G Protein-coupled Receptors Coupling Research Articles

Structural plasticity of arrestin-G protein coupled receptor complexes as a molecular determinant of signaling

G protein coupled receptors (GPCRs) are critically regulated by arrestins. In this study, high-resolution data was combined with molecular dynamics simulations to infer the determinants of β-arrestin 1 (βarr1)-GPCR coupling, using the V2 vasopressin receptor (V2R) as a model system.The study highlighted the extremely high plasticity of βarr1-GPCR complexes, dependent on receptor type, state, and membrane environment. The multiple functions of receptor-bound βarr1 are likely determined by the interplay of intrinsic flexibility and collective motions both as a bi-domain protein and as a whole. The two major collective motions of the whole βarr1, consisting in rotation parallel to the membrane plane and inclination with respect to the receptor main axis, are distinctly linked to the two intermolecular interfaces involved in tail and core interactions.The intermolecular dynamic coupling between βarr1 and V2R depends on the allosteric effect of the agonist arginine-vasopressin (AVP). In the absence of AVP the dynamic coupling concerns only tail interactions, while in the presence of AVP it involves both tail and core interactions. This suggests that constitutive and agonist-induced arrestin-receptor dynamic coupling is linked to distinct arrestin functions.

Quantifying Gq Signaling Using the IP1 Homogenous Time-Resolved Fluorescence (HTRF) Assay.

G protein-coupled receptors (GPCRs) play pivotal roles in cellular signaling and can regulate several cellular functions such as proliferation, secretion, protein expression, and cellular metabolism. Coupling of GPCRs to members of the Gq/11 protein family results in activation of inositol trisphosphate (IP3) and accumulation of calcium intracellularly. This protocol chapter outlines a step-by-step guide for utilizing the inositol phosphate-1 (IP1) accumulation assay, a time-resolved fluorescence resonance energy transfer (TR-FRET) method, to investigate Gq-IP3 signaling. The assay serves as a valuable tool for those conducting pharmacological investigations and compound screening targeting this critical cellular pathway. This protocol chapter covers experimental setup, sample preparation, and data analysis, providing researchers with an in-depth guide to explore the pharmacology of Gq-coupled receptors.

Effect of CBD on anti-tumor immune response and interaction with GPR55 on MAPK signaling in head and neck squamous cell carcinoma.

6060 Background: Head and neck squamous cell carcinoma (HNSCC) is a lethal malignancy and comprises two distinct etiology: human papillomavirus (HPV)+ve and -ve. Marijuana use is associated with HPV+ oropharyngeal infection and conflicting data show positive and negative associations of daily marijuana use with HPV+ and tobacco-associated HPV- HNSCC. However, cannabinoid use continues to increase in the US general population for recreational purposes as well as in cancer patients for palliative care. G-protein coupled receptors (GPCRs), including cannabinoid GPCRs, stimulated by specific ligands interact with multiple G proteins creating diversity and complexity of signaling. In this study, we aimed to investigate the CBD-induced GPCR coupling and its effect on anti-tumor activity by modulating immune response in HNSCC by using pre-clinical models. Methods: The anti-cancer effect of CBD was measured by cell proliferation, apoptosis and migration analysis. Next, TGF-α shedding assay was performed to identify the CBD-induced GPCR coupling with different cannabinoid receptors. The cannabinoid receptor GPR55 was subjected to silencing by siRNA and western blot analysis was performed to evaluate its role in activation of p38 MPAK pathway. Next, the anti-tumor immune response of CBD was evaluated by using an immunocompetent syngeneic C57BL/6 mEER RasG12D+/- HPV E6/E7 transformed tongue epithelial cell model that serves as a model for HPV+ HNSCC and the 4MOSC1 orthotopic, syngeneic, 4NQO (4-nitroquinoline-1 oxide)-induced murine oral tongue squamous cell carcinoma C57BL/6 mouse model that serve as HPV- HNSCC model as well as in immune-deficient Rag1 -/- and athymic nude mouse followed by measurement of immune cell infiltration by IHC analysis. Results: We found that 10μM of CBD treatment inhibited cell proliferation and migration in HNSCC cells by promoting apoptosis. Interestingly, we observed that CBD resulted in GPCR coupling for Gαi with GPR55 and Gαq/o and Gαq/s for CNR1, CNR2 and GPR55. Furthermore, we silenced GPR55 and observed that 10μM of CBD treatment inhibits the activation of p38 MAPK pathway whereas it promotes activation in non-silenced HNSCC cells. Next, we observed that treatment with 10μM of CBD significantly inhibited tumor growth compared to control in both HPV- and HPV+ models in immune-intact animals whereas, no significant difference in the tumor growth was observed in immune-deficient (Rag1 -/- and athymic nude) mouse, indicating role of CBD in modulating tumor immune microenvironment. We also observed CBD treatment enhanced CD4+ and CD8+ T cell infiltration into the primary tumors of HPV+ and HPV- mice models, implicating a downstream cytotoxic T cell response. Conclusions: Our study suggests that CBD promotes anti-tumor activity by modulating the immune microenvironment and interacts with GPR55 to activate intrinsic MAPK signaling pathway.

Gα12 and Gα13: Versatility in Physiology and Pathology.

G protein-coupled receptors (GPCRs), as the largest family of receptors in the human body, are involved in the pathological mechanisms of many diseases. Heterotrimeric G proteins represent the main molecular switch and receive cell surface signals from activated GPCRs. Growing evidence suggests that Gα12 subfamily (Gα12/13)-mediated signaling plays a crucial role in cellular function and various pathological processes. The current research on the physiological and pathological function of Gα12/13 is constantly expanding, Changes in the expression levels of Gα12/13 have been found in a wide range of human diseases. However, the mechanistic research on Gα12/13 is scattered. This review briefly describes the structural sequences of the Gα12/13 isoforms and introduces the coupling of GPCRs and non-GPCRs to Gα12/13. The effects of Gα12/13 on RhoA and other signaling pathways and their roles in cell proliferation, migration, and immune cell function, are discussed. Finally, we focus on the pathological impacts of Gα12/13 in cancer, inflammation, metabolic diseases, fibrotic diseases, and circulatory disorders are brought to focus.

Decoding Partner Specificity of Opioid Receptor Family.

This paper describes an exciting big data analysis compiled in a freely available database, which can be applied to characterize the coupling of different G-Protein coupled receptors (GPCRs) families with their intracellular partners. Opioid receptor (OR) family was used as case study in order to gain further insights into the physiological properties of these important drug targets, known to be associated with the opioid crisis, a huge socio-economic issue directly related to drug abuse. An extensive characterization of all members of the ORs family (μ (MOR), δ (DOR), κ (KOR), nociceptin (NOP)) and their corresponding binding partners (ARRs: Arr2, Arr3; G-protein: Gi1, Gi2, Gi3, Go, Gob, Gz, Gq, G11, G14, G15, G12, Gssh, Gslo) was carried out. A multi-step approach including models’ construction (multiple sequence alignment, homology modeling), complex assembling (protein complex refinement with HADDOCK and complex equilibration), and protein-protein interface characterization (including both structural and dynamics analysis) were performed. Our database can be easily applied to several GPCR sub-families, to determine the key structural and dynamical determinants involved in GPCR coupling selectivity.

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins provide insight into mechanisms of G protein selectivity.

G protein–coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11–GPCR complexes, the Gq–α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from “unwinding” of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs.

Functional approaches to the study of G-protein-coupled receptors in postmortem brain tissue: [35S]GTPγS binding assays combined with immunoprecipitation.

G-protein-coupled receptors (GPCRs) have an enormous biochemical importance as they bind to diverse extracellular ligands and regulate a variety of physiological and pathological responses. G-protein activation measures the functional consequence of receptor occupancy at one of the earliest receptor-mediated events. Receptor coupling to G-proteins promotes the GDP/GTP exchange on Gα subunits. Thus, modulation of the binding of the poorly hydrolysable GTP analog [35S]GTPγS to the Gα-protein subunit can be used as a functional approach to quantify GPCR interaction with agonist, antagonist or inverse agonist drugs. In order to determine receptor-mediated selective activation of the different Gα-proteins, [35S]GTPγS binding assays combined with immunodetection by specific antibodies have been developed and applied to physiological and pathological brain conditions. Currently, immunoprecipitation with magnetic beads and scintillation proximity assays are the most habitual techniques for this purpose. The present review summarizes the different procedures, advantages and limitations of the [35S]GTPγS binding assays combined with selective Gα-protein sequestration methods. Experience of functional coupling of several GPCRs to different Gα-proteins and recommendations for optimal performance in brain membranes are described. One of the biggest opportunities opened by these techniques is that they enable evaluation of biased agonism in the native tissue, which results in high interest in drug discovery. The available results derived from application of these functional methodologies to study GPCR dysfunctions in neuro-psychiatric disorders are also described. In conclusion, [35S]GTPγS binding combined with antibody-mediated immunodetection represents an useful method to separately evaluate the functional activity of drugs acting on GPCRs over each Gα-protein subtype.

The human GPCR signal transduction network

The eukaryotic cell surface G protein-coupled receptors (GPCRs) interact with a wide spectrum of ligands. The intracellular transmission of the extracellular signal is mediated by the selective coupling of GPCRs to G proteins, which, in turn, activate downstream effectors. GPCRs are of paramount pharmacological importance, with approximately 40% of all commercial drugs targeting these proteins. Herein, we have made an effort to unravel the molecular mechanisms underlying the GPCR-mediated signaling pathway and the way this pathway is associated with diseases. Network-based approaches were utilized to delineate the GPCR pathway, incorporating data from gene expression profiles across eleven healthy tissues and disease–gene associations from three diverse resources. The associations between the tissue-specific expression profiles of the disease-related genes along with the relative risk of disease development were further investigated. In the GPCR-activated pathway, the signal was found to be amplified at the successive steps of the pathway so that the effector molecules are highly expressed compared to ligands. This amplification effect was more pronounced when the respective genes encoding the particular proteins were associated with diseases. It was also found that co-expressed genes, corresponding to interacting molecules in affected tissues, may constitute powerful predictive markers for disease development. A disease risk prediction model based on tissue-specific expression profiles of the disease-associated genes was also generated. These findings could be applied to clinical settings.

A Native PAGE Assay for the Biochemical Characterization of G Protein Coupling to GPCRs.

G protein-coupled receptors (GPCRs) are a large family of membrane-embedded receptors that have diverse roles in physiology and are major drug targets. GPCRs transduce an agonist binding signal across the membrane to activate intracellular heterotrimeric G proteins. The dynamic nature of the receptors and the complexity of their interactions with agonists and G proteins present significant challenges for biochemical studies. Most biochemical/biophysical methods that have been employed to study GPCR-G protein coupling require purified receptors and are technically difficult. Here, we provide a protocol for a relatively simple and time- and cost-effective membrane protein native PAGE assay, to visualize and biochemically characterize agonist-dependent coupling of detergent-solubilized GPCRs to purified G protein surrogate "mini-G" proteins, which stabilize the receptor in an active state. The assay was developed for our studies of the calcitonin receptor-like receptor, a class B GPCR that mediates the actions of calcitonin gene-related peptide and adrenomedullin peptide agonists. It does not require a purified receptor and it can be used in a screening format with transiently-transfected adherent mammalian cell cultures, to quickly identify detergent-stable complexes amenable to study, or in a quantitative format with membrane preparations, to determine apparent affinities of agonists for the mini-G-coupled receptor and apparent affinities of mini-G proteins for the agonist-occupied receptor. The latter provides a partial measure of agonist efficacy. The method should be applicable to other GPCRs, and has the potential to be adapted to the study of other challenging membrane proteins and their complexes with binding partners. Graphic abstract: Visualizing agonist-dependent mini-G protein coupling and determining apparent binding affinities using the native PAGE assay quantitative formats.

PRECOG: PREdicting COupling probabilities of G-protein coupled receptors.

G-protein coupled receptors (GPCRs) control multiple physiological states by transducing a multitude of extracellular stimuli into the cell via coupling to intra-cellular heterotrimeric G-proteins. Deciphering which G-proteins couple to each of the hundreds of GPCRs present in a typical eukaryotic organism is therefore critical to understand signalling. Here, we present PRECOG (precog.russelllab.org): a web-server for predicting GPCR coupling, which allows users to: (i) predict coupling probabilities for GPCRs to individual G-proteins instead of subfamilies; (ii) visually inspect the protein sequence and structural features that are responsible for a particular coupling; (iii) suggest mutations to rationally design artificial GPCRs with new coupling properties based on predetermined coupling features.

Variable G protein determinants of GPCR coupling selectivity

G protein-coupled receptors (GPCRs) activate four families of heterotrimeric G proteins, and individual receptors must select a subset of G proteins to produce appropriate cellular responses. Although the precise mechanisms of coupling selectivity are uncertain, the Gα subunit C terminus is widely believed to be the primary determinant recognized by cognate receptors. Here, we directly assess coupling between 14 representative GPCRs and 16 Gα subunits, including one wild-type Gα subunit from each of the four families and 12 chimeras with exchanged C termini. We use a sensitive bioluminescence resonance energy transfer (BRET) assay that provides control over both ligand and nucleotide binding, and allows direct comparison across G protein families. We find that the Gs- and Gq-coupled receptors we studied are relatively promiscuous and always couple to some extent to Gi1 heterotrimers. In contrast, Gi-coupled receptors are more selective. Our results with Gα subunit chimeras show that the Gα C terminus is important for coupling selectivity, but no more so than the Gα subunit core. The relative importance of the Gα subunit core and C terminus is highly variable and, for some receptors, the Gα core is more important for selective coupling than the C terminus. Our results suggest general rules for GPCR-G protein coupling and demonstrate that the critical G protein determinants of selectivity vary widely, even for different receptors that couple to the same G protein.

Biased signaling of G protein coupled receptors (GPCRs): Molecular determinants of GPCR/transducer selectivity and therapeutic potential.

G protein coupled receptors (GPCRs) convey signals across membranes via interaction with G proteins. Originally, an individual GPCR was thought to signal through one G protein family, comprising cognate G proteins that mediate canonical receptor signaling. However, several deviations from canonical signaling pathways for GPCRs have been described. It is now clear that GPCRs can engage with multiple G proteins and the line between cognate and non-cognate signaling is increasingly blurred. Furthermore, GPCRs couple to non-G protein transducers, including β-arrestins or other scaffold proteins, to initiate additional signaling cascades. Receptor/transducer selectivity is dictated by agonist-induced receptor conformations as well as by collateral factors. In particular, ligands stabilize distinct receptor conformations to preferentially activate certain pathways, designated 'biased signaling'. In this regard, receptor sequence alignment and mutagenesis have helped to identify key receptor domains for receptor/transducer specificity. Furthermore, molecular structures of GPCRs bound to different ligands or transducers have provided detailed insights into mechanisms of coupling selectivity. However, receptor dimerization, compartmentalization, and trafficking, receptor-transducer-effector stoichiometry, and ligand residence and exposure times can each affect GPCR coupling. Extrinsic factors including cell type or assay conditions can also influence receptor signaling. Understanding these factors may lead to the development of improved biased ligands with the potential to enhance therapeutic benefit, while minimizing adverse effects. In this review, evidence for ligand-specific GPCR signaling toward different transducers or pathways is elaborated. Furthermore, molecular determinants of biased signaling toward these pathways and relevant examples of the potential clinical benefits and pitfalls of biased ligands are discussed.

CaMello-XR enables visualization and optogenetic control of Gq/11 signals and receptor trafficking in GPCR-specific domains

The signal specificity of G protein-coupled receptors (GPCRs) including serotonin receptors (5-HT-R) depends on the trafficking and localization of the GPCR within its subcellular signaling domain. Visualizing traffic-dependent GPCR signals in neurons is difficult, but important to understand the contribution of GPCRs to synaptic plasticity. We engineered CaMello (Ca2+-melanopsin-local-sensor) and CaMello-5HT2A for visualization of traffic-dependent Ca2+ signals in 5-HT2A-R domains. These constructs consist of the light-activated Gq/11 coupled melanopsin, mCherry and GCaMP6m for visualization of Ca2+ signals and receptor trafficking, and the 5-HT2A C-terminus for targeting into 5-HT2A-R domains. We show that the specific localization of the GPCR to its receptor domain drastically alters the dynamics and localization of the intracellular Ca2+ signals in different neuronal populations in vitro and in vivo. The CaMello method may be extended to every GPCR coupling to the Gq/11 pathway to help unravel new receptor-specific functions in respect to synaptic plasticity and GPCR localization.

Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells

G protein–coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase–mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype–biased ligands.

Role of G protein-coupled receptors-microRNA interactions in gastrointestinal pathophysiology.

G protein-coupled receptors (GPCRs) make up the largest transmembrane receptor superfamily in the human genome and are expressed in nearly all gastrointestinal cell types. Coupling of GPCRs and their respective ligands activates various phosphotransferases in the cytoplasm, and, thus, activation of GPCR signaling in intestine regulates many cellular and physiological processes. Studies in microRNAs (miRNAs) demonstrate that they represent critical epigenetic regulators of different pathophysiological responses in different organs and cell types in humans and animals. Here, we reviewed recent research on GPCR-miRNA interactions related to gastrointestinal pathophysiology, such as inflammatory bowel diseases, irritable bowel syndrome, and gastrointestinal cancers. Given that the presence of different types of cells in the gastrointestinal tract suggests the importance of cell-cell interactions in maintaining gastrointestinal homeostasis, we also discuss how GPCR-miRNA interactions regulate gene expression at the cellular level and subsequently modulate gastrointestinal pathophysiology through molecular regulatory circuits and cell-cell interactions. These studies helped identify novel molecular pathways leading to the discovery of potential biomarkers for gastrointestinal diseases.

Fingerprinting G protein-coupled receptor signaling.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) control a wide diversity of cellular responses throughout biology. Each GPCR couples to a distinct array of members of the G protein family to control the specificity and diversity of these responses and ultimately determines the therapeutic efficacy of drugs that target these receptors. In this issue of Science Signaling, Masuho et al. developed an approach to broadly defining these GPCR coupling fingerprints.

Calcium Channel Signaling Complexes with Receptors and Channels.

Voltage-gated calcium channels are not only mediators of cell signalling events, but also are recipients of signalling inputs from G protein coupled receptors (GPCRs) and their associated second messenger pathways. The coupling of GPCRs to calcium channels is optimized through the formation of receptor-channel complexes. In addition, this provides a mechanism for receptorchannel co-trafficking to and from the plasma membrane. On the other hand, voltage-gated calcium channel activity affects other types of ion channels such as voltage-and calcium-activated potassium channels. Coupling efficiency between these two families of channels is also enhanced through the formation of channel-channel complexes. This review provides a concise overview of the current state of knowledge on the physical interactions between voltage-gated calcium channels and members of the GPCR family, and with other types of ion channels.

Identification of amino acids that are selectively involved in Gi/o activation by rat melanin-concentrating hormone receptor 1

Many G-protein-coupled receptors (GPCRs) are known to functionally couple to multiple G-protein subfamily members. Although promiscuous G-protein coupling enables GPCRs to mediate diverse signals, only a few GPCRs have been identified with differential determinants for coupling to distinct Gα proteins. Mammalian melanin-concentrating hormone receptor 1 (MCHR1) couples to dual G-protein subfamilies. However, the selectivity mechanisms between MCHR1 and different subtypes of Gα proteins are unclear. Our previous studies demonstrated that mammalian MCHR1 couples to both Gi/o and Gq, whereas goldfish MCHR1 exclusively couples to Gq. In this study, we analyzed multiple sequence alignments between rat and goldfish MCHR1s, and designed three multisubstituted mutants of rat MCHR1 by replacing corresponding residues with those in goldfish MCHR1, focusing on regions around the cytosolic intracellular loops. By measurement of intracellular Ca2+ mobilization, we found that two MCHR1 mutants, i2_6sub and i3_6sub, which contained six simultaneously substituted residues in the second intracellular loop or a combination of substituted residues in the third intracellular loop and fifth transmembrane domain, respectively, significantly reduced Gi/o-sensitive pertussis toxin responsiveness without altering Gq-mediated activity. Analyses of 10 other substitutions revealed that the multiple substitutions in i2_6sub and i3_6sub were necessary for Gi/o-selective responses. As judged by Gi/o-dependent GTPγS binding and cyclic AMP assays, i2_6sub and i3_6sub elicited phenotypes for impaired Gi/o-mediated signaling. We also monitored the dynamic mass redistribution (DMR) in living cells, which reveals receptor activity as an optical trace containing activation of all GPCR coupling classes. Cells transfected with i2_6sub or i3_6sub exhibited reduced Gi/o-mediated DMR responses compared with those transfected with MCHR1. These data suggest that two different regions independently affect the Gi/o-protein preference, and that multiple residues comprise a conformation favoring Gi/o-protein coupling and subsequently result in Gi/o-selective signaling.

Fingerprinting of GPCR activity across multiple G protein substrates unveils complex profiles of functional selectivity (1066.12)

Despite the tractability of G protein‐coupled receptors (GPCRs) as drug targets, there are substantial challenges in understanding the mechanisms of drug action at these receptors. Since more than 800 GPCRs and 16 Gα subunits are identified in human genome and are capable to generate enormous array of GPCR‐G protein combinations, G protein‐coupling profiles of GPCRs are a critical missing piece of the GPCR‐signaling puzzle. Here, we have undertaken an attempt to comprehensively characterize G protein‐coupling selectivity and its modulation by agonists and intracellular regulatory proteins. We employed a single‐platform BRET assay to evaluate GPCR responses via 14 different Gα subunits using dynamic association/dissociation of Venus‐tagged Gβγ with NanoLuc‐tagged GRK3‐derived sensor in living cells. Distinct G protein‐coupling profiles for different receptors in both maximal amplitudes and kinetics of responses were observed and used as a basis for modeling “fingerprints” for each of the examined receptors. These fingerprints in many cases differed between physiological agonists and synthetic ligands. We further found that members of Regulators of G protein Signaling (RGS) family shape GPCR fingerprints. These results reveal underappreciated complexity of GPCR coupling to different G protein substrates and suggest a new avenue for designing signaling pathway‐specific therapeutics.Grant Funding Source: Supported by the NIH grants HL105550 and NS081282.

The Couple G Protein-Coupled Receptor-G Protein: Preassembly versus Agonist-Induced Interaction

The investigation of the physical and functional interaction of the G protein-coupled receptors (GPCRs) with their cognate heterotrimeric G proteins constitutes one of the most evolved aspects in this family of cell surface receptors. The development of the energy transfer-based approaches, bioluminescence or fluorescence resonance energy transfer (BRET or FRET, respectively), has strongly contributed to such advance. Using protease-activated receptors 1 and 2 (PAR1 and 2) as model for their multiple G protein coupling profiles, we revealed a preassembly between the two receptors and Gαi1 and Gαo proteins whereas their association with Gα12 occurred only upon receptor activation. Kinetic analysis in real-time and live cells showed important differences in the activation between these two modes of coupling which may be consistent with their implication in the physiological responses of PARs. Together, our finding indicate that preassembly or agonist-promoted association depend on receptor-G protein pair representing the key mechanisms of temporal and spatial regulation and integration of the multiple G protein coupling and signaling of GPCRs.