immunoassays have been 3 q 1

3 3 Specific

 33 dyes generating fluorescence upon binding of free Ca have been developed since almost 30 years. Most of them derivate from 2 + calcium chelators EGTA or BAPTA fused with an additional acetoxymethylester (AM) group to allow cell penetration. Once in the cell, AM is cleaved by endogenous esterases and the intracellular probe then becomes active. A number of chemical calcium indicators are now available and the investigator must consider the Ca affinity of the probe which must be compatible with the intracellular concentration of 2 + Ca to measure. Spectral properties of the indicators can also differ, varying from single wavelength to ratiometric indicators. The 2 +

2 + 2 +

 22 cannot be controlled or targeted. The other alternative is the use of genetically encoded luminescent proteins. Aequorin was the first Ca 2 +

2 +

 2 close physical relationship with mitochondria and the release of Ca from ER exposes mitochondria to very high Ca2 concentrations 2 + + [58

2 +

 2 biosensors were initially based on tandem repeats of mutants fluorescent proteins (Blue or Cyan mutant GFP and Green or Yellow mutant GFP) used as FRET donors and acceptors, interconnected by a Ca -sensitive linker of calmodulin fused to peptide M13 (a calmodulin 2 +

12 / 13 [ 72 73 ]

 12137273 YM-254890, a cyclic depsipeptide isolated from culture of Chromobacterium sp. QS3666, appears as a potent and specific inhibitor of the G family . It prevents the GDP/GTP exchange reaction on G , G and G isoforms by inhibiting the GDP release. Recent q/11

[ 35 S

 35 GTP S binding] γ

  in heterologous expression systems stably or transiently expressing receptor and/or different G protein subtypes of interest; however, competition with endogenously expressed G proteins may confuse the issue. Thus, cell lines expressing low levels of mammalian G protein such as Sf9 insect cells have also been used , . In all cases, changing both the receptor and G protein[88 89 ]

  103 ]relies on the fusion of the G -protein subunit N-terminus to the receptor C-terminus in a single open reading frame, leading to the α expression of a unique polypeptide containing both functionalities. G protein activation is then assessed according to S GTP S binding [ 35 ] γ performed on cell membranes expressing the R-G fusion construct, allowing evaluation of the role of the G protein isoform on the potency/efficacy of different ligands. Using this technique, different -adrenergic agonists showed different pharmacological profiles β 2

1 2 coelenterazine 2 the 2 Fig

 1222 Fig. (5)

  -G protein dissociation and G -G dissociation, iv/G -GTP hydrolysis, v G -GDP and G reassociation. Thus, this α βγ α / α βγ biological process obviously provided an ideal template for RET-based assays development. During the last ten years, several groups have developed different RET-based probes to monitor real -time dynamics of receptor mediated-G protein activation cycle in living cells.

  Fig. (6A) in one of the different subunits of the G protein and the RET -fusion proteins are then overexpressed in mammalian cells. BRET or αβγ FRET were measured between receptor and G or G subunits tagged at their N-terminus with the BRET partner and further between the β γ receptor and G subunit . Generating G -BRET probes was not an easy task given the complex structure of the protein α -124 ] α compared to G or G subunits and the relative high size of the BRET partner (around 26 40 Kda) to insert. Actually, several probes were β γ generated based on the G crystal structures available (unpublished data), but only a few potential insertion sites did not disrupt trafficking α and functional properties of the G subunit. In fact, all studies used intramolecular G -BRET probes with the RET partner generally α α introduced in the helical domain of the protein or the linker region connected the helical from the GTPase domain. Although FRET assays failed to detect basal interactions between receptor and G protein subunits , by opposition several BRET-based studies clearly [122 ]monitored constitutive R-G complexes, thus highlighting the existence of preformed R-

γ 2 [ 121 .

 121 Partial agonist led to a partial BRET modulation compared to the maximal RET signals obtained in the presence of full agonists, 122 ] while antagonists completely blocked the agonist response. Moreover, RET monitoring of agonist-induced receptor-G conformation γ changes shows high selectivity for the coexpressed G isoforms, despite all G protein subunits being overexpressed (which could favour α unspecific coupling), thus demonstrating the intrinsic coupling selectivity of each receptor , . Indeed, agonist-induced BRET [121 125 ]

2 γ 2 [ 21 ]

 2221 that when assessing the G protein activation using RET-based methods, the position of the RET partners in the G protein complex appears to be crucial especially for ligand efficacy evaluation. The insertion of the luc at different positions in the G highlighted that insertion R α i1 after amino acid 91 within the helical domain (G -91 luc) led to a potent and unique direct sensor of G protein activation when α i1 R measuring its interaction with the energy acceptor GFP10 tagged-G subunit at its N-terminus (GFP10-G ) ( ). This G γ Fig. (7A) α i1 -91 luc/GFP10-G BRET probe allowed measurement of the greater separation between the G helical domain and the G N-terminus R /GTP exchange that is translated by the BRET sensor as a decrease in BRET following receptor activation (Fig. (7B) ). Indeed, BRET modulations measured in the presence of various -adrenergic ligands correlated perfectly with their intrinsic signaling α 2A efficacy. Agonists induced a potent BRET decrease, partial agonists induced only a fraction of the signal promoted by full agonist while antagonists had no effect. No other probe position used to detect changes within the G complex provided such a direct correlation αβγ between signaling efficacy and BRET changes . Thus, the G -91 luc/GFP10-G BRET probe proved to be a potent sensor to of the G helical domain and the G N-terminus occurring during G protein activation and thus to evaluate receptor α γ ligands efficacy.

Fig. 1

 1 Fig. 1Schematic complex in the plasma membrane between rhodopsin (gray; PDB code 1GZM) and the inactive heterotrimeric G protein composed of , , and subunits (light blue/violet, red and yellow respectively; PDB code 1GG2). G N-terminal helix ( N) is shown in brown,α i1 β 1 γ 2 α i1 αwhile G -GTPase and G -helical domains ( H) are in light blue and violet respectively. Linker 1 connecting G -GTPase to the G Hα i1 α i1 α i1 α i1 α i1is represented in green. Both G N and G C-terminal helix ( C) are anchored to the membrane through lipid modification.

α i1 γ 2 γ 2 Fig. 2 2 receptor

 222 Fig. 2 Heterotrimeric G protein activation cycle. In the absence of agonist ( , basal state), G GDP-heterotrimeric G protein forms a tight inactive 1 α βγ complex dissociated from the receptor. The activation of the receptor by the agonist promotes recruitment of G to the receptor and the αβγ subsequent GDP/GTP exchange at the level of the G subunit ( , association). This nucleotide exchange then leads to the dissociation of the α

Fig. 3

 3 Fig. 3 Schematic representation of heterotrimeric G protein canonical pathways. G -coupled receptors usually promote direct activation of adenylyl α s cyclase (AC) leading to intracellular cAMP production which can directly bind and activate Protein Kinase A (PKA) or Exchange Protein directly Activated by cAMP (Epac) effectors. On the contrary, G -coupled receptors counteract the actions of G -GPCRs and inhibit AC α i/o

Fig. 5 1 and

 51 Fig. 5 The basics of BRET and BRET . BRET and BRET are based on the use of different coelenterazine substrates (Coelenterazine for BRET 1 2 1 2

  Renilla

1 2 Fig. 6 i1 β 1 γ 2 B α i1 β 1 γ 2 activation

 12622 Fig. 6 Configurations of the different BRET assays used to probe receptor-mediated G protein activation. Schematic representation of a GPCR (purple, Rhodopsin PDB code 1L9H) and a heterotrimeric G protein composed of , , and subunits (light blue, red and yellow α i1 β 1 γ 2 respectively; PDB code 1GG2) interacting at the plasma membrane, fused to luciferase (blue; PBD code 1LC1) or to GFP (green; PDB code 1GFL) as indicated. BRET monitoring receptor-G interaction. ( ) BRET monitoring G /G interaction. (A) α

  Also included in this group and with restricted distribution, are G which can be found in platelets and neurons, G

												α z	α
	t1	and G expressed in retina and G α t2	α gust	in taste buds. They all inhibit adenylyl cyclase activity and decrease intracellular cAMP levels.
	Beyond AC, most of the isoforms can also activate K channels or inhibit Ca channels. All these subunits can be ADP-ribosylated and + 2 +
	inactivated by pertussis toxin.
		The G	α q/11	family, including G , G α q	α 11	, G	α 14	, G	α 15	and G	α 16	isoforms, stimulates membrane-bound phospholipase C-, which

β

hydrolyses phosphatidyl 4,5-diphosphate (PIP2) into two second messengers, inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG).

IP3 is responsible for Ca liberation from intracellular stores, while DAG activates protein kinase C. Others effectors are also cited in the 2 + literature as, for example, p63-RhoGEF and K channels.

+

The G family comprises only 2 members to date (G and G ) with a ubiquitous expression profile. They regulate Rho family α 12/13 α 12 α 13

  assays using endogenous G proteins, the main drawback of the R-G fusion activation measurement is probably competition by endogenous receptors and/or G proteins which can result in a poor signal to noise ratio. To bypass this problem, G PTX-resistant isoforms can beα i/oused, coupled with PTX pre-treatment of cells to neutralize endogenous G isoforms . However, this issue still persists for all other

	α	[103 ]
	strategy to study G protein transactivation mechanisms mediated by GPCR dimers	[105 -107 ]	. Also true for classical S GTP S binding [ 35 ] γ
		α i/o	[102 ]

  There is abundant literature dealing with the physical principle of

	'
	this technique	, [108 109 ]	. Briefly, a polarized continuous wave laser is used to excite the resonator which consists in a thin silver film

  measurement of the real time kinetics of the activation process, on a milli-second time scale following immediate agonist stimulation , activation process, it remains an indirect sensor which monitors conformational rearrangements occurring within preformed R-G complexes. It follows that RET-based monitoring does not necessarily corroborate G protein activation state. For instance, the α 2

	[121 ]	i		βγ	α i
	G	. G -specificity of RET changes detected between receptor and G subunits was also confirmed for G -coupled-receptors by α s [125 ] α βγ i
	specific blockage with pertussis toxin pre-treatment	[121 ]	. Finally, RET analysis of receptor-mediated G protein activation allowed
					[21
	, 121 122 125 ] ,	. Although RET-based assays monitoring R-G interactions may provide accurate information about the G protein
	-adrenergic antagonist RX821002 increases the BRET signal between G and α i1	α 2A	-adrenegric receptor but is unable to promote G	α i1

  -arrestin to the receptor and initiate consecutive signaling pathways in the absence of G protein activation.Interestingly, very few ligands have been yet identified as perfect G protein-biased ligands, namely inducing G protein signal transduction without any -arrestin recruitment . For instance, GMME1 ligand binding to the CCR2 receptor led to calcium as G protein-biased are less potent for -arrestin recruitment than for G protein activation but they do activate the -arrestin pathway ,

			β		β	β
		β	[135 -137 ]
	mobilization, caspase-3 activation and consecutive cell death, but did not recruit beta-arrestin2	[138 ]	. Indeed, most of the ligands classified
		β			β	[134
	, 135 139 ]	. Interestingly, some ligands are biased in regard to the different G protein families	, [140 141 ]	. Thus, atosibans electively

  Most of biased ligand screening has focused on evaluation of G protein and -arrestin pathways separately. Many sensitive assays are monitoring of G protein activity in the context of biased activity is generally based on the evaluation of its downstream signaling by" "measuring G protein effectors activation (phosphorylation or second messengers measurement) or by direct evaluation of the G protein activity using the low sensitive assay S GTP S binding, which is the most classical method to directly analyse G protein activation but

								β	[135 142 ]
	angiotensin II receptor	, -[143 ] β 1 [144 ]	and -adrenergic receptors β 2	[145 ]	, or the CXCR7 decoy receptor	[146 ]	.
								β
	available to measure different levels of the -arrestin pathway activation in living cells β	[147 ]	, including -arrestin translocation assays β
	evaluating -arrestin recruitment to the receptor β	[148 -151 ]	, or measures of different conformational changes of the -arrestin protein β
	occurring during its activation process	, [152 153 ]	, to confocal analysis of the spatial redistribution of the receptor/ -arrestin complex β	[143
	,	154 -156 ]	. On the contrary, direct and accurate evaluation of G protein activation still remains elusive as discussed above. The
							[ 35 ]	γ
	exhibits a poor signal to noise ratio, even with technical improvements, and is not sensitive enough to monitor activity of all G protein
	families	, [48 157 ]	. Given the general low efficacy of biased ligands, S GTP S binding assay cannot be adapted to evaluate G protein [ 35 ]
	agonists	, [123 158 159 ] ,	, the low-sensitivity of current assays monitoring G protein activation appears to be a limiting step in the global

γ biased activity. The difficulty to measure G protein biased-activity comes also from the existence of a large panel of G protein subunits αβγ combinations compared to the existence of only two -arrestin ( -arrestin1 and -arrestin2) that are almost impossible to evaluate β β β individually. Recent development of RET-based probes monitoring the activation of specific G combinations should certainly help in αβγ that direction. This raises the question whether -arrestin biased ligands are truly unable to activate G proteins or if the assays were simply β not sensitive enough to detect low levels of G protein activation. Taking into account that biased ligands are generally less potent than full

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]

[START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]